Inhibition of Amino Acid Decarboxylases*

CHAPTER 9

William Gilbert Clark

I. Introduction 316 II. Pyridoxal Kinase 318 III. Apoenzyme-Coenzyme Dissociation 319 IV. Transport, Uptake, Binding, Release 320 V. Inanition 322 VI. Tissue Damage, Growth, Neoplasms, Organectomy 322 VII. Apoenzyme Synthesis 323 VIII. Inhibition by Apoenzyme Antibody 326 IX. Metals, Chelators, and Metal Complexers 327 X. Cyanide 330 XI. Pyridoxal-5-Phosphate (Codecarboxylase), Vitamin B6, Be-Deficien- cy, Be-Antagonists 331 A. Ββ-Deficiency and Antagonists 331 B. B6-P04 Hydrazones 334 C. Toxopyrimidine 335 D. Steroids 337 XII. Activators, Stabilizers, and Cofactors Other Than Be-P04 337 A. Metals 337 B. Surfactants 337 C. Solvents 337 D. Phosphates and Arsenate 338 E. Miscellaneous 338 XIII. Carbonyl Reagents and Inhibitors Acting on Coenzyme Other Than Cyanide, Substrate Analogues, and Pyridoxine Antagonists 339 A. Hydroxylamine, Hydrazides, Semicarbazide, Sulfite, Hydrazine, Oximes, etc 339 B. Cycloserine (4-Amino-3-isoxazolidone) 343 C. Penicillamine 345 D. Sulfonylureas 345 E. Cysteine, 2,3-Dimercapto-l-propanol (BAL), Glutathione 346 F. Ascorbic Acid 346

* Supported by Grants from the National Mental Health Association, U.S. Public Health Service, American Heart Association, Los Angeles County Heart Association, U.S. Army Chemical Center, American Cancer Society, Office of Naval Research, Helen Hay Whitney, Jr. Foundation, Life Insurance Medical Research Fund. 315 316 W. G. CLARK

XIV. Hormones 346 A. Insulin 347 B. Pituitary 347 C. Adrenal Cortex 347 D. Thyroid 349 E. Sex Hormones 352 XV. Miscellaneous 354 A. Antibiotics 354 B. Antihistamines 354 C. Reserpine 354 D. Tranquilizers 354 E. Tetrahydroisoquinolines 354 F. Folic Acid Antagonists 355 XVI. Substrate Analogues 356 A. Bacterial and Plant Decarboxylases 356 B. Mammalian (and Fowl) Decarboxylases in vitro 357 C. Mammalian Dopa Decarboxylase in vitro 358 D. Mammalian 5-HTP Decarboxylase in vitro 358 E. Glutamic Acid Decarboxylase in vivo 359 F. Dopa Decarboxylase in vivo 359 G. Quinones and Potential Quinoids 360 H. Ketonuria 361 I. α-Alkyl Substrate Analogues 363 References 366

I. INTRODUCTION

Although enzymic decarboxylation plays a minor role quantitatively ni metabolism fo amino acids, ti si na important one because fo the critical nature, marked pharmacological activity, and function fo the end products. The physiological significance fo most, fi not all, fo these end products still remains ot eb clarified, but many fo them probably are essential for a num• ber fo homeostatic regulatory and adaptive mechanisms ni all living organ• isms. tI was not until 1936-1937 that amino acid decarboxylases were de• scribed yb Okunuki (1937) ni plants (glutamic acid decarboxylase) and yb Werle (1936) and Holtz and Heise (1937, 1938) [histidine and 3,4-dihy- droxyphenylanine (dopa) decarboxylase]. This and other work, including data no inhibitors fo these decarboxylases, have been reviewed yb Gale (1940b, 1946, 1953), Holtz (1941), Storck (1951), Janke (1951), Karrer (1947), Mardashew (1949), Schales (1951), Mèister (1955, 1957), Werle (1943a,b, 1947, 1951), and Ruiz and Zaragoza (1959). It si the purpose fo this review ot discuss inhibition fo the amino acid decarboxylases ni general, with some emphasis no more recent contribu• tions (up ot early 1961 ni most cases). Inhibition yb general enzyme "poisons" ro dénaturants in vitro, such sa trichloroacetate and alkylat- 9. INHIBITION OF AMINO ACID DECARBOXYLASES 317 ing agents, will not be included. Clark (1959) and Clark and Pogrund (1961) recently reviewed the subject of dopa decarboxylase inhibition in vitro and in vivo, and Sourkes and D'lorio discuss the subject in Volume II of this treatise. Since many inhibitors of these enzyme systems exert their effects directly or indirectly through pyridoxal-5-phosphate (B6-P04), the coenzyme of amino acid decarboxylases, some aspects of the vitamin B6-dependent enzymes in general must be considered. Ref erence should be made to the extensive reviews available of the pyridoxine-dependent enzymes by Blaschko (1945a), Gunsalus (1951), Wil liams et al. (1950), Sherman (1954), Tower (1956, 1959, 1960), Umbreit (1954), Meister (1957), Mathews (1958), Snell (1958), Snell and Jenkins (1959), Siliprandi (1960), Roberts and Eidelberg (1959), Roberts et al (1960), Braunstein (I960), and Axelrod and Martin (1961). Braunstein's review, "Pyridoxal Phosphate" (1960), and that of Snell (1959), "Chemical Structure in Relation to Biological Activities of Vitamin B6," are particu larly exhaustive. The amino acid decarboxylases described so far catalyze the following reactions:

(1 Glycine —> methylamine (2; L-Alanine —> ethylamine (3: L-Serine —> ethanolamine (4; α-Aminobutyric acid —• propylamine (5; L-Methionine —> 3-methylthiopropylamine (β: L-Valine —> isobutylamine (7; L-Norvaline —• butylamine (s: L-Leucine —> isoamylamine (9: L-Isoleucine —* 3-methylbutylamine do: L-Aspartic acid —> L-alanine (11 L-Arginine —> agmatine (12: L-Histidine —> histamine (13 L-Aspartic acid —• /3-alanine (14 L-Ornithine —> putrescine (15 L-Lysine —> cadaverine (16 meso-δ-6-Diaminopimelic acid —> L-lysine (17 α-Aminomalonic acid —* glycine (is: δ-Hydroxy-L-lysine —» hydroxycadaverine (i9: L-Glutamic acid —> 7-aminobutyric acid (20 Allo-jS-hydroxy-L-glutamic acid —• 7-amino-/3-hydroxybutyric acid (21 7-Hydroxy-L-glutamic acid —• a-hydroxy-7-aminobutyric acid (22 7-Methylene-L-glutamic acid —> 7-amino-a-methylene butyric acid (23 L-Cysteic acid —> taurine (24 L-Cysteinesulfinic acid —> hypotaurine (25: L-Tryptophan -> tryptamine (26 4-Hydroxy-L-tryptophan —* 4-hydroxytryptamine (27 5-Hydroxy-L-tryptophan (^δ-ΗΤΡ") -> 5-hydroxytryptamine (serotonin, "5HT") 318 W. G. CLARK

(28) 6-Hydroxy-L-tryptophan —» 6-hydroxytryptamine (29) α-Methyl-L-tryptophan —» a-methyltryptamine (30) a-Methyl-5-hydroxy-L-tryptophan —» a-methyl-5-hydroxytryptamine (31) a. L-Phenylalanine —> phenylethylamine Ring-substituted hydroxy-L-phenylalanines to corresponding amines, e.g. : b. L-Tyrosine —» tyramine c. Diiodo-L-tyrosine —* diiodotyramine (Werle, 1947; not confirmed) d. 2-Hydroxy-L-phenylalanine (o-tyrosine) —> 2-hydroxyphenylethylamine (o-tyramine) e. 3-Hydroxy-L-phenylalanine (ra-tyrosine) —• ra-tyramine f. a-Methyl-3-hydroxy-L-phenylalanine (α-methyl-m-tyrosine) —> a-methyl- 3-hydroxyphenylethylamine (a-methyl-ra-tyramine) g. 3,4-Dihydroxy-L-phenylalanine (dopa) —• 3,4-dihydroxytyramine (dopa mine) (32) a. L-Phenylserine —• phenylethanolamine b. 2-Hydroxy-L-phenylserine —• 2-hydroxyphenylethanolamine c. 3-Hydroxy-L-phenylserine —> 3-hydroxyphenylethanolamine d. 4-Hydroxy-L-phenylserine —• 4-hydroxyphenylethanolamine (octopamine, norsynephrine) e. 3,4-Dihydroxy-L-phenylserine (dops) —> 3,4-dihydroxyphenylethanol- amine (arterenol, norepinephrine) Some of these reactions have been shown to be catalyzed by one and the same enzyme, and possibilities of this kind should be borne in mind in considering this section. Further discussion of this point, classifying and documenting the individual enzymes by their distribution in micro organisms, plants, and animals, the structures and stereospecificity of their substrates, their kinetics, apoenzyme-coenzyme affinity and dissoci ation must be sought in the reviews cited. Since the introduction of tracer techniques and amine catabolic enzyme inhibitors, many decarboxylations formerly thought to be absent in animals are being announced.

II. PYRIDOXAL KINASE

Snell (1959) and McCormick et al (1961; McCormick and Snell, 1961) have discussed codecarboxylase kinase (pyridoxal phosphokinase) and have reviewed the literature. Chevillard and Thoai (1951) and Thoai and Chevillard (1951a,b) showed that Mg+ + and Mn+ + activate it, ATP or ADP are necessary, and thiamine inhibits it. Hurwitz (1952, 1953, 1955) showed that some pyridoxine analogues inhibit tyrosine decarboxylase in bacteria, while others inhibit the phosphorylation of pyridoxal in the presence of ATP. Some adenine and purine derivatives inhibit competi tively and several metal cations activate (cf. McCormick et al., 1961; McCormick and Snell, 1961). Hurwitz suggested that the adenines and purines act through the activating metallic ions. McCormick and Snell 9. INHIBITION OF AMINO ACID DECARBOXYLASES 319

(1959, 1961) and McCormick et al. (1960, 1961) showed that purified pyridoxal phosphokinase from brain is markedly inhibited by a variety of condensation products formed from pyridoxal and hydroxylamine, O-substituted hydroxylamines, hydrazine, and substituted hydrazines; they questioned former explanations of the convulsive effects of such carbonyl reagents which causally related the seizures to lowered brain 7-amino butyric acid (gaba) through interaction with B6-PO4. Dubnick and co workers (1960c) postulated that pyridoxal hydrazones are formed from hydrazines and B6-P04 in vivo and exert toxic effects by inhibiting the phosphokinase (cf. Balzerei al., 1960a,b,c; and Baxter and Roberts, 1960). Recently, Wada and Snell (1961), Turner and Happold (1961), and Wada et al. (1961) described an enzyme which oxidizes pyridoxine and pyridoxamine phosphates to B6-PO4. Evidently, the primary pathway of the formation of B6-P04 is phosphorylation by the kinase, followed by the action of the oxidase. The oxidase, a flavoprotein with riboflavin-5'- phosphate as a cofactor, is sensitive to thiol reagents, heavy metals, and some phosphorylated analogues of Be, especially pyridoxal phosphate oxime. Greenberg et al. (1959) observed that chlorpromazine had little or no effect on glutamic acid decarboxylase of rat brain, but that the kinase in brains of B6-deficient rats has a greater susceptibility to inhibition by chlorpromazine than that in- normal rats. It is anticipated that many compounds acting on B6-enzyme systems, including the decarboxylases, will be shown to do so by effects on the kinase and the oxidase.

III. APOENZYME-COENZYME DISSOCIATION

Mason (1957) showed that phosphate ions may, at somewhat higher concentrations, inhibit transamination of kynurenine by rat kidney and that this can be reversed by B6-PO4. Certain keto acids in low concentra tions also prevented the inhibition by decreasing dissociation and main taining the holoenzyme in a more stable apoenzyme-coenzyme form. Hartman et al. (1955) showed that inorganic phosphate and arsenate re activated dialyzed dopa decarboxylase of hog kidney when added to gether with B6-P04, but that none of these three substances reactivated alone. It is possible that this effect, like that in Mason's preparation, can also be explained by an effect of phosphate or arsenate on the apoenzyme- coenzyme dissociation. A systematic study of this should be undertaken on the effect on decarboxylations. Werle and Aures (1959) showed that the reaction rates of purified dopa, 3,4-dihydroxyphenylserine (dops), and 5-HTP decarboxylases are differ ent, but that when the differences in requirement, sensitivity, and affinity 320 W. G. CLARK of coenzyme-apoenzyme in the presence of the different substrates are taken into account, the activity ratios of the decarboxylations of these three substrates remain constant through all purification steps, thus indicating that the same enzyme decarboxylates all three substrates. Other evidence supporting this contention now is fairly conclusive and has been summarized elsewhere by this reviewer (1959; Clark and Pogrund, 1961); and it is discussed later in this chapter. Ekladius and co-workers (1957) showed that leucine competitively inhibits valine decarboxylase in bacteria, depending on the amount of coenzyme present. Leucine decarboxylation is less dependent on this factor. When coenzyme is not present in excess, the decarboxylation of either amino acid is completely inhibited by the other.

IV. TRANSPORT, UPTAKE, BINDING, RELEASE

Werle (1947) was the first to include among decarboxylase inhibitors substances which might inhibit uptake by cells of the amino acid sub strates. The subject of the active absorption of amino acids has been reviewed and investigated by Christensen (1955, 1960), Meister (1957), Guroff and Udenfriend (1960), Neame (1961), Edelman (1961), and Gagnon (1961). The uptake can be blocked in some instances by inhibitors of respiratory metabolism or associated phosphorylative enzyme systems. Schanberg and Giarman (1960a, b) found that the active uptake of labeled 5-HTP by brain and other tissue slices is inhibited by 02 lack, low tem perature, and 2,4-dinitrophenol (DNP). The uptake was most active in those tissues where 5-HTP decarboxylation is greatest. Schanberg et al. (1961) also showed that the active uptake of 5-HTP by brain slices is markedly inhibited by tryptophan, tyrosine, and dopa, but not by a-methyldopa, glutamic acid, and gaba. This correlated with a decrease of brain 5-HT in rats fed large doses of tryptophan. Active uptake o+ f +dopa by+ mitochondrial particles of guinea pig brain is inhibited by Ca , Mg+ , and nonionic detergents, but not by inhibitors of oxidative phosphorylation (Iwamoto and Nukada, 1961). Neame (1961) reported active uptake of six amino acids by brain slices, inhibited by 02 lack and cyanide. Weiss- bach et al. (1960b) arid Weissbach and Redfield (1960) reported that tryptophan, but not tyrosine or 5-HTP, is actively absorbed by blood platelets. The process is not inhibited by fluoride as is the active uptake of 5-HT, a glycolytic enzyme-dependent system (vide infra). Christensen and others (cf. Moldave, 1958) found that pyridoxine is involved. Jacobs et. al. (1960) found that the inhibitory effect of DNP on active uptake of L-methionine from the intestine is partially counteracted by Be-P04 9. INHIBITION OF AMINO ACID DECARBOXYLASES 321 but not B6. They also showed the active intestinal absorption of L-tyrosine is B6-P04-dependent [but cf. Guroff and Udenfriend (I960)]. They reviewed work which supports the B6-dependent concept and mechanisms proposed by Pal and Christensen (1958), Riggs and Walker (1958), Christensen et al. (1960), Christensen (1960), Shishova (1956) and others (cf. Riggs and Walker, 1960, and related studies mentioned in Section XIV, B). Akedo et al. (1960) and Ueda et al. (1960) showed impaired active intestinal absorption of L-amino acids in B6-deficient rats and found that B6-PC>4 prevents the in hibitory effect of DNP. Tsukada et al. (1960) showed that the active uptake of gaba by brain slices is augmented by pyridoxal. One mechanism proposed for the participation of pyridoxal in, for example, the transport of glycine is that it increases potassium efflux, which in turn increases glycine uptake. Hemplin42g (1961) in a preliminary abstract tested this theory by measur ing K fluxes in the presence and absence of pyridoxal during glycine uptake, and found that pyridoxine decreased the influx and efflux, but not if glycine was present, since glycine stimulates potassium fluxes. Oxender and Royer (1961) found that the stimulation of active amino acid uptake by Ehrlich ascites cells by pyridoxal and B6-P04 was not inhibited by carbonyl reagents; hence, the B6 substances may not be directly involved as carriers. Christensen (1955) showed that α-methylamino acids were often par ticularly active in the competitive inhibition of active uptake of amino acids by cells. If a methyl group was introduced on the α-carbon, the degree of accumulation was greater, due perhaps to the formation of a Schiff base with Be-derivatives. It was suggested that the absence of an α-methyl group would prevent electromeric shifts of the double bond which characterized Be-derivatives. Thus, the α-methyl group might pre vent the diversion of portions of the hypothetical carrier amino acid complex into forms which would not redissociate readily. The active up take of amino acids is competitively inhibited by other amino acids (Chris tensen, 1955, 1960; Wiseman and Ghadially, 1955; Hagihira et al., 1960, 1961; Lin and Hastings, 1960; Tenenhouse and Quastel, 1960; Jacquez, 1961), and Lin et al. (1961) showed that a free carboxyl group is essential for the transport, that substitutions on the α-amino group which prevent Schiff base formation block the transport, and that the α-hydrogen seems important, since replacement by a methyl group decreases uptake, for example, a-methyl-DL-tyrosine. a-Methylphenylalanine analogues have been shown to inhibit the "nonspecific" aromatic amino acid decarboxylase of mammals (see Sec tion XVI, I). Goldberg et al. (1960), Porter et al. (1961), Hess et al. (1961), and Sourkes et al. (1961) have shown, however, that the effects of these analogues on tissue amine levels in vivo are due much more to a defect 322 W. G. CLARK

in an uptake-binding-release mechanism than to inhibition of the enzyme. In connection with uptake, Oates and associates (1960b) found no effect of α-methyldopa on the active absorption of tyrosine by the intestine, brain, or muscle.

V. INANITION

Since starvation can affect synthesis of proteins, including apoenzymes and coenzymes, in vivo, this factor must be controlled by paired feeding and similar measures in experiments involving B6- and other vitamin de ficiencies, febrile diseases, thyrotoxicosis, neoplasms, and other debilitating states possibly affecting amino acid decarboxylation in vivo (cf. Blaschko, 1945b, Armstrong et al, 1950; Holtz et al, 1956; Blaschko et al, 1948; and Weil-Malherbe, 1956). Failure to consider such nutritional factors by some biochemists casts doubt on many reports in the literature.

VI. TISSUE DAMAGE, GROWTH, NEOPLASMS, ORGANECTOMY

Hawkins and Walker (1952), in studying the effect of rapid nuclear and cellular division on soluble and insoluble enzyme activity, examined dopa decarboxylation by rat liver and adrenal medulla after partial hepatectomy. Before compensatory hypertrophy was initiated, the enzyme activity de creased, but was restored nearly to normal with compensatory hyper trophy. This increase, however, was no1t4 initiated at the same time as growth. Kahlson et al, using histidine-C on the other hand, found histi dine decarboxylase (presumably the "specific" L-histidine decarboxylase discussed in Section VII) markedly increased in such livers as well as in wound healing (Kahlson, 1960; Kahlson et al, 1960a) and in embryos (Kahlson et al, 1958, 1959, 1960b; Kahlson and Rosengren, 1959b), and believe that histamine plays a role in growth (Kahlson, 1960) and neo plasms. Kizer and Chan (1961) found a correlation between impairment of 5-HTP decarboxylation and carcinogenesis in rat liver. Aures, in this laboratory (unpublished), could not confirm Kahlson's report of increased histidine decarboxylase in the rapidly growing liver after partial hepatec tomy in rats. Clark (1959; Clark and Pogrund, 1961) showed that if cats and rats are kept in good physiological condition after acute nephrectomy, gastrectomy, enterectomy, or indeed total evisceration, blood pressure responses to intravenously administered dopa remain nearly normal, indicating an 9. INHIBITION OF AMINO ACID DECARBOXYLASES 323 ubiquitous distribution of dopa decarboxylase. This does not mean, of course, that effects are not seen some time after castration, ovariectomy, pancreatectomy, thyroidectomy, or adrenalectomy (see Section XIV, E). Of importance is the observation of Gey and Pletscher (1960) that 5-HTP decarboxylase activity of brains removed from rats and left at 37°C shows a decrease during the first half-hour. The enzyme activity continues for some time, however. This observation subsequently led the authors (Pletscher and Gey, 1961) to develop a useful assay for 5-HTP decarboxylase activity and its inhibitors by analyzing for brain 5-HT in incubated heads of rats decapitated after injection of 5-HTP and inhibitor.

VII. APOENZYME SYNTHESIS

When discussing inhibitors of enzyme systems, consideration must be made of the effects of agents and conditions on the synthesis of apoenzymes, some of which can be through adaptive or induced mechanisms, both in microorganisms and in higher animals. The former has been reviewed by Pollock (1959) and the latter by Knox et at. (1956). Gale (1940a) and others have shown that many bacterial enzymes, including amino acid decarboxylases, are deficient or absent if their sub strates are absent and are adaptively induced when these are added. The story is complicated by cross induction and many other factors; for ex ample, an uncharacterized inhibitor of several amino acid decarboxylases is produced by Pseudomonas reptilivora, when assays are carried out with heavy cell suspensions (Seaman, 1960). A single inducer may affect more than one enzyme (Ando, 1959b). Further, substrate analogues may com petitively inhibit induced synthesis. Mandelstam (1956) showed that in duced syntheses of ornithine and lysine decarboxylases in Bacillus cadaveris are reversibly inhibited by β-phenylserine and 5-methyltryptophan. Pre sumably many reports of bacterial growth inhibition by substrate analogues, if re-examined experimentally with methods now available, might reveal such effects on induced syntheses of other amino acid decarboxylases, as well as other enzymes. D-Chloramphenicol (but not the L-isomer), a phenylserine analogue, exerts part, if not all, of its antibiotic action on the ATP-dependent syn thesis of proteins, including certain bacterial apoenzymes (cf. Section XV, A). Thus, Grunberger and Sorm (1954), Grunberger et al. (1948, 1955), and Sorm and Grunberger (1953) showed that this and other anti biotics (chlortetracycline, oxytetracycline) in subgrowth-inhibitory con centrations inhibit the production of glutamic acid, lysine, and arginine decarboxylases and aspartic acid oxidase in Escherichia coli, but not that 324 W. G. CLARK

of tyrosine decarboxylase yb Streptococcus faecalis. Sorm et al. (1955) also showed that strains fo E. coli made slightly resistant ot chloramphenicol, synthesize less glutamic acid decarboxylase than those made highly re• sistant, which have normal levels. Further, Grunberger and Sorm (1954) showed that the drug has on inhibitory effect no four transaminases, pyridoxal kinase, ro adaptive enzymes which participate ni carbohydrate metabolism; thus ti si fairly specific. tI si interesting that Lagerborg and Clapper (1952) found arginine, glutamic acid, tyrosine, and histidine de• carboxylases present ni some but not all fo thirty-three strains fo lacto• bacilli examined. Four strains fo Streptococcus mitis and two fo S. faecalis which lacked these enzymes produced arginine and tyrosine decarboxylases when made resistant ot sulfathiazole. No explanation si given. Inhibitors fo nucleic acid metabolism can prevent the adaptive synthesis of apoenzymes. Thus, Ando (1959a) showed that 8-azaguanine, ni concen• trations insufficient ot affect cellular growth, strongly inhibits the induced synthesis fo histidine decarboxylase ni Proteus morganii. Excessive amounts of purines and pyrimidines may also inhibit induced apoenzyme synthesis, as shown yb Bellamy and Gunsalus (1946) with uracil and guanine no tyrosine decarboxylase fo S. faecalis. Melnykovych and Johansson (1955) and Melnykovych et al. (Melnyko• vych and Johansson, 1958, 1959; Melnykovych and Snell, 1958), ni studies of the mechanism fo growth-stimulating effects fo antibiotics ni animals, found that subgrowth-inhibitory levels fo several but not all antibiotics studied, ni addition ot inhibiting enzymic decarboxylation fo several amino acid decarboxylases yb E. coli, stimulate their inductive synthesis. They speculate no the possibility that these antibiotics may favorably influence animal growth yb preventing the production fo toxic amines yb intestinal bacterial flora but that, insofar sa the inductive syntheses are concerned, they may ni some way favorably affect the integrity fo the holodecar- boxylases. Ehrismann and Werle (1948) found that the nonadaptive synthesis fo histidine decarboxylase yb strains fo E. coli, B. parasarco- physematos, and aClostridium si inhibited yb semicarbazide. Sher and Mallette (1954) found that bacteriophage infection fo E. coli blocks the adaptive synthesis fo lysine decarboxylase. The phage ghosts also are active. An explanation si lacking. Rajewsky et al. (1959) and Bùcker et al. (1960) studied the ultraviolet irradiation inactivation spectrum fo the inductive synthesis fo lysine decarboxylase ni Bacterium cadaveris. The spectrum si identical with that of nucleic acids, and the spectrum fo inactivation for the maximal attain• able enzyme activity si similar ot the action spectrum fo a cysteine-rich protein. They found a satisfactory correlation with the action spectrum fo ribonuclease and suggest that the velocity fo the induced enzyme synthesis 9. INHIBITION OF AMINO ACID DECARBOXYLASES 325

s controlled by a nucleic acid but that the final activity is controlled by mother mechanism. This reviewer could find no reports in the literature of "feedback con trol" of amino acid decarboxylase synthesis (nor of decarboxylation rate tself for that matter) similar to those discussed by Doy and Pittard ^1960). These authors found that in Aerobacter aerogenes tryptophan may ;ontrol its own biosynthesis by inhibiting ("negative feedback control") he action of an enzyme necessary for a stage prior to anthranilic acid; they review nine publications on other feedback controls of enzyme syn thesis. Such considerations have not been made in the case of animal enzymes, but this should be taken into account in studies of inhibitors in intact cells and animals, especially if specificity is claimed. Knox and associates (1956) in their review on enzymic adaptations in animals do not list decarboxylases among the enzyme systems induced by treatment with substrates. Shishova and Gorozhankina (1959) found in creased histidine decarboxylase in the blood of rats fed large amounts of histidine in the diet. They also reported an increase in histidine decar boxylation by livers of rats injected with cortisone. Several enzymes in animals have been reported to be induced adaptively by adrenal cortical hormones (Knox et al, 1956). Schayer, who introduced a new era in the biochemistry, physiology, and pharmacology of the catechol amines and histamine by first using radio isotope techniques for the study of the metabolism of these compounds, has made the remarkable discovery that histidine decarboxylase synthesis can be rapidly and markedly stimulated in intact animals by a number of nonspecific "stressful" treatments. These include injections of histamine releasers, histamine itself, endotoxin, adrenaline, 5-HT, burns, exposure to low temperature, development of the tuberculin reaction, and sensitiza tion to pertussis vaccine (Schayer et al., 1959, 1960; Schayer and Ganley, 1959a, b, 1960, 1961 ; Schayer, 1960, 1961, 1962). It is possible that the sea sonal variations of dopa decarboxylase activity of guinea pig kidney ex tracts, shown by Polonovski and co-workers (1946) to be due to temperature effects on the whole animal, are caused by a nonspecific effect on the adaptive synthesis of apoenzyme. In one paper, Schayer et al. (1959) sug gests that the effect may be related to the production of new, resistant mast cells, active in forming the "specific" L-histidine decarboxylase (vide infra). It would be interesting to see if the enzyme in basophilic leucocytes is adaptively affected because of their similarity to mast cells and because they are the only source of unreleased histamine in most species except the rabbit and contain the enzyme (Hartman et al., 1961). The adaptive enzyme-induced production of histamine by other tissues is much greater than in blood, however, and Schayer et al. (1959; Schayer and 326 W. G. CLARK

Ganley, 1959a, b, 1960, 1961 ; Schayer, 1959, 1960, 1961, 1962) have in voked it in the homeostasis of the microcirculation. If true, the physio logical implications will be profound. Kahlson et al. (1960a; Kahlson, 1960) found that the inhibition of histidine decarboxylase activity in intact rats, caused by B6-deficiency and/or semicarbazide inactivation of the coenzyme, is followed by an overshoot in activity, caused by adaptive biosynthesis of new apoenzyme when the inhibition is terminated. It is possible that the histidine decar boxylase of mast cells (Weissbach 1961), rat fundus (Schayer, 1956c, et al.y 1957), embryonic rat organs (Ganrot et al., 1961), and probably the adaptive enzyme of Schayer (see previous paragraph) and of Kahlson (Section VI) is an L-histidine decarboxylase, unrelated to the general aromatic amino acid decarboxylase of most mammalian tissues (Lovenberg et al., 1962). An adaptive increase in the nonspecific decarboxylase has not been reported. It would be of great interest to find effective inhibitors of the "specific" histidine decarboxylase, since those which inhibit the "nonspecific" enzyme do not inhibit the "specific" one (see Section XVI, I).

VIII. INHIBITION BY APOENZYME ANTIBODY

Happold and Ryden (1952) attempted to obtain immune rabbit serum by using tyrosine apodecarboxylase of S. faecalis prepared by the method of Epps (1944, 1945) as antigen, and although a precipitin test was ob tained against the original antigen, the enzyme activity remained un changed or was slightly increased after removing the precipitate. Howe and Treffers (1952) were more successful with lysine decarboxylase par tially purified from E. coli used as antigen in rabbits, obtaining 90% inhibition of homologous enzyme and no inhibition of glutamic acid de carboxylase. All activity was found in the precipitate obtained by the precipitin reaction. Gubarev (1960), using glutamic acid decarboxylase of Bacillus dysenteriae purified by starch column electrophoresis as antigen, obtained an active rabbit antiserum which partially inactivated the original homologous enzyme. If apoenzymes could be sufficiently purified, it would be of interest for purposes of classification to attempt similar experiments with the other decarboxylases, especially in higher organisms. Whether or not inhibitions could be obtained by such immunological approaches in higher animals remains to be seen, but the physiological implications would be of great interest. The general subject of possible inhibitions by immunological approaches in higher animals was reviewed some years ago by Marrack (1951). If the 9. INHIBITION OF AMINO ACID DECARBOXYLASES 327 antigenic groups of the enzyme molecule are not situated in the active centers, presentation of antibody might cause the immune reaction, such as precipitation, with no loss of activity. If the groups are at active centers, then reaction with antibody will inhibit in competition with substrate.

IX. METALS, CHELATORS, AND METAL COMPLEXERS

Heavy metal inhibition of amino acid decarboxylases (they will be listed below by amino acid only) has been studied extensively.

A. Ferric Ion

With the exception of one report which omits methods (Martin et al.} 1942), most amino acid decarboxylase+ + s studied are inhibited by relatively high concentrations of Fe +: bacterial (Epps, 1944, 1945; Gale and Epps, 1944; Taylor and Gale, 1945; Krebs, 1950; Saito, 1957); plant glutamic acid (Eggleston, 1958; Bottger and Steinmetzer, 1960); mam malian dopa only slightly (Fellman, 1959).

B. Ferrous Ion

Bacterial (Taylor and Gale, 1945; Arjona et al, 1950; Eggleston, 1958); plant glutamic (Eggleston, 1958) ; mammalian glutamic (Eggleston, 1958) but not 5-HTP (Buzard and Nytch, 1957a, b).

C. Cupric Ion

All studied. Bacterial (Epps, 1944, 1945; Gale and Epps, 1944; Taylor and Gale, 1945; Ishikawa and Obata, 1955; Eggleston, 1958; Koizumi et al., 1958; Yamagami, 1958); plant glutamic (Okunuki, 1943; Bôttger and Steinmetzer, 1960) ; mammalian, cysteic, cysteinesulfinic, and glutamic, which may be identical (Davison, 1956b), and 5-HTP (Buzard and Nytch 1957a, b).

D. Silver Ion

Bacterial (Epps, 1944, 1945; Gale and Epps, 1944; Taylor and Gale, 1945; Ekladius et al, 1957; Saito, 1957; Eggleston, 1958; Yamagami, 1958; Sutton and King, I960); plant glutamic (Okunuki, 1943). 328 W. G. CLARK

E. Mercury Ion

AU studied. Bacterial (Epps, 1944; Gale and Epps, 1944; Taylor and Gale, 1945; Oliver, 1952; Saito, 1957; Ekladius et al, 1957; Koizumi et al, 1958; Yamagami, 1958; Sutton and King, 1960); but not plant glutamic (Okunuki, 1943); mammalian dopa (Fellman, 1959).

F. Lead (Plumbic) Ion

Bacterial histidine and tyrosine (Epps, 1944, 1945).

G. Nickel Ion

Mammalian glutamic (Eggleston, 1958).

H. Cobaltic Ion

No effect on bacterial (Krebs, 1950); plant glutamic (Okunuki, 1943); and mammalian dopa (Fellman, 1959), but inhibits mammalian glutamic (Eggleston, 1958).

I. Cobaltous Ion

No effect on mammalian dopa (Perry et al, 1955).

J. Aluminum Ion

Slight (Krebs, 1950) or no effect (Arjona et al, 1950) on bacterial (Eg gleston, 1958); inhibits plant glutamic (Eggleston, 1958).

K. Stannic Ion

Bacterial in some only (Eggleston, 1958).

L Zinc Ion

Slight or no inhibition of mammalian dopa (Fellman, 1959).

M. Cadmium Ion

Bacterial glutamic (Koizumi et al, 1958); no effect on mammalian dopa (Fellman, 1959). 9. INHIBITION OF AMINO ACID DECARBOXYLASES 329

N. Manganic Ion

Slightly inhibits bacterial at relatively high concentration (Krebs, 1950; Nash, 1952); no effect on mammalian dopa (Buzard and Nytch, 1957a, b) nor on histidine (Werle and Krautzun, 1938) except at neutrality, where it does inhibit.

O. Metal Chelating Agents

Little or no effect on bacterial (Gale and Epps, 1944; Krebs, 1950; Dewey et al., 1954; Eggleston, 1958); inhibits bacterial (Ekladius et al., 1957; Koizumi et al., 1958); no effect on plant glutamic (Eggleston, 1958); no effect on mammalian dopa (Hartman et al., 1955; Perry et al., 1955; Fell- man, 1959); cysteic, cysteinesulfinic, glutamic (Davison, 1956b; Sorbo and Heyman, 1957), inhibits 5-HTP (Beiler and Martin, 1954).

P. Carbon Monoxide

No effect on bacterial enzymes tested (Gale and Epps, 1944), plant glutamic (Beevers, 1951); nor on mammalian histidine (Werle and Heitzer, 1938) and dopa (Blaschko, 1942a), suggesting an absence of copper and iron requirement.

Q. Azide

Little effect on bacterial, except lysine (Gale and Epps, 1944; Taylor and Gale, 1945; Krebs, 1950); nor on mammalian dopa (Blaschko, 1942a; Perry et al, 1955).

R. Metaphosphate and Pyrophosphate

May inhibit bacterial tyrosine at relatively high concentrations (Sloane- Stanley, 1949a; Krebs, 1950). No effect on mammalian histidine (Werle, 1943b) or dopa (Perry et al, 1955).

S. Sulfides

Inhibit bacterial glutamic (Krebs, 1950) and tyrosine, but histidine only slightly, and lysine not at all (Epps, 1944, 1945; Gale and Epps, 1944); inhibit mammalian histidine (Werle, 1947), and dopa slightly (Blaschko, 1942a). 330 W. G. CLARK

T. Dimercaprol (2,3-Dimercapto-l-Propanol; BAL)

Wenzel and Beckloff (1958) present data suggesting that dimercaprol inhibits kidney dopa decarboxylase in renal hypertensive rats more than in normal rats and speculate on a possible role of a metal activator (cf. also Section XII). In some of these studies intact cells were used and in others, cell-free extracts and semipurified preparations. This could be critical, as could dialysis. In general, it may be concluded that the amino acid decarboxylases are sensitive to heavy metals but that most, but not all, undialyzed prepara tions are not metal-dependent. Thus, the general inhibitions seen with heavy metals probably are due to inactivation of essential thiol groups, and the lack of effect of heavy metal binding and chelating agents suggests that most, but not all (see Section XI, A), amino acid decarboxylases, especially mammalian, are not metal-dependent (cf. Section XII).

X. CYANIDE (cf. Sections XI, Β; XIII)

All amino acid decarboxylases studied are cyanide-sensitive but in widely different degrees, including bacterial (Gale, 1941; Gale and Epps, 1944; Epps, 1944, 1945; Taylor and Gale, 1945; Krebs, 1950; Dewey et al, 1954; M0ller, 1954, 1956; and Kauffmann and M0ller, 1955, who used growth and decarboxylation inhibition by cyanide for bacterial classification; Ekladius et al, 1957; Ekladius and King, 1956; Gupta et al, 1960, who used it for classification); plant (Okunuki, 1937, 1942, 1943; Werle and Raub, 1948; Werle and Peschel, 1949; Morrison, 1950; Cheng et al, 1960); animal, including insect (venom gland of bee) histidine de carboxylase (Werle and Gleissner, 1951); and aminomalonic acid decar boxylase of silk gland in silkworms (Shimura et al, 1956); and mammalian histidine (Werle and Hermann, 1937; Werle and Krautzun, 1938; Werle, 1940-1941); dopa (Holtz et al, 1939; Holtz and Heise, 1938; Imiya, 1941; Blaschko, 1942a; Clegg and Sealock, 1949; Schayer and Kobayashi, 1956); dops (Werle and Peschel, 1949); aminomalonic (Shimura et al, 1956); and cysteic, cysteinesulfinic, and glutamic acids [Blaschko, 1942b; Wingo and Awapara, 1950; Davison, 1956b; Tursky, 1960 (in vivol);2 Simmonet et al, I960]3 . Werle and Peschel (1949) reported that 10~ M CN inhibits 4and 10~ M stimulates the decarboxylation of p-hydroxyphenylserine ; 10~ M has no effect. No explanation was offered, but the very low rates of reac tion, measured manometrically, make the data questionable. The effect should be re-examined by spectrofluorimetric or radioisotopic methods. 9. INHIBITION OF AMINO ACID DECARBOXYLASES 331

The inhibition does not occur by removing an essential metal and is reversible in some cases by dialysis. Cyanide is generally classified as a carbonyl group inhibitor (see Braunstein, 1960). Bona vita (1959, 1960a, b), Bonavita and Scardi (1958a, b, 1959b), and Scardi and Bonavita (1957a, b, 1958) demonstrated that cyanide reacts with the 5-formyl group of B6-P04 to form a cyanhydrin. The addition of cyanide to apotransaminase or apodecarboxylase preincubated with B6-P04 caused no inhibition, and Β6-Ρθ4 cyanhydrin is ineffective in activating the apoenzymes. Bonavita (1960b) reviews the enzymatic implications. Their work provides an ex planation of apoenzyme-coenzyme site reaction in B6-dependent enzymes by claiming that the 4-formyl group of B6-PO4 is involved in binding to the apoenzyme, rather than forming a Schiff base with the amino acid substrate, as previously suggested by Schlenk and Fischer (1947) and elaborated upon by Metzler, Ikawa and Snell (1954), Braunstein (1960), and others (cf. Sections XI and XIII).

XI. PYRIDOXAL-5-PHOSPHATE (CODECARBOXYLASE), VITAMIN B6/ B6-DEFICIENCY, B6-ANTAGONISTS (cf. Section XIII)

Since all amino acid decarboxylases are B6-P04-dependent, all agents and conditions which inhibit by inactivating, limiting, or removing the coenzyme must be considered, including B6-deficiency.

A. B6-Deficiency and Antagonists

Most amino acid decarboxylations carried out by bacteria are inhibited in B6-deficient media, the individual differences being due to different apoenzyme-coenzyme affinities. The exquisite sensitivity of tyrosine apodecarboxylase of bacteria grown in a Be-deficient medium has been used by most investigators for estimating B6-P04 (Sloane-Stanley, 1949a). Inhibition of amino acid decarboxylations by phosphorylated pyridoxine analogues in microorganisms and animals in vitro and in vivo has been demonstrated repeatedly. Beiler and Martin (1947) showed that phos phorylated 4-deoxypyridoxine inhibits tyrosine decarboxylase in S. faecalis partially purified by the method of Epps (1944, 1945), but because Martin and Beiler (1947) found that 4-deoxypyridoxine phosphate did not inhibit dopa decarboxylation by rat kidney in vitro, while certain folic acid ana logues did (cf. Section XV, E), they suggested that a folic acid derivative rather than B6-P04 might be the coenzyme. Umbreit and Waddell (1949) 332 W. G. CLARK

showed that 4-deoxypyridoxine inhibits tyrosine decarboxylase of bacteria by first being converted to its phosphorylated analogue, which then com petes with B6-PO4 for the apoenzyme. Meadow and Work (1958) showed that ω-methylpyridoxamine phos phate partially inhibits decarboxylation of diaminopimelic acid by Bacillus sphaericus asporogenes in the presence of B6-PO4, but activates it in the absence of the latter. Snell (1958) has reviewed chemical structure in relation to the biological activities of B6 and its analogues and the mechanism of action of Ββ-Ρ04 in B6-dependent enzymes. Original data are given, which show that of several derivatives tested on S. faecalis tyrosine apodecarboxylase, those which inhibited include pyridoxine phosphate, 4-deoxypyridoxine phos phate, 3-amino-4,5-dihydroxymethyl-2-methylpyridine phosphate, and pyridoxamine phosphate. Olivard and Snell (1955) showed that ω-methyl- pyridoxal phosphate can replace B6-PO4 in alanine-glutamic acid trans aminase of bacteria but not cysteine desulfhydrase. Recently, Matsuda and Makino (1961b) showed that pyridoxal-5-si^/ate competitively in hibits the B6-P04 activation of glutamic acid decarboxylase of brain homogenates of B6-deficient mice. Present evidence suggests that the affinity of tyrosine-glutamic transaminase for its substrate, like that of alanine race- mase, is decreased when the ω-analogue replaces B6-PO4 as coenzyme. Blaschko et al (1948, 1951, 1953; Blaschko, 1950) were the first to show decreased amino acid decarboxylase activity in B6-deficient ani mals (rats). Cysteic acid decarboxylase is more sensitive than dopa, and activity is not completely restored by the addition of B6-PO4 in vitro, implying an impairment of apcenzyme synthesis (cf. Sections V and VII). Although Be-deficiency itself had no effect on adrenal medullary cate chol amine content, resynthesis was slower than normal after depletion by insulin hypoglycemia. Dietrich et al. (Dietrich and Shapiro, 1953; Dietrich and Borries, 1956) confirmed this and found that dopa decar boxylase in mouse liver is less sensitive to B6-deficiency than glutamic- aspartic transaminase and cysteine desulfurase. West (1953) also found that Be-deficiency decreases dopa decarboxylase in rat liver and kidney. In confirmation of Blaschko et al. (1951), this causes a slower resynthesis of adrenal medullary catechol amines after depletion by insulin hypo glycemia. Sourkes et al. (1960) confirmed Blaschko et al. and West that Be-deficiency does not alter catechol amine levels in the adrenals and extended this to show the same lack of effect in other organs. Concomitant B2-deficiency caused a marked decrease, especially in brain and liver. Sourkes et al. (1960) also showed less excretion of dopamine after injecting dopa in Ββ-deficient rats. Pogrund and associates (1955, 1961; cf. Clark, 1959 and Clark and Pogrund, 1961) showed that B6-deficient rats exhibit subnormal responses 9. INHIBITION OF AMINO ACID DECARBOXYLASES 333 to dopa and to 3-hydroxy- and 3,4-dihydroxyphenylpyruvic acids, which are transaminated in vivo to m-tyrosine and dopa, respectively. Dopamine responses remained normal. Kidney apodopadecarboxylase remained normal, liver apoenzyme decreased, and coenzyme was low in kidney and liver, the former more so, in confirmation of Beaton and McHenry (1953) and Wachstein and Moore (1958). Roberts et al. (1951) found a 50% decrease in saturation of glutamic acid apodecarboxylase with coenzyme in B6-deficient rat brains. This could be completely reversed by feeding B6. This has been confirmed by Bergeret et al. (1955), Rosen et al. (1959), and others, and the literature is reviewed by Elliott and Jasper (1959), Roberts (1960), Roberts and Eidelberg (1959), Roberts et al. (1960), and Burns and Shore (1961). Chatagner and co-workers (1954), Bergeret et al. (1955), and Fromageot (1956) confirmed Blaschko et al. that B6-deficiency decreases cysteic acid and cysteinesulfinic acid decarboxylation in rats and rabbits, and both Fromageot (1953-1954) and Chatagner (1959) cover this work in their re views on sulfur metabolism. Hope (1955, 1957) also confirmed this and reviews the field (1959). Hope presented evidence that glutamic, cysteic, and cysteinesulfinic acids are decarboxylated by the same enzyme in mammals. Marco (1957) observed that in comparison with in vitro observations, the normal rat brain B6-PC>4 content is lower than that which will allow maximum decarboxylation of glutamic, cysteic and cysteinesulfinic acids. Massive doses of B6 given in vivo increase it somewhat. Holtz (1959, 1960a) noted a linear activation of dopa decarboxylase activity by B6-P04 in brain homogenates, which does not occur in homogenates and extracts of other organs. Addition of brain extracts to dopa decarboxylase prepara tions from liver or kidney reactivate the decarboxylation after it has leveled off, in the presence of B6-P04. He postulated that the ethanolamine moiety of brain cephalin, through its amine group, forms a Schiff base with B6-P04, which acts as a coenzyme, much as the B6-PO4 hydrazones studied by Gonnard (see Section XI, B, below). Buxton and Sinclair (1956) found low 5-HTP decarboxylase activity in Be-deficient rats, restored in vitro by B6-P04. This was confirmed indirectly by Weissbach et al. (1957), who found lower levels of 5-HT in tissues of Be-deficient chicks. Exogenously administered 5-HTP was decarboxylated at a much lower rate in B6-deficiency (cf. Udenfriend et al, 1957). Buzard and Nytch (1957a, b) showed that supplementary B6 in the diet increases 5-HTP decarboxylation by rat kidney, and B6-deficiency reversibly de creases both apoenzyme and coenzyme, especially the latter. Schrodt et al. (1960) attempted to induce 5-HTP decarboxylase in sufficiency with 4-deoxypyridoxine in two patients with a carcinoid syn drome. There was no change of urinary 5-HIAA excretion, nor in the 334 W. G. CLARK symptoms in one case, while in the other the excretion was less. It does not follow, however, that 5-HTP decarboxylation was necessarily decreased in this patient. Kizer and Chan (1961) found that 5-HTP decarboxylase activity is absent in transplanted and primary rat hepatoma tissue and in pre cancerous liver tissue of rats treated with carcinogens, the rate of its loss in the latter being correlated with carcinogenesis. Schayer (1959) showed that tissues of B6-deficient rats have a lowered histidine decarboxylase activity which is partially restored by feeding B6 14 14 or by adding B6-P04 in vitro. These B6-deficient rats excreted less histamine-C after the injection of histidine-C . Nadkarni and Sreenivasan (1957), using rat liver homogenate, could obtain no decrease in decar boxylation of serine to ethanolamine in B6-deficiency in comparison with controls. The results would have been more significant if data on the capacity of these livers to decarboxylate other amino acids had been examined. Indirect reports of possible impairment of decarboxylation in Be-deficiency by measurements of tissue amine levels and/or excretion are fraught with uncertainty because of possible effects on apoenzyme syn thesis, pyridoxal kinase, and on uptake, transport and binding of amines (e.g., Yeh et al., 1959; Ferrari et al., 1957). Also, claims of decreased de carboxylation in Be-deficiency by even more indirect methods are open to criticism. Thus, Martin (1946) postulated that tyrosine is less toxic in Be-deficiency because of less decarboxylation to tyramine, a toxic amine, yet no direct measurements were made. Similar doubt is cast upon at tempts to correlate decreased tissue amines with decreased amino acid decarboxylase activity induced by other types of treatment, such as lethal X-irradiation (Anderson et al., 1951). The reviewer wishes to mention the elegant work of Shukuya and Schwert (1960a, b, c), who have purified bacterial glutamic acid decar boxylase to the extent that its molecular weight could be estimated. Their studies of its characteristics and kinetics have thrown much light on the mechanism of apoenzyme-coenzyme dissociation and the nature and reactivity of the apoenzyme molecule.

B. B6-P04 Hydrazones

Certain hydrazides may inactivate B6-P04 by carbonyl group reaction (cf. Section XIII). Gonnard et al. (Gonnard, 1958; Gonnard and Boigné, 1961; Gonnard and Chi, 1958, 1959a, b; Gonnard and Nguyen-Philippon, 1959, 1961) have found that synthetic hydrazones, formed by reacting B6-P04 with the hydrazides of isonicotinic acid (for methods, cf. Curry and Balen, 1960; Testa et al., 1961), benzoic acid, nicotinic acid, picolic 9. INHIBITION OF AMINO ACID DECARBOXYLASES 335

acid, p-aminosalicylic acid, and thienic acid, all activate mammalian dopa decarboxylase, glutamic-aspartic transaminase, and kynureninase, all of which are B6-P04-dependent enzymes. Unlike B6-P04 alone, which permits only incomplete decarboxylation of dopa, these hydrazones, also acting as true cofactors (without dissociating), allow the reaction to go to com pletion, though at a somewhat lower rate. Palm (1958), working with Β6-Ρθ4 isonicotinoylhydrazone, believed that the product is slowly hy drolyzed to give B6-P04, which allows the reaction to go to completion, although more slowly. Since the aldehyde moiety of the B6-P04 is blocked in the reaction product, Gonnard believes this may be additional evidence against the concept of a mechanism of transamination and decarboxylation involving the formation of Schiff bases between coenzyme and substrate. The phenomenon also was confirmed by Bonavita and Scardi (1959a, b), who found that synthetic B6-PO4 isonicotinoylhydrazone activated glutamic-oxalacetic transaminase of pig heart, although longer incubations were required than with B6-P04 alone. Biehl and Vil ter (1954) and Davison (1956c) had shown that isonicotinic acid hydrazide (INH) inhibits B6-dependent enzymes due to hydrazone formation with B6-P04. Hence, Bona vita and Scardi believed the hydrazone should be inactive as a cofactor, like the B6-P04 cyanhydrin they had studied previously (cf. Section X), and were surprised when activation occurred. In seeking an explanation, they considered that (1) B6-PO4 is liberated from the hydrazone, but pointed out that this is unlikely because of the stability of the complex; and although Youatt (1958) found some release of unphosphorylated pyridoxal from its isonicotinoylhydrazone by tubercle bacilli, there is no evidence that the hydrazone is enzymatically split; (2) the B6-P04 hydra zone itself is bound as such to the apoenzyme with a subsequent displace ment reaction with substrate to yield holoenzyme and isonicotinic acid. This does not rule out the possibility of its reactivity with amino acids. To explain the differences between the B6-P04 hydrazone and B6-P04 cyan hydrin, it was pointed out that although their fluorescent spectra are similar, the pif

C. Toxopyrimidine

Toxopy rimidine (2-methyl-4-amino-5-hy droxymethylpy rimidine), the pyrimidine component of thiamine, was first described as a convulsant in animals by Abderhalden (1939a, b, 1940, 1954). This compound and 336 W. G. CLARK

several of its analogues compete with B6-PO4, and Snell (1958) classifies it with Be-analogues which are antimetabolites after being phosphorylated by pyridoxal kinase. This classification is undoubtedly correct, since its phosphate has been synthesized and found to inhibit semipurified B6-dependent apoenzyme systems whereas the unphosphorylated form does not (Koike, 1954; Makino and Koike, 1954a, b; Haughton and King, 1957). The toxopyrimidine analogues also act as competitive B6-antagonists in general, both in the growth of microorganisms and on their amino acid decarboxylases (Sakuragi and Kummerow, 1957; Scheunert et al, 1957; Shintani, 1956). A considerable literature has grown on the essential chemi cal structure for activity of these compounds because of their potency (Abderhalden, 1954; Kawashima, 1957; Shintani, 1957a, c; Miyake, 1957; Hayashi, 1957; Nishizawa et al, 1958a; Rindi et al, 1959b). A methyl group appears necessary in the 2-position, an amino in the 4-position, and a radical easily changed to a hydroxymethyl in the 5-position. Abderhalden (1954) originally found antitoxopyrimidine activity in extracts of grains, yeast, etc., and the active factor later was shown to be pyridoxamine. Papers on "atoxopyrimidine" activity of various B6-derivatives have ap peared (Morii, 1941; Abderhalden, 1954; Makino et al, 1954; Makino and Koike, 1954a, b; Makino and Kinoshita, 1955; Sakuragi and Kummerow, 1957; Scheunert et al, 1957; Konishi, 1957; Hayashi, 1957; Nozaki, 1958; Rindi et al, 1959a, b; Rindi and Ferrari, 1959). In addition to pyridoxine, pyridoxal, pyridoxamine, and B6-P04, antagonists of toxopyrimidine effects in vivo include 2-methyl-4-hydroxy-4-hydroxy(or amino) me thy lpyrimidine, and 2-methyl-4-amino-5-f ormylaminomethyl (or 5-aminomethyl) pyrimidine, which Hayashi (1957) designated the "atoxopyrimidine" group of ana logues. A clue to the metabolism of such compounds was afforded by Shintani (1957b), who found that hydroxymethylpyrimidine administered to rabbits is metabolized and excreted as the 5-carboxy analogue. In animals, these substances are highly potent agents, producing convul sions ("running fits") and death in small doses. Yamanaka (1957) claims that the site of the convulsive effect is centered in the nuclei lenticu- laris and caudatus of the brain stem. Liver pathology is caused in doses as small as 4 Mg/gm in mice (Kooka, 1957; Nishizawa et al, 1957), which is correlated with decreased pyridoxine, pyridoxamine, and pyridoxal in the liver. It is prevented by treatment with these compounds, and also by such agents as methionine and iV-carbobenzoxyglutamylcholine. Among the various B6-enzymes which are competitively inhibited by toxopyrimidine in vivo are glutamic acid decarboxylase of liver and brain, and several transaminases (Mandokoro, 1957; Namba, 1957; Yamanaka, 1957; Konishi, 1957; Moriya, 1958; Kobayashi, 1958; Sagawara, 1958; Nozaki, 1958; Nishizawa, 1958; Nishizawa et al, 1958b, c, d, 1959a, b, c, d, 1960; Rindi et al, 1959a, b, c), and the convulsive symptoms correlate 9. INHIBITION OF AMINO ACID DECARBOXYLASES 337 better with decreased decarboxylation to gaba than with other metabolic pathways examined. Purified pyridoxal kinase of yeast is activated by toxopyrimidine (Moriya, 1958) and thus does not compete with pyridoxal, although it also is a substrate.

D. Steroids

Steroids may be powerful inhibitors of Ββ-dependent enzymes through interaction with B6-P04 (cf. Section XIV).

XII. ACTIVATORS, STABILIZERS, AND COFACTORS OTHER THAN B6-P04 A. Metals

Heavy metal ions have been reported to activate some amino acid decarboxylases in plants and animals (Guirard and Snell, 1954; Happold, 1956; Eggleston, 1958; Mazelis, 1959), but there are conflicting reports on their activation of amino acid decarboxylases in mammals (Sorbo and Heyman, 1957; Steensholt et al., 1956). The subject has been reviewed by Dixon and Webb (1958, p. 447) and Braunstein (1960), the latter con cluding that the experimental evidence for an involvement of metals in activating Ββ-enzymes in general is highly contradictory.

B. Surfactants

Ionic detergents have been reported to inhibit some amino acid de carboxylases, to stimulate some, and to have no effect on others. In some, but not all cases, these effects may be through permeability effects when intact cells are involved (Krebs, 1948; Nossal, 1952; Oliver, 1952; Hughes 1949, 1950; Storck, 1951; Cosin, 1955, 1956; Yabe et al, 1957; Eggleston, 1957; Crawford, 1958; Baker et al, 1941), and Hughes postulated that surfactants increase the affinity of apoenzyme for substrate or remove an inhibitor by complex formation.

C. Solvents

The effect of solvents in general have been studied on various amino acid decarboxylases (cf. Krebs, 1948, 1950; Mardashew, 1949; Mardashew and Etinghof, 1948; Holtz and Heise, 1938; Shimada et al, 1954; Blaschko, 1942b, c). 338 W. G. CLARK

Waton (1956a, b) made the interesting observation that many organic solvents activate nonspecific mammalian histidine decarboxylation by rabbit kidney mince and extracts. Pyridine, toluene, benzene, chloro- benzene, and petroleum ether accelerated 6-9 times; chloroform, cyclo- hexane, nitrobenzene, aniline, ether, and carbon tetrachloride 2-3 times; butanol, tetrachlorethane, pentan-l-ol, cyclohexanone, and acetone slightly; amyl alcohol not at all. The amount used was one drop in the incubation mixture. The mechanism is postulated to be independent of antibacterial action since the incubations were usually an hour or less. Most of the active solvents have aromatic rings, and the action is inde pendent of the formation of two phases. Waton speculated that perhaps there is a physical effect on the enzyme, such as a breaking up of micelles and making previously unaccessible sites available for activity. Hughes' (1949, 1950) suggestion of the micellar mode of action of surfactants on enzymes is not acknowledged, and a full explanation of the phenomenon remains to be established. It is possible that the effect is due in part to exposing available enzyme sites by removal of lipoprotein barriers. Schayer (1957) confirmed Waton that benzene markedly enhances histidine decarboxylase by rat kidney, in this case cell-free, but strongly inhibits that of rat stomach. Rothschild and Schayer (1959) also found that histidine decarboxylase of rat peritoneal mast cells was similar to that of stomach and was different from that of kidney in that benzene markedly inhibits its activity. This, in addition to finding different pH optima, led Schayer to postulate that there are two different enzymes, as discussed elsewhere in this chapter.

D Phosphates and Arsenate

Sloan-Stanley (1949a) and Sorm and Tursky (1954) showed that phos phate and pyrophosphate, among other anions, affect the reaction between bacterial tyrosine decarboxylase apoenzyme and coenzyme. Several workers have shown that inorganic phosphate and arsenate may accelerate some bacterial, plant, and mammalian amino acid decarboxylases (Gon nard, 1951; Krebs et al., 1955; Hartman et al., 1955; Eggleston, 1957), and the possibility of an intracellular organic phosphorylated regulator, other than B6-P04, of the B6-dependent decarboxylases should be considered.

E. Miscellaneous

Sankar and Bender (1960) found that D-lysergic acid diethylamide (LSD) stimulates the decarboxylation of glutamic acid by cerebral cortex homogenates, and suggest that LSD may help channel the metabolism of glutamate via the gaba pathway. 9. INHIBITION OF AMINO ACID DECARBOXYLASES 339

XIII. CARBONYL REAGENTS AND INHIBITORS ACTING ON CO ENZYME OTHER THAN CYANIDE, SUBSTRATE ANALOGUES, AND PYRIDOXINE ANTAGONISTS.

A. Hydroxylamine, Hydrazides, Semicarbazide, Sulfite, Hydrazine, Oximes, Etc.

As is usual with Independent enzymes, all amino acid decarboxylases studied so far (fourteen) in bacteria and other microorganisms including yeast are inhibited, usually reversibly, through B6-P04 inactivation, by the usual carbonyl reagents, such as bisulfites, thiosulfate, hydroxylamine, hydrazides, oximes, hydrazines, carbazides, and their derivatives, including the hydrazide amine oxidase inhibitors. The degree of inhibition varies widely, depending on the apoenzyme-coenzyme affinity (Cattanéo-Lacombe and Senez, 1956; Crawford, 1958; Dewey et al., 1954; Ehrismann and Werle, 1948; Ekladius and King, 1956; Ekladius el al, 1957; Epps, 1945; Gale, 1941; Gale and Epps, 1944; Gangadharam and Sirsi, 1956; Hicks and Clarke, 1959; Hoare, 1956; Kating, 1954; Knivett, 1954; Krebs, 1950; Krishnaswamy and Giri, 1956; Aoki, 1957a; Mauron and Bujard, 1960; Miyaki et al, 1959; Nash, 1952; Roberts, 1952b; Saito, 1957; Schormuller and Leichter, 1955a, b; Senez et al., 1959; Shukuya and Schwert, 1960a; Sutton and King, 1960; Taylor and Gale, 1945; Wachi et al, 1959; Willett, 1958; Yamagami, 1958; Yoneda and Asano, 1953). Plant glutamic acid decarboxylase also is inhibited by hydrazines, hydrazides, hydroxylamine, and bisulfite (Okunuki, 1942, 1943; Werle and Bruninghaus, 1951; Schales and Schales, 1946a; Beevers, 1951; Matsuda et al, 1955; Rohrlich and Rasmus, 1956; Rohrlich, 1957; Cheng et al, 1960), as are γ-methyleneglutamic acid decarboxylase (Fowden, 1954) and histidine decarboxylase (Werle and Raub, 1948). Similarly, hydroxylamine and semicarbazide inhibit insect aminomalonic (Shimura et al, 1956) and histidine (Werle and Gleissner, 1951) decar boxylases; they and hydrazine also inhibit bird (embryo) (Simonnet et al, 1960) and mammalian cysteic and cysteinesulfinic acid decarboxylation in vitro and in vivo (Werle and Bruninghaus, 1951; Canal and Garattini, 1957; Davison, 1956a, c). Davison (1956c) also showed the latter to be inhibited by isonicotinoyl hydrazide (INH). Nadkarni and Sreenivasan (1957) reported that hydroxylamine inhibits serine decarboxylation by rat liver homogenate. Werle and Heitzer (1938) and Werle (1940), who were the first to describe the effect on such enzymes by carbonyl reagents, re ported the inhibition of histidine decarboxylase of mammalian tissues (which Werle discovered) by hydroxylamine, semicarbazide, bisulfite, a series of hydrazides including Girard's reagents, hydrazine, guanylhydrazone derivatives including the antihypertensive drug 1-hydrazinoph- 340 W. G. CLARK

thalazine (Apresoline) and some of its analogues (cf. dopa decarboxylase below). Schayer and Kobayashi (1956), Schayer (1957), and Rothschild and Schayer (1959) found that histidine decarboxylase of rabbit platelets, rat mast cells, and other tissues is reversibly inhibited by such agents as hydroxylamine and semicarbazide; Kahlson et al. (1958, 1960a, b; Kahlson and Rosengren, 1959a, b; Kahlson, 1960) found that hydrazine and semi carbazide inhibited the enzyme in vitro and in vivo in rats, including embryos. Pyridoxine deficiency increases the inhibition. They causally invoke histamine in embryonic, regenerative, and malignant growth, using such inhibition as a tool in their thesis (cf. Section VII). Mammalian dopa decarboxylase has been known to be inhibited by carbonyl reagents since Imiya (1941) demonstrated the effect with bi sulfite in tissues of three species in vitro. Semicarbazide inhibits in vitro (Werle, 1943b; Clegg and Sealock, 1949; Langemann, 1951; Fellman, 1959), as do hydroxylamine (Schales and Schales, 1949; Davison, 1956c), hydrazine (Gonnard, 1951), and INH (Davison, 1956c; Palm, 1958), although Canal and Garattini (1957) could obtain no inhibition with the latter on guinea pig and rat kidney dopa decarboxylase in vivo, in contrast with decarboxylation of cysteic acid by liver, and glutamic acid by brain. Hydrazine derivatives known to inhibit monoamine oxidase also have been shown to inhibit dopa decarboxylation in vitro, including iproniazid by chicken adrenal gland (Hagen and Welch, 1956) and l-phenyl-2- hydrazinopropane ("JB-516," Catron) by rabbit brain (Brodie et al, 1958, 1959). Levy and Michel-Ber (1960a, b), from indirect pharmacological evidence based on the reversal by B6-P04 of the potentiation of hypnotics by iproniazid and hydrazino-2-octane when 5-HTP or dopa are given, conclude that these monoamine oxidase inhibitors block dopa (and 5-HTP) decarboxylase in vivo. Direct measurements in vivo, however, by Brodie et al. (1958, 1959) and others have shown that iproniazid and l-phenyl-2- hydrazinopropane do not inhibit these enzymes in vivo (but cf. Canal and Maffei-Faccioli, 1959, 1960). In the reviewer's opinion, however, this sub ject deserves a systematic study, since inhibition by Catron and iproniazid of aromatic amino acid decarboxylation in vitro and in vivo has been observed repeatedly in this laboratory. Perry et al. (1955) reported the inhibition of mammalian dopa decar boxylase in vitro by low concentrations of the. antihypertensive agents 1-hydrazinophthalazine (Apresoline), 1,4-dihydrazinophthalazine (Nep- resol), and 2-hydrazino-5-phenylpyridizine. This was confirmed and ex tended by Werle et al. (1955), who examined the effects of twelve hydrazine and guanylhydrazone derivatives, including aminoguanidine, on mono amine and diamine oxidases and on histidine and dopa decarboxylases by mammalian preparations in vitro. Schayer and associates (1955) found no 9. INHIBITION OF AMINO ACID DECARBOXYLASES 341 effect of aminoguanidine on histidine decarboxylase in vitro, although Yuwiler et al. (1959) reported that it inhibits 5-HTP decarboxylase in vitro. This reviewer could find no reports of a similar effect of this agent in vivo. Bygdeman and Stjàrne (1959) found a depression of catechol amines by 1,4-dihydrazinophthalazine in various organs, including the adrenal medulla, and suggested that one explanation might be the blocking of dopa decarboxylase. Sano et al. (1960) confirmed the effect of 1-hydrazinophthalazine on dopa decarboxylase of brain and kidney in vitro, and found that it also markedly depressed endogenous brain noradrenaline and dopamine in vivo, the latter as much as 20-30 times. They suggested, as had Bygdeman and Stjârne, that this might be one explanation of the depressor effect of these compounds. Schuler and Wyss (1960), in an ex tensive study of 75 hydrazine derivatives, reported that about half of 37 compounds examined for dopa decarboxylase inhibition in vitro exhibited a weak to moderate inhibition, and confirmed Werle et al. (1955) in that there was no correlation with pharmacological activity such as depressor effects in rabbits and reserpine antagonism in mice. Since 5-HTP, dopa, and possibly other aromatic amino acid decarboxyla tions are due to the same enzyme in mammals, it is not surprising that carbonyl reagents affect them similarly. Carbonyl reagents which inhibit 5-HTP decarboxylation by mammalian tissues in vitro, reversibly in most cases, include hydroxylamine, thiosemicarbazide, bisulfite, thiocyanate, aminoguanidine, isonicotinyl hydrazide (INH), iproniazid, and 1-hydrazinophthalazine. Recently Schumann et al. (1961) listed 16 ori/io-substituted phenylhydrazines which inhibited 5-HTP decarboxylase in vitro. In general, the inhibition correlates with their inhibition of monoamine oxidase (C. T. Clark et al, 1954; Beiler and Martin, 1954; Weissbach et al, 1957; Buzard and Nytch, 1957b; Ozaki, 1959). Weissbach et al. (1957) demon strated inhibition of 5-HTP (and o-tyrosine) decarboxylase by thio semicarbazide in chicks and mice, in vivo, which was confirmed by Wiss and Weber in rats (1958). Dubnick and associates (1962) showed an inhibition of 5-HTP decarboxylation in mice with high doses (16-80 mg/kg IV) of the monoamine oxidase inhibitor β-phenylethylhydrazine (Phenalzine), although with doses adequate for monoamine oxidase in hibition it apparently has no effect (Dubnick et al, 1960a). Dubnick et al. (1960c) also found the expected antidotal effect of pyridoxine and pyridox amine against the convulsive effect of hydrazines in mice (vide infra), but pyridoxal enhanced the toxicity. The pyridoxal hydrazones of β-phenylethylhydrazine and phenylisopropylhydrazine are more toxic than equimolar doses of the parent compounds. The authors postulate that these hydrazines form B6-PO4 hydrazones in vivo, with subsequent inhibition of pyridoxal kinase (cf. Sections II, XI, and the end of this section). Canal 342 W. G. CLARK and Maffei-Faccioli (1959, 1960) reported inhibition of 5-HTP decarboxy lase in liver and brain of rats by INH in vitro and in vivo, reversed by B6-P04 in vitro. It is interesting that Ludwig (1960) found that 400 mg INH daily in two carcinoid patients showed some indications of decreased blood and platelet 5-HT and urinary 5-HIAA, with some improvement in gastro intestinal symptoms. The effect could, of course, be on transport mech anism, pyridoxal kinase, and/or monoamine oxidase, although the author suggests it may occur through decarboxylase inhibition. Glutamic acid decarboxylation to gaba by mammalian tissue prepara tions in vitro is inhibited by hydroxylamine and semicarbazide, as first shown by Wingo and Awapara (1950) and Roberts and Frankel (1951). Isonicotinyl hydrazide also inhibits the enzyme in vitro, as shown by Davison (1956c), Killam and Bain (1957a), Yoshimura (1958), Hado (1959), and others. Heilbronn (1960), however, examined the effects on glutamic acid decarboxylase of brain in vitro of a number of monosubsti- tuted and Ν, iV-disubstituted hydrazines, hydrazides, and hydrazines and found no effect. He concluded that a reaction between the hydrazines or hydrazides and B6-P04 cannot alone be the cause of inhibition by such compounds of a B6-P04-dependent enzyme system. In vivo, many investigators have shown reversible inhibition of glutamic acid decarboxylation and/or gaba formation by a variety of carbonyl re agents, including thiosemicarbazide, thiocarbohydrazide, semicarbazide, furoic acid hydrazide, isonicotinic acid hydrazide, and hydroxylamine (Killam and Bain 1957a, b; Canal and Garattini, 1957; Roberts et al, 1958; Pietra et al., 1959; Caspers, 1960; Baxter and Roberts, 1960; Balzer et al., 1960a, b, c). A large literature has grown from these observations, as possibly correlated with brain gaba levels, electrophysiological changes, and epileptiform seizures, which has been reviewed elsewhere (Tower, 1956, 1959, 1960; Elliott and Jasper, 1959; Roberts and Eidelberg, 1959; Roberts, 1960; Roberts et al, 1960; and Holtz, 1960a, b). The hypothesis that the carbonyl-induced seizures are causally cor related with brain levels of gaba has, however, been challenged, since Baxter and Roberts (1960) showed that although semicarbazide-induced seizures in rats correlate with lowered brain gaba levels, rats injected with both thiosemicarbazide and hydroxylamine may have seizures when the brain gaba is normal or elevated. They suggest that the seizure-inducing carbonyl agents may thus be affecting other Bo-dependent enzymes in volved, nerve membranes, or by preventing the formation of a physiologi cally active derivative of gaba. Furthermore, Balzer et al (1960a, b, c) showed that although convulsive doses of thiocarbohydrazide and other hydrazides lower brain gaba levels in mice, convulsive doses of thiosemi- 9. INHIBITION OF AMINO ACID DECARBOXYLASES 343 carbazide may not do so unless given along with anticonvulsive doses of pyridoxine. They suggest that the pyridoxine may catalyze gaba-a-keto- glutaric acid transamination more than it does glutamic acid decarboxyla tion, and that the seizures are not caused by decreased levels of the brain gaba pool but by a decreased turnover rate in glutamic acid-gaba-a-keto- glutaric metabolism. Kamrin and Kamrin (1961) agreed with Baxter and Roberts and with Balzer et al. that the absolute level of brain gaba cannot be used as an index of the degree of seizures or state of excitability of the brain. The B6-antagonist, 4-methoxymethylpyridoxine, decreased mouse brain gaba 50% following seizures, but protection from the seizures by B6 did not affect this gaba decrease. Seizures caused by other convulsants caused no amino acid changes, and in general there were no amino patterns reflecting either convulsions nor protection from them. Finally, McCormick and Snell (1959) and McCormick et al. (1960) point out that several hydrazides, hydrazones, pyridoxal oxime, and hydroxyl amine may inhibit pyridoxal kinase of liver and brain in vitro 100-1000 times more than they do glutamic acid decarboxylase, that hydrazine inhibits the kinase more than the decarboxylase in vivo, and that the di- hydrazone of pyridoxal (azine) causes a marked decrease of kinase of liver and brain without affecting the decarboxylase at all. Thus, they raise the question that the seizure-gaba level correlation (also questioned by Baxter and Roberts and by Balzer et al., 1960a, b, c) may be related to kinase inhibition rather than to decarboxylase inhibition. This view is shared by Dubnick et al. (1960c), who postulate hydrazone formation in vivo with pyridoxal and the convulsive hydrazines, with consequent inhibition of pyridoxal kinase.

B. Cycloserine (4-Amino-3-lsoxazolidone)

Aoki (1957a, b, c), Yamada et al. (1957), Kochetkov et al. (1959), Vyshe- pan et al. (1959), Braunstein and Hsu (1960), and Azarkh et al. (1960) (cf. Braunstein, 1960) found that the tuberculostatic drug cycloserine in hibits such bacterial Ββ-enzymes as transaminases, tryptophanase, and glutamic acid decarboxylase. Glutamic acid decarboxylase of mouse brain homogenate also is inhibited (Aoki, 1957a). Aoki (1957c) and GoFdshtein (1960) also showed that it inhibits the nonenzymic formation of indole from tryptophan in the presence of pyridoxal and cupric ions, and sug gested that the mechanism was due to the formation of a Schiff base be tween cycloserine and B6-P04. Synthesis of the base was successful. GoPd- shtein (1960) reported a marked effect of cycloserine on nicotinic acid metabolism in man. Yamada et al. (1957) made a similar suggestion and proposed that the clinical side effects of the drug might be due to such a 344 W. G. CLARK

reaction in vivo. Ito et al. (1958) obtained data to support this in studies which showed that administration of cycloserine increased B6-excretion in mice. Kochetkov et al. (1959) proposed that cycloserine forms an azo- methine with B6-P04 as well as labile Schiff base intermediates which are converted to stabilized isomeric complexes. Cascio and Monaco (1957), Benigno et al. (1958), La Grutta and Monaco (1958), Monaco (1958a, b), Monaco and Cascio (1959), Monaco and La Grutta (1959, 1961), Benigno and Monaco (1960), and Crane (1961) have studied the central nervous symptoms seen clinically during cycloserine therapy and induced later experimentally. Both Azarkh et al. (1960) and Monaco et al. found the L-isomer more potent. The psychic alterations, epileptiform seizures, marked sedation, and "tranquilization" seen after administration of the L-isomer were correlated with electroencephalographic changes. The compound also enhances narcosis by ethanol, barbiturates, and ether, and is used for preanesthesia induction (Monaco and Cascio, 1959). Dengler (1961) and Dengler et al. (1962) have analyzed the kinetics of the reaction of cycloserine and B6-P04, using dopa and glutamic acid decar boxylase systems of guinea pig kindey and rat brain, respectively, as model enzymes because they might be correlated with the seizure threshold and also brain amine levels. The r>- and L-isomers of cycloserine were equally in hibitory; the inhibito3r concentration giving 50% inhibition under his con ditions was 6 Χ 10~ M. It was shown that it does not affect apodopadecarboxylase. Adding more B6-P04 reactivates the inhibited system. Evidence was obtained indicating that cycloserine forms a complex with B6-P04 which competes with free B6-P04 for the apoenzyme and that this reaction product is not a Schiff base but a substituted oxime (I). The evidence is based on

CH=N—Ο—CH2—CH—NH2 o- coo- —CH2OP03H2 N CH3 I the following facts: (a) its UV absorption maximum is 330 ηΐμ, close to that of B6-P04 oxime prepared according to Heyl et al. (1948,1951,1952), and (b) it gives a negative test with Jones' reagent, which suggests fission of the isoxazolidine ring. With higher concentrations of cycloserine, a second product was obtained which, like the oxime, has a strong fluorescence, activated at 380 m/z and emitting at 455. Paper chromatography showed a 9. INHIBITION OF AMINO ACID DECARBOXYLASES 345 different R/ than that of the oxime. It may be the condensation product of B6-P04 with 2,5-bis(aminooxymethyl)-l,4-dioxopiperazine, which can be formed spontaneously from cycloserine. The dioxopiperazine derivative at doses of 50 mg/kg intravenously in rats gave 100% inhibition of dopa decarboxlyation by preparations made from the subsequently removed kidneys.

C. Penicillamine

Roberts (1960) lists penicillamine as an inhibitor of glutamic acid decarboxylase in mammalian tissues. This amine induces B6-deficiency symptoms in rats when fed in the diet and inhibits the B6-dependent transaminases and kyureninase in the liver, which is restored to normal when extra B6-P04 is added in vitro (Wilson and Du Vigneaud, 1950; Du Vigneaud et al., 1957; Kuchinskas et al., 1957; Kuchinskas and Du Vigneaud 1957; cf. Ueda et al. 1960). Bonavita (1959), however, found no inhibition by penicillamine of a partially purified glutamic-oxalacetic transaminase preparation of human brain. Kuchinskas (1961) in a preliminary abstract points out that penicillamine is a potent heavy metal chelating agent, bonding the metal between the amino and thiol groups, as in the case of cysteine; hence, its action on enzymes can be complex. Matsuda and Makino (1961a) correlated the running fits in mice injected with penicilla mine with decreased glutamic acid decarboxylase in the brain, both being prevented by simultaneous injection of pyridoxine. The decarboxylase was restored to normal by Be-P04 in vitro, and the inhibition in vitro was competitive by the Lineweaver-Burk analysis. Wilson et al. (Wilson and Du Vigneaud, 1950; Du Vigneaud et al., 1957) and Mardashew and Semina (1961) showed that the penicillamine inhibition is due to a nonenzymic reaction with the carbonyl group of B6-P04 to form a thiazolidine.

D. Sulfonylureas

The hypoglycemic agents, such as carbutamide (N-sulfanilyl-iV-butylcarbamide) and tolbutamide [iV-(p-tolylsulfonyl)-AT-butylcarbamide3 have been shown to inhibit B6-enzymes such as alanine transaminase of liver (Bornstein, 1957) and dopa decarboxylase of guinea pig kidney (Gonnard et al, 1958, 1959). The latter was not modified by adding B6-P04; hence, the authors believe the effect is not on the coenzyme. In vivo, as in insulin hypoglycemia, these compounds deplete the adrenal medulla of its catechol amines, and it was suggested that the mechanism might in part be through this inhibition and not exclusively a release-secretion phenomenon. Sulfacetamide (AT-sulfanilylacetamide) and sulfadiazine (2-sulfanilamido- pyrimidine) did not inhibit the liver enzymes (Bornstein, 1957). 346 W. G. CLARK

E. Cysteine, 2,3-Dimercapto-l-Propanol (BAL), Glutathione

Higher concentrations of these agents can inhibit by combining with Β6-Ρθ4, although they can als4o combine with metals. Werle and Heitzer (1938) found that 4.2 X 10~ M cysteine inhibited mammalian histidine decarboxylase almost 100% in vitro. Cystine had little effect. Cysteine also was shown by Clegg and Sealock (1949) to inhibit dopa decarboxylase in vitro. Gonnard (1949, 1951) showed that reduced glutathione inhibits dopa decarboxylase of guinea pig kidney in vitro. Bergeret and co-workers (1956) found that both cysteine and BAL inhibit cysteic, glutamic acid, and cysteinesulfinic acid decarboxylases of mammalian tissues in vitro, and postulated that cysteine complexes nonenzymically with B6-PO4 to form an oxazolidine. Mardashew and Chao (1960) reported an inhibition of transamination in tissue cultures of rat liver by cysteine, reversed by B6-PO4. Mardashew and Semina (1961) showed that inhibition of bacterial decarboxylases by L-cysteine and DL-homocysteine is due to a nonenzymic reaction with the carbonyl group of B6-PO4 to form a thiazolidine and thiazane, respectively, and succeeded in synthesizing 2-(3-hydroxy-2- methyl-5-hydroxymethyl-pyridyl-4)-l ,3-thiazancarboxylic-4-acid.

F. Ascorbic Acid

It is not clear how ascorbic acid may inhibit decarboxylase systems. Gonnard (1949) postulated an intermediate oxidative step in amino acid decarboxylations in which a quinoid form of the substrate is proposed as a catalyst in the formation of a Schiff base and suggested that ascorbic acid, glutathione, and aldehydes inhibit decarboxylases at this step. This theory has received no confirmation. In any event, several workers have reported mild inhibition by relatively high concentrations of ascorbic acid of the decarboxylation of dopa (Gonnard, 1949, 1951) and histidine (Martin et al., 1949; Gâbor et al., 1952a, b; Parrot and Reuse, 1954) by mammalian tissues preparations. Hartman et al. (1955) found no effect of moderate concentrations of ascorbic acid, nor for that matter cysteine or glutathione on dopa decarboxylase of hog kidney.

XIV. HORMONES

Villee (1960) recently has reviewed the general subject of the inter relations between enzymes and hormones. 9. INHIBITION OF AMINO ACID DECARBOXYLASES 347

A. Insulin

Gonnard et al. (1958; Gonnard and Chi, 1959a) found no effect of insulin on dopa decarboxylation by guinea pig kidney extract in vitro. Yamada et al. (1959) also found no in vitro effect of insulin on bacterial trypto phanase, another B6-dependent enzyme. Costa and Himwich (1959), how ever, reported a marked effect of hypoglycemic doses of insulin on rabbit kidney and brain 5-HTP decarboxylase in vivo. They did not rule out effects on 5-HTP uptake, monoamine oxidase, or pyridoxal kinase, which insulin may affect (cf. Manchester and Young, 1960; Akedo and Christen- sen, 1961; on uptake of amino acids).

B. Pituitary

Schayer (1957) reported a marked decrease of histidine decarboxylase in hypophysectomized rats within the first day, which persisted 4 days, then gradually returned to normal by the eighth day. Page and Reed (1945) found no effect of hypophysectomy on the pressor effect of intravenously administered dopa in rats. Yeh and associates (1959) reported no change in brain levels of 5-HT in hypophysectomized rats, but if they also injected adrenocorticotropic hormone (ACTH), brain 5-HT levels dropped. Since 5-HTP decarboxylase was not measured, no conclusions can be drawn regarding an effect on 5-HT synthesis until an effect on transport is ruled out, since it is known that, e.g., somatotropic hormone and hypophysectomy markedly affect amino acid transport in cells (Noall et al., 1957; Riggs and Walker (1960); and Kostyo et al. (1959; Kostyo and Knobil, 1959a, b, c; Kostyo and Engel, 1960; Kostyo and Schmidt, 1962). It is possibly of interest here to point out that the anterior pituitary exerts some control on adaptive enzyme formation, at least in the case of the rat liver tryptophan peroxidase-oxidase system (Geschwind and Li, 1953; cf. also Section XIV, C below; Schayer et al., and Kahlson et al., Section VII).

C. Adrenal Cortex

Page and Reed (1945) found that the pressor response to intravenously administered dopa was abolished initially after adrenalectomy in rats, with a gradual restoration to normal after two weeks. This was confirmed by Clark et al. (1956; Clark, 1959). 348 W. G. CLARK

An extensive literature exists on the effect of the adrenal cortex on blood histamine levels and histamine effects, particularly as related to allergic phenomena. Goth et al. (1951a, b) reported data suggesting that cortisone inhibits the resynthesis of histamine following its release and depletion by the surfactant Tween-20 in dogs. Mitchell et al. (1954) reported that free and conjugated urinary histamine are markedly increased in allergic patients by cortisone treatment, in confirmation of Grob (1952), who found also that urinary histidine is markedly increased, probably because of a decrease in tubular reabsorption and/or a decreased extrarenal uptake. Kurotsu et al. (1955) found that adrenalectomized rabbits have an increased blocd histamine. Beiler and co-workers (1958) extended Goth's experi ments on histamine resynthesis after its release by Tween-20 in dogs and observed that cortisone treatment prevented the reaction to a second injection of Tween-20. Schayer et al. (1954b, 1955; Schayer, 1956a, c, d, 1957) found that cortisone and its analogues prevent the decarboxylation of histidine in the intact rat as well as in isolated tissues. In addition, it inhibits the binding of the newly formed histamine. Adrenalectomy had an opposite effect (except on the enzyme from rat fundus, presumably the specific L-histidine decarboxylase). He was careful to point out that the inhibitory effect of cortisone might also be due to an increase in the destruction of histamine, the formation of inhibitors, decrease in cofactor, or combinations of these. Halpern and Briot (1956), Halpern et al. (1957), Halpern (1957), and Neveu (1960) presented evidence indicating that histamine synthesis is enhanced after treatment with histamine releasers (cf. Schayer's later work on adaptive increase in histidine decarboxylase apoenzyme synthesis, re viewed in Section VII), but that cortisone blocks the resynthesis. Yeh et al. (1959) showed that adrenalectomy only slightly alters rat brain 5-HT, but nearly doubles the liver level, and that cortisone lowers the liver level. The decarboxylase was not measured, so that the mechanism remained unexplained. Although cortisone injections decrease rat skin 5-HT levels, 5-HTP decarboxylase itself is unaffected (West, 1958), and adrenalectomy increases the tissue levels of 5-HT as well as histamine (Hicks and West, 1958a, b). Brains from rats treated chronically with cortisone or prednisolone have a normal 5-HTP decarboxylase level (Price and West, 1960). Telford and West (1960) made a careful study of the effect of adrenal cortical steroids on the histamine and 5-HT content of rat tissues and discuss the possibility that histidine decarboxylase of rat fundus (specific L-histidine decarboxylase) is stimulated by glucocorticoids. Presumably the adrenal corticosteroids do not affect the enzyme in cell- free preparations (Schayer et al., 1954b, 1955; Schayer, 1956a, c, d, 1957). See also Yamada et al. (1959), who found no effect of ACTH or hydro cortisone on cell-free bacterial tryptophanase, another B6-dependent enzyme. 9. INHIBITION OF AMINO ACID DECARBOXYLASES 349

Lin et al. (1958) showed an accelerating effect of hydrocortisone on synthesis of several adaptive apotransaminases in rat liver. Whether or not the glucocorticoids affect the decarboxylases through such a mechanism has not been clarified.

D. Thyroid

Thyrotoxicosis may decrease the availability of essential metabolites (see Section V); hence, in investigations directed toward elucidating a direct effect of the thyroid on enzyme systems, careful control of the dietary intake must be made. Thus, Drill and Overman (1942) found that rats on an adequate but minimal yeast supplement lost weight readily when fed thyroid. Thiamine injections blocked the weight loss but did not restore weight unless supplemented with a rich source of vitamin Β complex. Pantothenate and Ββ injections effectively replaced the latter, indicating an increased requirement of all of these factors in thyrotoxicosis. Horvath et al. (1961; Horvath^ 1957, 1958, 1959) were aware of such possibilities in studies in vivo and in vitro of the effect of thyroxine on B6-dependent enzymes, such as cysteine desulfhydrase, serine and threonine dehydrases and transaminases, and obtained paper chromatographic and spectro scopic evidence that thyroxine may form a Schiff base with the coenzyme by interaction of the amino group with the formyl group. They (1961) realized, however, the necessity for consideration of possible interference at the pyridoxal kinase level. Wachstein and Lobel (1956) found less xanthurenic acid in the urine of hyperthyroid patients, restored to normal by extra B6. They suggested that tryptophan metabolism was impaired due to a decreased availability of Be. This was confirmed and extended by Wohl et al. (1960), who found a greater urinary output of xanthurenic acid after a tryptophan test load in 14 hyperthyroid patients than in 14 euthyroid controls, which was re stored to normal by extra B6, indicating either a decreased availability of the latter in hyperthyroidism, or a defective incorporation of B6 into the enzyme systems required for normal tryptophan metabolism, or both. That the thyroid influences dopa metabolism in vivo was first suggested by the detection by Goodall (1950) of endogenous dopa on paper chro- matograms of extracts of adrenal medullary tissue from thyroidectomized sheep. No measurements of dopa decarboxylase itself were made. Samiy (1952; see Clark et al., 1956; Clark, 1959) in this laboratory found that prolonged administration of L-thyroxine to rats resulted in a marked and fairly sustained pressor response to intravenously administered L-dopa. This seemed to be due largely, if not altogether, to enhanced receptor sensitivity to the formed dopamine, as might be expected from the extensive literature on enhanced sensitivity to sympathomimetic 350 W. G. CLARK

drugs in hyperthyroidism. At first Samiy thought decarboxylation to dopamine by the intact animal was enhanced, as judged by chemical analyses and bioassays of the urine, but this remains to be established unequivocally. Analyses of all the visceral organs by manometric and microbiological assays (Clostridium tyrosine decarboxylase, manometrically; Lactobacillus delbriicki, growth; direct dopa decarboxylation, manometrically) showed that hyperthyroidism had no measurable effect on the apoenzyme or coenzyme content of any organs except liver and kidney. Hyperthyroidism decreased dopa decarboxylation in liver and increased it in kidney, the former apparently due to an effect on the apoenzyme rather than coenzyme. Since normal liver and kidney tissues contain 4-5 μg B6 per gram wet tissue, experiments using liver and kidney tissue as a source of B6 for bacterial tyrosine decarboxylase were performed to show the relative amounts of B6 in normal and hyperthyroid tissues. The results showed the hyperthyroid tissues contained as much or more B6 than normal tissues, thus providing additional evidence that the increases and decreases in dopa decarboxylase shown previously were due to changes in apoenzyme rather than to changes in the amount of cofactor present (Clark, 1959). These observations are supported by the work of Mascitelli-Coriandoli and Boldrini (1959), who found that B6-P04 is decreased 50-60% in livers of thyrotoxic rats (cf. also Bergeret et al. below). Thyroxamine and triiodothyonamine also enhanced the pressor re sponses to dopa, but without the long lag period required for the thyroxine effect (Clark, 1959). Thibault (1950, 1952a, b) had previously shown a similar lack of a lag period in the effect of these decarboxylated thyroid hormones on adrenaline responses. Westermann (1956) and Holtz et al. (1956) showed that in hyperthyroid guinea pigs and rats, dopa decarboxylase decreased in liver, as it also did in starvation (cf. Section V), and that this was reversed in the latter case but not the former by B6-P04 added to the liver in vitro. Spinks and Burn (1952) and Trendelenburg (1953) previously reported that hyperthyroidism in rabbits deceased monoamine oxidase and that hypothyroidism in creased it in liver; they postulated that the enhanced sensitivity to pressor amines in hyperthyroidism is in part caused by this decrease. Holtz et al. (1956) questioned this, since they found hyperthyroidism increased monoamine oxidase in rats and guinea pigs. Litwin and Kordecki (1958) fed large doses of thyroid to dogs for a month, and because they obtained no difference in pressor responses to adrenaline whether injected into the portal vein or into the femoral vein, argued that hyperthyroidism had no effect on liver moncamine oxidase. Since they did not measure the latter, and since we now know that injected adrenaline is metabolized chiefly by catechol-O-methyl transferase, conclusions based upon such indirect evi- 9. INHIBITION OF AMINO ACID DECARBOXYLASES 351 dence are not justified. Similar criticism can eb made fo conclusions drawn from measurement fo tissue amine levels such sa those fo Kato and Valzelli (1959), who postulated na inhibition fo monoamine oxidase ni brain yb thyroxine injections, since the latter caused na increased rat brain 5-HT, although thyroidectomy ro thiouracil treatment had on effect. Zile et al. (Zile and Lardy, 1959; Zile, 1959, 1960) more recently obtained direct evidence ot support the work fo Spinks and Burn yb measuring mono• amine oxidase ni brain, liver, and heart mitochondria fo hyper- and hypo• thyroid rats. Hypothyroid rats had twice the activity fo hyperthyroid ones. They suggest that the inhibition yb hyperthyroidism si not a direct effect, since on inhibition occurred in vitro and on inhibitor could eb de• tected ni the liver tissue preparation. Thus, they postulate that there may be na effect no the synthesis fo the apoenzyme ro no other enzymes which may regulate monoamine oxidase activity ni liver. This work si supported by the recent report fo Dubnick et al. (1960b) that thyroidectomized rat hearts show a marked decrease ni monoamine oxidase, accompanied yb slight increases ni heart levels fo 5-HT and noradrenaline. They point out that ti si established that amine depletion, sa obtained with reserpine, increases the sensitivity fo the heart ot catechol amines and that, con• versely, higher amine levels may desensitize it, sa ni hypothyroidism. Regarding the in vitro effects, other workers have observed inhibition fo other B6-dependent enzymes in vitro yb thyroxine (Horvath, 1957, 1958, 1959; Yamada et al, 1959). Bergeret et al. (1958), Chatagner (1959), Chatagner et al. (1959), Labouesse-Mercouroff et al. (1960), and Jollès- Bergeret et al. (1960) recently reported that thyroxine injections decrease dopa and cysteinesulfinic acid decarboxylations and cysteine desulfuration by rat liver. Thyroidectomy increased the rates ni livers fo female rats but had on effect no decarboxylation fo glutamic acid yb brain, the former being ni agreement with Holtz et al. and Clark. They also found that liver B6-P04 decreased ni hyperthyroidism and increased after thyroidectomy, in agreement with Clark (1959). Canal and Garattini (1957), Canal and Tessari (1957), and Canal et al. (1958; Canal and Maffei-Faccioli, 1959) reported a marked decrease ni cysteic acid decarboxylase fo livers fo hyperthyroid rats, on effect no glutamic-pyruvic transaminase, na increase in glutamic-oxalacetic transaminase fo liver, and on effect no glutamic acid ro 5-HTP decarboxylation yb brain and liver. Goldstein et al. (1958) found that hyperthyroidism increased the urinary excretion fo radioactive adrenaline and noradrenaline after injecting labeled dopamine and decreased what apparently were its O-methylated metabolites. DTorio and Leduc (1960) reported that hyperthyroid rat livers have a 45% decrease ni catechol-O-methyltransferase and that hyperthyroid rats excrete less labeled metanephrine after injection fo 352 W. G. CLARK radioactive adrenaline. There was, however, no direct effect of thyroxine on the transferase system in vitro. It is not yet known whether the effect in vivo is a competitive inhibition, and although 3,5-diiodo-4-hydroxy- benzoic acid (MacLagan, and Wilkinson, 1951) and thyroxine (Roche et al., 1961) are O-methylated in vivo, and injected estradiol (Kraychy and Gallagher, 1957) is excreted as 2-methoxyoestrone, DTorio and Leduc (1960) suggest that the action of thyroid hormone is an indirect one, possibly interfering with the general mechanism of transmethylation (cf. Sourkes and D'lorio, Volume II).

E. Sex Hormones

Sloane-Stanley (1949b) reported that cysteic acid decarboxylase of rat liver is twice as active in males as in females, the difference being abolished by spaying and partially restored to the normal lower level by injecting estrone into the spayed females, but there was no effect on the higher level in males following castration. He speculates that perhaps the female, be cause of the lower cysteic acid decarboxylase activity in the liver, has a lower rate of fat absorption because of less taurine incorporation into the taurocholic acid in bile, which participates in fat emulsification in the intestine. He also suggests that ovariectomy, by increasing the liver de carboxylase activity, might increase fat absorption to levels which cause ketosis or fatty liver. Samiy (1952) observed a marked sex difference, in favor of the male, on the enhancement by thyroxine of the hypertensive effect of injected dopa in rats. Dietrich and Shapiro (1955) found no effect of testosterone injections on dopa decarboxylase, cysteic acid desulfhydrase, and two transaminases in enzyme preparations or tissue slices of normal or adenocarcinomatous tis sues in mice. However, testosterone in conjunction with deoxypyridoxine injections increased the sensitivity of these enzymes to Bo-deficiency by the deoxypyridoxine. An extensive study was made on the effect of sex hormones on dopa, cysteinesulfinic acid, and glutamic acid decarboxylases in rats by Chatagner et al. (1959; Chatagner and Bergeret, 1956, 1957), Labouesse et al. (1959), Labouesse-Mercouroff et al. (1960), and Bergeret et al. (1958), which con firmed and extended the observations of Sloane-Stanley on the sex differ ence of cysteinesulfinic acid decarboxylation by rat liver and which may explain the greater taurine levels in the female liver. There were no sex differences in cysteic or glutamic acid decarboxylases of brain. Ovariectomy abolished the lower activity of cysteinesulfinic acid decarboxylase in the female, estradiol partly restored the difference, and progesterone com- 9. INHIBITION OF AMINO ACID DECARBOXYLASES 353 pletely restored it. Ten micrograms of estradiol benzoate injected daily for 8-12 days decreased cysteinesulfinic acid decarboxylase activity in livers of both males and females, markedly so after the latter were ovariectomized. The sex difference in dopa decarboxylation was much less, but still in favor of the male, an observation which has been borne out by Clark (1959). In this case, however, ovariectomy or estradiol injections had no effect. Estradiol increased dopa decarboxylase activity which had been lowered in liver by thyroidectomy. Since "physiological" amounts of thyroxine de crease both decarboxylases in ovariectomized and in thyroidectomized females, they postulate two independent hormonal regulators of the two enzymes. Kato (1960) found that male rats have slightly lower brain 5-HT levels than females and that estradiol injections increase the levels in both young females and adult males. Testosterone had no effect. These observations are difficult to reconcile with the work of Sloane-Stanley and of Chatagner et al. (Section IV) on uptake and binding and should be borne in mind in studying estrogen effects on amino acid and amine levels, since it is known they may have profound effects on amino acid transport (Noall et al., 1957; Noall, 1960; Daniels and Kalman, 1961). 5 6 Labouesse et al. (1959) found that 3.2 Χ 10~ M to 10~ M estradiol benzoate had no effect on cysteinesulfinic acid decarboxylase activity of a dialyzed rat liver preparation in vitro. Pertinent to this discussion are the recent reports by Mason and Gullek- son (1959, 1960) of marked inhibitory effects on kynurenine transaminase of rat kidney in vitro by7 estradiol sulfate and diethylstilbestrol disulfate at levels as low as 5 X 10~ M. Nonanionic steroids, such as estradiol, diethyl stilbestrol, and estrone, were inactive. Estrone sulfate and pregnanediol glucuronide inhibited but were much less active. Several bile acids were inhibitory at higher concentrations, in confirmation of Werle (1940, 1943b, and Okunuki, 1943). The inhibition by these estrogens varied inversely with the concentration of B6-P04, and was completely reversed by dialysis, which indicates that the inhibition results from competition between the estrogen sulfates and the coenzyme for the apoenzyme. Reconstitution of the enzyme by adding B6-P04 was blocked by diethylstilbestrol disulfate, but once reconstitution occurred, low concentrations of the conjugate no longer inhibited. Inhibitory effects also were observed on other B6-dependent systems. The reader is referred to these papers and to the review by Chatagner (1959) for discussions of the possible relations of these effects to the physiological actions of estrogens. Interpretations of possible effects of sex hormones on histamine production in vivo which are based on measurements of the amine or its metabolites without direct measure ments of histidine decarboxylase itself are unacceptable because of effects on histamine catabolism (Marshall, 1961). 354 W. G. CLARK

XV. MISCELLANEOUS

A. Antibiotics

Antibiotics and sulfonamides inhibit some but not all amino acid de carboxylases (Schayer et al., 1954a; Matsuda et al., 1955; Melnykovych and Johansson, 1955; Michel and Francois, 1956; Alexander, 1960; Yamagami, 1958; Werle and Aures, 1960; Martin et al, 1942; Gale and Epps, 1944; Epps, 1944, 1945; Taylor and Gale, 1945; Gale 1946; Arjona et al, 1950, 1951; Schreus and Stuttgen, 1950; Werle, 1951, Bornstein, 1957; Wachi et al, 1959). The growth-stimulating effect of antibiotics fed to animals has been ascribed by some of these workers to a decrease in the production of toxic amines by intestinal flora.

B. Antihistamines

It is important to record that these agents do not inhibit bacterial (Sterzl and Krecek, 1949, 1950; Kfecek et al, 1950) or mammalian (Uchida, 1954; Schayer et al, 1955) histidine decarboxylases.

C. Reserpine

Because this drug markedly depletes tissue amines, it is important to know that most (Haverback et al, 1956; Erspamer and Ciceri, 1957; Brodie et al, 1957; Dubnick et al, 1960a, b, c; Bartlet, 1960), but not all (West, 1958), investigators have found that it has no effect on dopa and 5-HTP decarboxylase in vitro or in vivo.

D. Tranquilizers

Since it was thought possible that chlorpromazine might exert its de pressive effect by inhibiting the formation of brain amines (and gaba), its effects on the decarboxylases responsible for their formation have been examined, with conflicting results (West, 1958; Bartlet, 1960; Ehringer et al, 1960; Pletscher and Gey, 1960; Gey et al, 1961; Gey and Pletscher, 1962; Greenberg et al, 1959). Chlorpromazine does, however, markedly inhibit pyridoxal kinase, especially in B6-deficiency (Greenberg et al, 1959).

E. Tetrahydroisoquinolines

Parrot and Laborde (1955, 1956, 1959) have observed that certain de rivatives of isoquinolines inhibit histidine decarboxylase of mammalian 9. INHIBITION OF AMINO ACID DECARBOXYLASES 355 tissues in vitro and in vivo orally administered, and clinically in allergies. Compounds which were active are benzyl-l-analogues of l-phthalidyl-2- methyl-6,7-methylenedioxy-8-methoxy-l ,2,3,4-tetrahydroisoquinoline. No data are given, however, on a direct measurement of inhibition in vivo, but only on protection of guinea pigs from histamine aerosol and blood levels of histamine.

F. Folic Acid Antagonists

Martin and Beiler (1947) found inhibition of dopa decarboxylation by mammalian tissue preparations in vitro by 30-300 Mg/ml final concentra tion of 7-methylfolic acid and by the aspartic acid analogue of folic acid. Tyrosine decarboxylase of S. faecalis also was inhibited, but ten times greater amounts were required. With the thought that this enzyme is in volved in the biosynthesis of endogenous catechol amines and their par ticipation in adrenergic nerve control of arterial blood pressure, Martin and co-workers (1947) were motivated to examine the effect of 7-methyl- pteroylglutamic acid on acute blood pressure changes of dogs.. They found that it was a powerful depressor when given intravenously in doses of 5-50 mg/kg (75-100 mg/kg was fatal). In 1948, Martin et al. followed this up with a systematic study of 30 pteroylglutamic acid-displacing agents, which were synthesized (details of the syntheses were given for three com pounds) and tested (a) as pteroylglutamic antagonists on S. faecalis growth, (b) by dopa decarboxylase inhibition of rat kidney extract in vitro, and (c) by acute depressor responses in an average of six dogs per compound tested, thus presumably in 180 dogs. A representative table permits one to examine these three correlates for 29 compounds, with data given for 13 on dopa decarboxylation, five of which were folic acid antag onists. The results are given as concentration per milliliter which caused a given percentage inhibition (no Qco2 or μΐ CO2 data are given). The amount of enzyme is not given, no B6-P04 was added, and it is not stated whether residual C02 is expelled by acids at termination of the run. Furthermore, rat kidney is a weak source of the enzyme. There was no apparent corre lation with the blood pressure effects, but the authors invoke the rationale of an effect on catechol amine synthesis being causally related to the re sults. Obviously, much more work should be done before such claims are made, and in any event, from what we know of the biosynthetic pathways, the acute depressor responses seen are unrelated to any effects on dopa decarboxylation. Schales and Schales (1949), working with guinea pig and rabbit kidney extracts, confirmed Martin et al. that folic acid antagonists inhibit dopa decarboxylase in vitro. Of five analogues tested, three gave mild to moder ate inhibition, namely 7-methylfolic, 2,4-diamino-6,7-bis-(p-sulfinomethyl- 356 W. G. CLARK aminophenyl)pteridine and 2-amino-4-hydroxypteridine aldehyde, at concentrations of 0.4-2.5 mmoles/liter. Their data are clear. They criticize Martin et al. by pointing out that the low levels of decarboxylase activity implied (from former references) do not permit an accurate assessment of inhibitory activity, since Martin et al. based their .calcula tions on differences of less than 4 μΐ C02 and did not correct for retained C02. In any event, the water-soluble bis analogue was most active (47% inhibition at 0.4 mmoles/liter). The mode of action remains to be explained, and there was no correlation with antifolic acid action. Gonnard (1951) also confirmed the observation that certain folic acid analogues inhibit mammalian dopa decarboxylase in vitro, namely, aminofolic, xanthopterin- carboxylic, and isoxanthopterincarboxylic acids. In none of these reports was a mechanism of action suggested. In the 14 years which have passed since Martin's first report, the reviewer has seen no papers on attempts to see if such compounds affect the decarboxylations in vivo using acceptable techniques. Recent experiments on this point in this laboratory were nega tive.

XVI. SUBSTRATE ANALOGUES

Like the entire subject of antimetabolites, this is a complex subject and is interrelated with many of the sections above. With B6-dependent en zymes it is peculiarly complex because of interreaction of substrate and substrate analogues with coenzyme. Inhibition by "mass action" is compli cated in interpretation because of this effect. Nevertheless, true com petitive inhibition by substrate analogues occurs, and for this reason this section is a highly important one. The subject is discussed in Volume II by Sourkes and DTorio.

A. Bacterial and Plant Decarboxylases

1. GLUTAMIC ACID Glutamic acid decarboxylation by whole cells or extracts of several species of bacteria is inhibited by aspartic acid (Storck, 1951). There is little or no effect on decarboxylation by plant extracts of other amino acids (Okunuki, 1937; Schales and Schales, 1946b).

2. HISTIDINE

Histidine decarboxylation by E. coli is unaffected by benzoylhistidine or acetylhistidine (Geiger, 1944). Plant histidine decarboxylase is not inhibited by D-histidine, unlike the mammalian enzyme (vide infra) (Werle and Raub, 1948). 9. INHIBITION OF AMINO ACID DECARBOXYLASES 357

3. TYROSINE

Tyrosine decarboxylation by S. faecalis preparations is not inhibited by 23 tyrosine analogues tested (Epps, 1944; McGilvery and Cohen, 1948; Frieden et al, 1951; Lestrovaya and Mardashew, 1960), even if present in amounts exceeding the substrate eightfold (McGilvery and Cohen, 1948). Blaschko and Stiven (1950) showed that o-, m-, and p-chlorophenylalanines act neither as substrates nor inhibitors of tyrosine decarboxylase in S. faecalis.

B. Mammalian (and Fowl) Decarboxylases in vitro (cf. also Section XVI, G)

1. CYSTEIC ACID Cysteic acid decarboxylase is not inhibited by asparagine (Werle and Bruninghaus, 1951), cysteine, phosphoserine3 , aspartic acid, or benzene- sulfinic acid, but is inhibited 76% by 10~ M cysteinesulfinic acid (Simon- net et al., 1960) and by homocysteic acid, a nonsubstrate (Blaschko, 1945b).

2. GLUTAMIC ACID Glutamic acid decarboxylase of brain homogenates is competitively in hibited by p-hydroxyphenylacetic > phenylpyruvic > p-hydroxyphenylpyruvic acids, but not by phenylacetic acid or phenylalanine (Hanson, 1958) (cf. Sections XVI, C and H below). Greig et al. (1959) reported that several derivatives of α-methyltryptamines inhibited (cf. Section XVI, I below, and substrate-coenzyme interaction, discussed by Sourkes and DTorio in Volume II of this treatise).

3. HISTIDINE Histidine decarboxylase (nonspecific) is not inhibited by other amino acids, with the exception of cysteine; equivocally by tyrosine and trypto phan; dopa, which strongly inhibits (Werle and Heitzer, 1938; Werle and Koch, 1949; Ganrot et al., 1961); and α-methyldopa (but cf. Section XVI, I below). Interestingly, D-dopa inhibits more than L-dopa, according to Werle (1941). Adrenaline and noradrenaline, D-histidine, imidazole, and benzylimidazole (Priscol) also inhibited. Beiler et al. (1949) confirmed the lack of effect of other amino acids, but found mild inhibition by 1.0 mg/ml iV-sulf anilyl-4-aminobenzimidazole, benzoxazolone, 3-benzothioph ene-a- aminopropionic acid, and β-2-thienylalanine. Schayer and Kobayashi (1956) and Schayer (1956b) confirmed the latter with histidine decar boxylase of rabbit blood platelets and rat peritoneal mast cells, and ex tended the list to β-3-thienylalanine, methylhistidine, tyrosine, tryptophan and 5-HTP. Phenylalanine, D-histidine, histamine itself, and phenylalanine 358 W. G. CLARK

were inactive, and thiolhistidine only slightly. Caffeic acid and catechol also inhibit histidine decarboxylase of rabbit kidney, but these agents (and α-methyldopa) do not inhibit the specific L-histidine decarboxylase of embryonic rat liver (Ganrot et al., 1961).

C. Mammalian Dopa Decarboxylase in vitro

This subject, as well as dopa decarboxylase inhibition in vivo, has been adequately reviewed by Clark (1959), Clark and Pogrund (1961), and Sourkes and DTorio (see Volume II) and will not be repeated here in extenso. 14 Drell (1957) incubated tyrosine-C with beef adrenal slices and found that the decarboxylase inhibitor, 5-(3-hydroxylcinnamoyl)salicylate (Clark, 1959; Clark and Pogrund, 1961), markedly decreased the incorporation of label into the catechol amine fraction and increased that in the catechol acid fraction, which includes dopa. Fellman (1959) found that 5-HTP competitively inhibits dopa decar boxylation. During purification, the ability to cleave dopa, o-tyrosine, and 5-HTP remained constant at each step, leading him to conclude that the same enzyme is involved. Rosengren (1960) confirmed this and, in addi tion, found that dopa and 5-HTP decarboxylations are cross-inhibited also by ra-tyrosine, o-tyrosine, and caffeic acid, the latter in confirmation of Hartman et al. (1955). Werle and Aures (1960) also found inhibition by caffeic acid, chlorogenic acid, and 5-(3-hydroxycinnamoyl)salicyclic acid, in confirmation of Hartman et al. Griesemer et al. (1961), in a preliminary abstract, recently extended the studies of Hartman et al. (1955) to add 45 more compounds to their original list of over 200 compounds. Sixteen of the 45 gave good inhibition, and the essential structural requirements formerly elucidated were confirmed and extended, the best inhibitors having the structure R—CH=CH—CO—R', where R = 3-hydroxyphenyl, 3,4-dihydroxyphenyl, or 5-hydroxyindole, and R' = OH, O-alkyl or aryl.

D, Mammalian 5-HTP Decarboxylase in vitro

C. J. Clark and associates (1954) found no inhibition o2f this enzyme by tryptophan, 7-hydroxytryptophan3 , or 5-HT up. to 10~ M. 5-Benzoxy- tryptophan inhibited at 10~* M. The list was extended in the same labora tory (Fréter et al., 1958) to include 10 other 5-HT and 5-HTP analogues, the most effective of which was 2,5-dihydroxytryptophan, which gave 68% inhibition at concentrations equimolar with substrate. Ozaki (1959) reported that iV-methyldopa inhibits 5-HTP decarboxylase of rat brain. 9. INHIBITION OF AMINO ACID DECARBOXYLASES 359

Yuwiler et al. (1959, 1960) examined the best dopa decarboxylase in hibitors reported by Hartman et al. (1955) on 5-HTP decarboxylase and found them equally active and competitive. In addition, they found com petitive inhibition by the analogue l-[5-hydroxyindolyl-3]-2-(3-carboxy- 4-hydroxylbenzoyl)ethylene. This compound is also a good inhibitor of dopa decarboxylase in vitro (cf. also Griesemer et al., 1961). /3-(3-Indolyl)- acrylic acid was a fair inhibitor, but 5-(3-indoleacryloyl)-salicylic acid and l-[5-oxyindolyl-(3)]-butenone-(3) were inactive, which is interesting since the m-hydroxyphenyl analogue of the latter, with the same side chain, —CH=CH—CO—CH3 (m-hydroxybenzalacetone), inhibits dopa decar boxylase (Hartman et al., 1955). Recently, Erspamer et al. (1961) examined 22 tryptophan analogues as substrates of 5-HTP decarboxylation by guinea pig kidney extracts and reported marked inhibition at pH 6.8 by 8 μηιοΐββ of caffeic acid and 1,4- bis-(3,4-dihydroxycinnamoyl)quinic acid (1,4-dicaffeylquinic acid, "eyna- rine"). Inhibition of decarboxylation of 5-HTP was more complete than that of 4-HTP (the only other analogue of the 22 examined which was a substrate) or of dopa.

Ε. Glutamic Acid Decarboxylase in vivo

This reviewer could find no work on this subject except some yielding indirect evidence (cf. Section XVI, I below).

F. Dopa Decarboxylase in vivo (cf. Clark, 1959; Clark and Pogrund, 1961; and Sourkes and D'lorio, Volume II)

The first clear-cut evidence of dopa decarboxylase inhibition by dopa analogues in vivo which also are active in vitro was presented in abstract form by Pogrund and Clark (1953) and Pogrund et al. [1955; cf. Clark and Pogrund (1961), which describes methods in detail]. In a review on dopa decarboxylase inhibitors, Clark (1959) listed 30 active compounds and 50 inactive analogues. In the meantime, Dengler and Reichel (1957) and Westermann et al. (1958) demonstrated the inhibition of dopa, dops, and 5-HTP in vivo with a-methyldopa. During this work, it was noted that if a Lineweaver-Burk analysis is made in vivo (for discussions of this, cf. Chen and Russell, 1950; Gaddum, 1957; Nickerson, 1959), the inhibitors appear to be competitive (Clark, 1959; Clark and Pogrund, 1961). It was also found that there is no strict parallelism between the inhibition in vitro and in vivo, but those substances which were inactive in vitro almost always were also inactive in vivo. All 360 W. G. CLARK

substances tested were rapidly metabolized, and the inhibitory effects disappear within 30-60 minutes, including α-methyldopa (vide infra). Because of this, further pharmacological, physiological, and clinical studies were not planned until compounds with more prolonged effects could be developed. In a few experiments (Clark and Pogrund, 1961), however, resynthesis of catechol amines by the insulin-depleted adrenal glands of rats seemed suppressed by repeated injections of two inhibitors which later were shown to be one-fifth as active as the best one, 5-(3- hydroxycinnamoyl)salicylic acid. Brodie et al. (1962), Drain et al. (1962), and Burkard et al. (1962), however, showed that nearly complete inhibition of 5-HTP/dopa decarboxylase in vivo by α-methyldopa, a-benzyloxy- amine, and α-benzylhydrazine do not affect the endogenous levels of catechol amines and 5-HT, possibly because only a small fraction of the nonrate-limiting decarboxylase activity available in normal tissues is sufficient for a physiological rate of decarboxylation. Indirect evidence of cross competition of dopa and 5-HTP decarboxyla tion was recently afforded by Kato (1959), who showed that 5-HTP po tentiation of barbiturate hypnosis in mice is completely reversed by dopa.

G. Quinones and Potential Quinoids

In addition to reacting with thiol groups, such compounds also may react with free amino groups. Many compounds described in the literature as inhibitors of amino acid decarboxylases may belong to this category rather than to those claimed by the authors. If compounds have potential quinoid structures, precautions should be taken to prevent oxidation to quinones by adding, e.g., cysteine to substrate and glutathione to enzyme before reacting under anaerobic conditions (even then, such reductants have not prevented quinone formation in some cases). If this and other precautions, such as performing a Lineweaver-Burk analysis, 1have not been taken, re ports of inhibition by "substrate competition/ "displacement," or by "metabolic antagonism" of various physiological functions in vivo are open to criticism. This is particularly true in the clinical literature. Examples are the clinical reports on the oral administration of flavonoids ("vitamin P") in allergies, based on the report that they were shown to inhibit histamine synthesis in vitro. To the reviewer's knowledge, the first ex amples which demonstrated unequivocally the alteration of a clinical entity by inhibiting an amino acid decarboxylase in human subjects are the recent reports of Oates, Gillespie, Sjoerdsma, Crout, and Udenfriend at the National Institutes of Health, Bethesda, Maryland (cf. Section XVI, I below). 9. INHIBITION OF AMINO ACID DECARBOXYLASES 361

Bacterial decarboxylation of basic amino acids is inhibited by tannins and their precursors, such as o-dihydroxyphenolearboxylic acids and poly phenols, the former being more effective (Kimura et al, 1958). The in hibition is not antagonistic to the coenzyme, is noncompetitive with the substrate, and is not reversed by cysteine. Inhibition of mammalian dopa and histidine decarboxylases in vitro has been reported for many quinones and potential quinoid compounds, including quinone, hydroquinone, catechol, pyrogallol, dopa, catechol amines, and o-dihydroxyphenols in general, including flavonoids, anthocyanins, hematoxylin, and related compounds (Werle, 1941; Werle and Koch, 1949; Malkiel and Werle, 1951; Werle and Aures, 1960; Imiya, 1941; Martin et al, 1942, 1949; Martin, 1951; Bargoni, 1946; Gonnard, 1951; Gâbor et al, 1952a, b; Parrot and Reuse, 1954; Hartman et al., 1955; Kimura et al., 1958), and glutamic acid decarboxylase by adrenochrome (Holtz and Westermann, 1956a, b, 1957). The latter may not be a purely nonspecific quinone inhibition, however, since Deltour et al. (1959a, b) found activation of the same enzyme by adrenochrome derivatives in which the quinone function is blocked. Schayer et al. (1955) found no inhibition of mammalian histidine de carboxylase by cf-catechin. Hartman and co-workers (1955) examined a series of flavonoids for their ability to inhibit dopa decarboxylase, includ ing flavones, flavans, flavanones, chalcones, coumarins, and related com pounds, and discuss structural requirements. The list was extended recently in a preliminary abstract by Griesemer et al. (1961) to include additional analogues. If cysteine is added to the side arm of the Warburg vessel with substrate, and glutathione with enzyme before mixing to start the reaction, many potential quinoid structures had markedly less inhibition, and a Lineweaver-Burk analysis showed the inhibition was competitive. With out such precautions, the inhibition was noncompetitive or pseudocom- petitive. Incubation of inhibitor with enzyme prior to starting the reaction markedly enhanced inhibition. Compounds with 3,4-dihydroxy groupings in the flavone type (or the analogous 3',4'-dihydiOxy groupings in the chalcones) were most active, but the 3-hydroxychalcones, which are non- quinoid, also were quite active.

H. Ketonuria

Weil-Malherbe (1955) reported low blood adrenaline levels in mentally defective patients including phenylketonurics (phenylpyruvic oligophrenia). This led Fellman (1956) to examine the effects of aromatic acids associated with the disease on dopa decarboxylation of beef adrenal medullary ex tracts. Phenylalanine had no effect, but phenylpyruvic, phenyllactic, and 362 W. G. CLARK

phenylacetic acids, in that order, inhibited at concentrations from 3 to 30 ^mole/ml, depending on the compound. The author speculated on whether this might be one reason for Weil-Malherbe's observation. These results were surprising in view of the results of Hartman et al. (1955), who found no appreciable effects of these compounds. However, Davison and Sandler (1958) confirmed the effect of these compounds on 5-HTP decarboxylase in vitro, except that phenylacetic acid was more active than either phenylpyruvic or phenyllactic acid at the same concentrations. Pare and associates (1957, 1958a, b) speculated that, in addition to a defective hydroxylation of phenylalanine in ketonurics, there might be a similar defect in 5-HT synthesis and found low blood 5-HT and urine 5-HIAA levels in such patients. Low phenylalanine diets increased the blood 5-HT in six of seven cases. 5-HTP tolerance tests in four cases showed subnormal urinary 5-HT and 5-HIAA excretion. However, therapy with 5-HTP was of no benefit. Sandler (1959a, b; Sandler and Close, 1959) reported that phenylacetic acid administered in doses of 5 gm by mouth to five ketonurics decreased urinary 5-HIAA in three of them, although there was no relief of symptoms. Phenylalanine had no such effect. Hanson (1958, 1959) examined the inhibitory effect of such compounds on glutamic acid decarboxylation by brain in vitro and found p-hydroxyphenylacetic > phenylpyruvic > p-hydroxyphenylpyruvic acids in con centrations of 25-100 /xmoles/ml. Phenyllactic acid and phenylalanine had no effect. Baldridge et al. (1959) found that, following the decrease of serum phenylalanine seen in ketonurics on a low phenylalanine diet, urinary 5-HIAA excretion increased. Oral administration of tryptophan or 5-HTP produced the same effect. Huang et al. (1961) found that rats on high phenylalanine and tyrosine diets excrete 5-10 times more phenylpyruvic acid; this was correlated with decreased 5-HIAA excretion and lowered plasma phenylalanine and 5-HT. Thus, such diets simulate the condition seen in clinical ketonurics. Tashian (1960) found increased urinary indoleacetic acid in normal sub jects fed phenylpyruvic, phenylacetic, or o-hydroxyphenylacetic acids, the latter being more effective. The administration of phenylpyruvic or o-hydroxyphenylacetic acids caused an increase in 5-HIAA excretion which was less than that of indoleacetic acid. Assuming that the same metabolites which can inhibit 5-HTP decarboxylase can also inhibit tryptophan de carboxylase, Tashian postulated that inhibition of tryptamine and 5-HT synthesis, therefore, could shunt an increased amount of tryptophan through transanimation to indolepyruvic acid and IAA, thus accounting for the observations. Subsequently (1961), he found that glutamic acid 9. INHIBITION OF AMINO ACID DECARBOXYLASES 363 decarboxylation by rat brain homogenates and E. coli powder was com petitively inhibited by phenylpyruvic, p-hydroxyphenylpyruvic, phenylacetic, p-hydroxyphenylacetic, o-hydroxyphenylacetic acids, and deriva tives of valine and leucine. He postulated that if compounds such as these, which are formed in greater amounts in phenylketonuria and branched- chain ketonuria (maple syrup disease), reach the developing brain, they might limit the formation of gaba and of amines possibly essential to normal neurological function.

I. a-Alkyl Substrate Analogues (see also Section IV)

This subject has been treated by Sourkes and DTorio in Volume II. The subject was also briefly reviewed up to November, 1960 (Clark and Pogrund, 1961). Pfister et al. (1955) and Stein et al. (1955) described the syntheses of some α-methyl homologues of glutamic acid, methionine, diaminopimelic acid, and phenylalanine, including those of tyrosine and dopa. These were prepared as potential antimetabolites, including amino acid decarboxylase antagonists. Roberts (1952a, 1953) examined some of these and other glutamic acid analogues for their ability to inhibit glutamic acid decarboxylation by acetone powders of E. coli and mouse brain. Noninhibitors of the bac terial enzyme included the diastereoisomer racemates of a-hydroxy- glutamic acid, methionine sulfoxide, various iV-substituted α-amides of glutamic acid, and pyrrolidonecarboxylic acid analogues. Inhibition was best with α-hydroxyiminoglutaric and DL-a-methylglutamie acid, the former probably because of its hydrolysis to HONH2 and the latter by competitive inhibition. Waksman (1957) found that α-methylglutamic acid inhibits glutamic acid decarboxylation by acetone powder of Torulopsis utilis. Roberts (1952a) reported that α-methylglutamic acid itself was not a substrate, at least by the method used. It also was the most potent of the series in inhibiting the utilization of glutamic acid for Lactobacillus ara binosus growth. It was a weak to moderate inhibitor of the brain enzyme. Evidence for inhibitory action in vivo was suggested by the observation that α-methylglutamic acid enhanced the incidence of audiogenic seizures in susceptible mice, which was counteracted by glutamic acid. Ginsburg and Roberts (1951) reported that, of the various enhancing agents, me thionine sulfoxide and α-methylglutamic acid aggravated the seizures in proportion to their potency as metabolic antagonists of glutamic acid in bacterial growth. Ginsburg (1954) demonstrated a seizure enhancement effect of α-methylglutamic acid, but enhancement was also induced by 364 W. G. CLARK other organic acids. Other examples of inhibitory effects of α-methyl metabolite analogues exist in the literature, such as the inhibition of B6-dependent active uptake of amino acids inhibition of the enzymic formation by α-methylglutamic acid of the transferase in sheep brain, which produces glutamohydroxamic acid from hydroxylamine and glu- tamine, and of glutaminase in dog kidney (Lichtenstein et al., 1953a, b). Other antimetabolite effects of α-methyl amino acids, as well as the mech anism of action, were reviewed briefly by Umbreit (1955) (cf. also Christen sen, Section IV). Sourkes (see Sourkes and DTorio, Volume II) tested a series of 22 phenylalanine analogues, including the α-methyl derivatives prepared by Pfister et al., on dopa decarboxylase in vitro. Among them, a-methyl-DL4 - dopa and α-methyl3 -DL-ra-tyrosinB e were good inhibitors at 1-5 Χ 10~ M and 5 X 10~ to 5 X 10~ M, respectively, when preincubated with enzyme before adding substrate. Subsequently, Hartman et al. (1955) found that, when added 3simultaneously, the inhibition by a-methyldopa is only fair, 35% at 10~ M. When preincubated 15 minutes with enzyme, in6 contrast, 5-(3,4-dihydroxycinnamoyl)salicylate inhibited 100% at 10~ M (cf. also Griesemer et al., 1961). Simmone3t and co-workers (1960) reported that DL-a-methylcysteic acid, 1.5 X 10~ M, inhibits cysteic acid decarboxylase of chick embryo tissue in vitro. Greig et al. (1959, 19612 ) examined the inhibitory effects of relatively high concentrations (10~ M) of a series of α-alkyltryptamines on 5-HTP decarboxylase in vitro and in vivo and found inhibition in vitro by 5-hydroxy-a-methyltryptamine-creatinine sulfate and α-methyltryptamine but not by α-ethyltryptamine (Etryptamine, Monase). The 5-hydrox3 y ana logue was most active. Iproniazid also inhibited at 5 X 10~ M. Both compounds also were active in intact mice (cf. Fréter et al., 1958 for other inhibitory 5-hydroxytryptamine analogues). Glutamic acid decarboxyla tion by brain in vitro was unaffected. The predominant action of these 5-HT analogues was, however, inhibition of monoamine oxidase. Yuwiler et al. (1959) did not confirm Greig et al. on an inhibitory effect of a-methyltryptamine (as the methanesulfonate) on 5-HTP decarboxylase in vitro. Van Meter et al. (1960) found that α-methyltryptamine inhibited 5-HTP decarboxylation by brain in vivo, as did methyltryptamine to a lesser degree. These compounds also increased 5-HT levels in brain, .probably by blocking monoamine oxidase. Effects on binding-release mechanisms were not examined. The effects were correlated with electroencephalographic and behavioral changes. Weissbach et al. (1960a, 1961) and Lovenberg et al. (1962) showed that α-methyldopa inhibits decarboxylation by a semipurified guinea pig 9. INHIBITION OF AMINO ACID DECARBOXYLASES 365 kidney preparation, of 5-HTP, dopa, tryptophan, phenylalanine, tyro sine, and histidine and that several of the α-methyl analogues are them selves decarboxylated, in proportion to their inhibitory power. This shows that there need not be a hydrogen atom on the α-carbon for de carboxylation. They propose that the same enzyme is involved in all of these reactions and propose to term it the "general aromatic amino acid decarboxylase." Previously, they had claimed that 5-HTP and dopa decarboxylases were distinct enzymes (C. J. Clark et al, 1954) but re tracted this in agreement with the conclusions made by Yuwiler et al. (1959, 1960), Werle and Aures (1959, 1960), Holtz (1959), Fellman et al. (1960), and Rosengren (1960). The original discrepancy was attributed to differences in B6-P04 requirement with different substrates (Werle and Aures), and subsequent work by Lovenberg et al. (1962) showed the ratios of activity with different substrates remained constant when more highly purified enzyme preparations were used. Erspamer et al. (1961) have confirmed the inhibitory effect of a-methyldopa on 5-HTP decarboxylase in vitro. Inhibition of the decarboxylation of 4-HTP, also a substrate, and dopa was less marked. Cooper and Melcer (1961) also confirmed the inhibition of 5-HTP decarboxylase in vitro, using intestinal mucosa. Griesemer et al. (1961) found inhibition of dopa decarboxylase in vitro by a-methyl-3,4-dihydrox}cinnamic acid, but it was not significantly greater than 3,4-dihydroxycinnamic acid (Hartman et al, 1955). Dengler and Reichel (1957), Westermann et al (1958), Smith (1960), Porter et al. (1961), Hess et al. (1961), Murphy and Sourkes (1961), Gold berg et al. (1960), Gillespie (1960), Sjoerdsma et al (1960), Gillespie and Sjoerdsma (1961), Sjoerdsma (1961), and Wilson et al (1961) (cf. references quoted in Section IV, and Sourkes and DTorio, Volume II) have extended the observations of inhibition of the "general decarboxylase" to intact animals, to normal human subjects, and to patients with essential hyper tension, pheochromocytoma, and malignant carcinoid. The hypotensive effects in essential hypertension, observed first by Oates et al. (1960a, b), have since been confirmed by others. The only central effect noticed has been drowsiness. Azima (personal communication) has observed no effects of large, repeated doses of α-methyldopa orally and intravenously in a series of neurotic and borderline psychotic patients with tension and anxiety. Murphy and Sourkes (1961) found that a-methyl-2,5-dopa and especially a-methyl-5HTP also are active in vivo. Pletscher and Gey (1961) have developed a method for assaying 5-HTP decarboxylation in intact brain by incubating the isolated rat head, which seems well adapted for screening inhibitors; they illustrate this application by the use of α-methyldopa injected in rats prior to 5-HTP. 366 W. G. CLARK

Werle (1961) confirmed Weissbach et al. (1961) that α-methyldopa inhibits "nonspecific" histidine decarboxylase in vitro and extended the work to intact animals. He found that the inhibition in vitro is reversed by B6-P04. This was confirmed by Burkhalter (1962) and by Ganrot et al. (1961), the latter stating that since α-methyldopa reacts so readily with Β6-Ρθ4, it still is not known whether or not the inhibition is due to a reac tion between α-methyldopa and histidine at the active center of the enzyme or to a lack of B6-PO4. Burkhalter (1962), Weissbach et al. (1961), and Ganrot et al. (1961) found, however, that α-methyldopa does not inhibit "specific" L-histidine decarboxylase of mast cells, rat fundus, and embryo*tissue (and presum ably the adaptive enzyme of Schayer; cf. Section VII). If such inhibitors are found, their physiological effects could be of great importance.

ACKNOWLEDGMENTS The author gratefully acknowledges the help of his associates Drs. David T. Masuoka, Herman F. Schott, and William J. Hartman, now de ceased, for critically editing the manuscript and wishes to point out that they, and his former associate, Dr. Robert S. Pogrund, were responsible for most of the experimental work done in this laboratory. The author also wishes to thank Dr. Theodore L. Sourkes and Dr. Julius Axelrod for their helpful criticisms and suggestions in revising the final manuscript.

REFERENCES

Abderhalden, R. (1939a) Klin. Wochschr. 18, 171. Abderhalden, R. (1939b) Arch. ges. Physiol., Pflugers 242, 199. Abderhalden, R. (1940) Arch. ges. Physiol, Pflûgers 243, 133. Abderhalden, R. (1954) Z. Vitamin-, Hormon-, u. Fermentforsch. ,6 295. Akedo, H., and Christensen, H. N. (1961) Federation Proc. 20, 140. Akedo, H., Sugawa, T., Yoshikawa, S., and Suda, M. (1960) J. Biochem. (Tokyo) 47, 124. cf. Akedo, H. (1960) Osaka Daigaku Igaku Zasshi 12, 841. Alexander, B. (1960) Appl. Microbiol. ,8 69. Anderson, C. T., Blaschko, H., Burn, .J H., and Mole, R. H. (1951) Brit. J. Phar• macol 6,342. Ando, S. (1959a) J. Biochem. (Tokyo) 46, 883. Ando, S. (1959b) J. Biochem. (Tokyo) 46, 1331. Aoki, T. (1957a) Kekkaku 32, 418. Aoki, T. (1957b) Kekkaku 32, 544. Aoki, T. (1957c) Kekkaku 32, 605. Arjona, E., Perianes, J., Lorente, L., Aguirre, M., and Jimenez-Diaz, C. (1950) Bull. Inst. Med. Research, Univ. Madrid ,3 .1 Arjona, E., Perianes, J., Jimenez-Diaz, C, Lorente, L., and Aguirre, M. (1951). Bull. Inst. Med. Research, Univ. Madrid ,4 131. 9. INHIBITION OF AMINO ACID DECARBOXYLASES 367

Armstrong, K. L., Feldott, G., and Lardy, H. A. (1950) Proc. Soc. Exptl Biol Med. 73, 159. Axelrod, A. E., and Martin, C. J. (1961) Ann. Rev. Biochem. 30, 383. Azarkh, R. M., Braunstein, A. E., Paskhina, T. S., and Hsu, T. (1960) Biokhimiya 25, 954. Baker, Z., Harrison, R. W., and Miller, B. F. (1941) J. Exptl. Med. 73, 249. Baldridge, R. C, Borofsky, L., Baird, H., Reichle, F., and Bullock, D. (1959) Proc. Soc. Exptl. Biol. Med. 100, 529. Balzer, H., Holtz, P., and Palm, P. (1960a) Arch, exptl. Pathol, u. Pharmakol, Naunyn-Schmiedeberg's 240, 23. Balzer, H., Holtz, P.7, and Palm, D. (1960b) Arch, exptl. Pathol, u. Pharmakol, Naunyn-Schmiedeberg s 239, 520. Balzer, H., Holtz, P., and Palm, D. (1960c) Biochem. Pharmacol. 5, 169. Bargoni, N. (1946) Boll. soc. ital. biol. sper. 22, 336. Bartlet, A. L. (1960) Brit. J. Pharmacol. 15, 140. Baxter, C. F., and Roberts, E. (1960) Proc. Soc. Exptl. Biol. Med. 104, 426. Beaton, G. H., and McHenry, E. W. (1953) Brit. J. Nutrition 7, 357. Beevers, A. (1951) Biochem. J. 48, 132. Beiler, J. M., and Martin, G. J. (1947) J. Biol. Chem. 169, 345. Beiler, J. M., and Martin, G. J. (1954) J. Biol. Chem. 211, 39. Beiler, J. M., Brendel, R., Graff, M., and Martin, G. J. (1949) J. Am. Pharm. Assoc., Sci. Ed. 38, 315. Beiler, J. M., Brendel, R., and Martin, G. J. (1958) Exptl. Med. Surg. 16, 138. Bellamy, W. D., and Gunsalus, I. C. (1946) Bacteriol. 50, 95. Benigno, P., and Monaco, P. (1960) Arch, intern, pharmacodynamic 124, 191. Benigno, P., La Grutta, G., and Monaco, P. (1958) Atti Accad. Sci. med. Palermo 337, 102. Bergeret, B., Chatagner, F., and Fromageot, C. (1955) Biochim. et Biophys. Acta 17, 128. Bergeret, B., Chatagner, F., and Fromageot, C. (1956) Biochim. et Biophys. Acta 22, 239. Bergeret, B., Labouesse, J., and Chatagner, F. (1958) Bull. soc. chim. biol. 40, 1923. Biehl, J. P., and Vilter, R. W. (1954) J. Am. Med. Assoc. 156, 1549. Blaschko, H. (1942a) J. Physiol (London) 101, 337. Blaschko, H. (1942b) Biochem. J. 36, 571. Blaschko, H. (1942c) Physiol (London) 101, 6. Blaschko, H. (1945a) Advances in Enzymol. 5, 67. Blaschko, H. (1945b) Biochem. J. 39, 76. Blaschko, H. (1950) Biochim. et Biophys. Acta 4, 130. .Blaschko, H., and Stiven, J. (1950) Biochem. J. 46, 88. Blaschko, H., Carter, C. W., O'Brien, J. R. P., and Sloane-Stanley, G. H. (1948) Physiol (London) 107, 18P. Blaschko, H., Burn, J. H., and Carter, C. W. (1951) /. Physiol. (London) 115, 37P. Blaschko, H., Datta, S. P., and Harris, H. (1953) Brit. J. Nutrition 7, 364. Bonavita, V. (1959) J. Neurochem. 4, 275. Bonavita, V. (1960a) Arch. Biochem. Biophys. 88, 366. Bonavita, V. (1960b) Ricerca sci. 30, 672. Bonavita, V., and Scardi, V. (1958a) Experientia 14, 8. Bonavita, V., and Scardi, V. (1958b) Experientia 14, 133. Bonavita, V., and Scardi, V. (1959a) Arch. Biochem. Biophys. 82, 300. 368 W. G. CLARK

Bonavita, V., and Scardi, V. (1959b) Biochem. Pharmacol. 2, 58. Bornstein, J. (1957) Nature 179, 534. Bôttger, S., and Steinmetzer, W. (1960) Z. Zuckerind. 10, 235. Braunstein, A. E. (1960) In "The Enzymes" (P. D. Boyer, H. Lardy, and K. Myr bâck, eds.), 2nd éd., Vol. 2, p. 113. Academic Press, New York. Braunstein, A. E., and Hsu, P. (1960) Biokhimya 25, 758. Brodie, B. B., Tomich, E. G., Kuntzman, R., and Shore, P. A. (1957) Pharmacol. Exptl. Therap. 119, 461. Brodie, Β. B., and Spector, S., Kuntzman, R. G., and Shore, P. A. (1958) Natur- wissenschaften 45, 243. Brodie, Β. B., Spector, S., and Shore, P. A. (1959) Pharmacol. Revs. 11, 548. Brodie, Β. B., Kuntzman, R., Hirsch, C. W., and Costa, E. (1962). Life Sciences 3,81. Bucker, H., Pauly, H., and Rajewsky, B. (1960) Strahlentherapie 111, 330. Burkard, W. P., Gey, K. F., and Pletscher, A. (1962) Experientia 18, 411. Burkhalter, A. (1962) Biochèm. Pharmacol. 11, .1 Burns, .J J., and Shore, P. A. (1961) Ann. Rev. Pharmacol. ,1 79. Buxton, J., and Sinclair, H. M. (1956) Biochem. J. 62, 27P. Buzard, .J Α., and Nytch, P. D. (1957a) J. Biol. Chem. 227, 225. Buzard, J. Α., and Nytch, P. D. (1957b) J. Biol. Chem. 229, 409. Bygdeman, M., and Stjàrne, L. (1959) Nature 184, 1646. Canal, N., and Garattini, S. (1957) Arzneimittel-Forsch 7, 158. Canal, N., and Maffei-Faccioli, A. (1959) Rev. espan. fisiol. 15, 225. Canal, N., and Maffei-Faccioli, A. (I960) Rev. espan. fisiol. 16 (Suppl. 2), 235-240. Canal, N., and Tessari, L. (1957) Boll. soc. ital. biol. sper. 33, 1472. Canal, N., Clementi, M. S., and Maffei-Faccioli, A. (1958) Boll. soc. ital. biol. sper. 34, 789. Cascio, G., and Monaco, P. (1957) Boll. soc. ital. biol. sper. 33, 1368. Caspers, H. (1960) Med. exptl. 2, 198. Cattanéo-Lacombe, J., and Senez, J. C. (1956) Compt. rend. soc. biol. 150, 748. Chatagner, F. (1959) Prov. IVth Intern. Congr. Biochem., Vienna, 1968 13, 5. Perga- mon Press, New York. Chatagner, F., and Bergeret, B. (1956) Bull. soc. chim. biol. 38, 1159. Chatagner, F., and Bergeret, B. (1957) Compt. rend. acad. sci. 244, 2322. Chatagner, F., Tabechian, H., and Bergeret, B. (1954) Biochim. et Biophys. Acta 13,313. Chatagner, F., Bergeret, B., and Labousse, J. (1959) Biochim. et Biophys. Acta 35, 231. Chen, G., and Russell, D. (1950) J. Pharmacol. Exptl. Therap. 99, 401. Cheng, Y., Linko, P., and Milner, M. (1960) Plant Physiol. 35, 68. Chevillard, L., and Thoai, Ng.-V. (1951) Bull. soc. chim. biol. 33, 1147. Christensen, H. N. (1955) In "Amino Acid Metabolism" (W. D. McElroy and B. Glass, eds.), p. 63. Johns Hopkins Press, Baltimore, Maryland. Christensen, H. N. (1960) Advances in Protein Chem. 15, 239. Christensen, H. N., Riggs, T. R., and Jones, J. C. (1960) Federation Proc. 19, 129. Clark, C. T., Weissbach, H., and Udenfriend, S. (1954) J. Biol. Chem. 210, 139. Clark, W. G. (1959) Pharmacol. Revs. 11, 330. Clark, W. G., and Pogrund, R. S. (1961) Circulation Research 9, 721. Clark, W. G., Pogrund, R. S., Akawie, R. L, Drell, W., and Hartman, W. J. (1956) Abstr. Comm. XXth Intern. Physiol. Congr., Brussels, p. 179. Clegg, R. E., and Sealock, R. R. (1949) /. Biol. Chem. 179, 1037. Cooper, J. R., and Melcer, I. (1961) /. Pharmacol. Exptl. Therap. 132, 265. Cosin, R. (1955) Rev. espan. fisiol. 11, 187. 9. INHIBITION OF AMINO ACID DECARBOXYLASES 369

Cosin, R. (1956) Proc. 3rd Intern. Biochem. Congr., Brussels, p. 36. Costa, E., and Himwich, H. E. (1959) Proc. IVth Intern. Congr. Biochem., Vienna, 1968 3, 283. Pergamon Press, New York. Crane, G. E. (1961) Compr. Psychiat. 2, 51. Crawford, L. V. (1958) Biochem. J. 68, 221. Curry, H. M., and Balen, W. A. (1960) Org. Chem. 25, 209. Daniels, J. R., and Kalman, S. H. (1961) Federation Proc. 20, 2. Davison, A. N. (1956a) Biochem. J. 64, 546. Davison, A. N. (1956b) Biochim. et Biophys. Acta 19, 66. Davison, A. N. (1956c) Biochim. et Biophys. Acta 19, 131. Davison, A. N., and Sandler, M. (1958) Nature 181, 186. Deltour, G. H., Ghuyssen, J. M., and Claus, A. (1959a) Proc. J$h Intern. Congr. Biochem., Vienna, 1958 15, 184. Pergamon Press, New York. Deltour, G. H., Ghuyssen, J. M., and Claus, A. (1959b) Biochem. Pharmacol. 1, 267. Dengler, H. (1961) Dissertation, Univ. Heidelberg. Dengler, H., and Reichel, G. (1957) Arch, exptl. Pathol, u. PharmakoL, Naunyn- Schmiedeberg's 232, 324. Dengler, H. J., Rauch, E.1, and Rummel, W. (1962) Arch, exptl. Pathol, u. PharmakoL, Naunyn-Schmiedeberg s 243, 366. Dewey, D. L., Hoare, D. S., and Work, E. (1954) Biochem. J. 58, 523. Dietrich, L. S., and Borries, E. (1956) Arch. Biochem. Biophys. 64, 512. Dietrich, L. S., and Shapiro, D. M. (1953) Proc. Soc. Exptl. Biol. Med. 84, 555. Dietrich, L. S., and Shapiro, D. M. (1955) Cancer Research 15, 133. D'lorio, Α., and Leduc, J. (1960) Arch. Biochem. Biophys. 87, 224. Dixon, M., and Webb, E. C. (1958) "Enzymes," Academic Press, New York. Doy, C. H., and Pittard, A. J. (1960) Nature 185, 941. Drain, D. J., Harlington, M., Lazare, R., and Poulter, G. A. (1962) Life Sciences No. 1, 93. Drell, W. (1957) Federation Proc. 16, 174. Drill, V. Α., and Overman, R. (1942) Am. J. Physiol. 135, 474. Dubnick, B., Leeson, G. Α., and Phillips, G. E. (1962) Biochem. Pharmacol. 11, 45. Dubnick, B., Leeson, G. Α., Chessin, M., and Scott, C. C. (1960a) Arch, intern. pharmacodynamic 126, 194. Dubnick, B., Leeson, G. Α., and Leverett, R. (1960b) Pharmacologist 2, 67. Dubnick, B., Leeson, G. Α., and Scott, C. C. (1960c) Toxicol. Appl. Pharmacol. 2, 403. Du Vigneaud, V., Kuchinskas, E. J., and Horvath, A. (1957) Arch. Biochem. Biophys. 69, 130. Edelman, I. S. (1961) Ann. Rev. Physiol. 23, 37. Eggleston, L. V. (1957) Biochem. J. 65, 735. Eggleston, L. V. (1958) Biochem. J. 68, 557. Ehringer, H., Hornykiewicz, O., and Lechner, K. (1960) Arch, exptl. Pathol, u. PharmakoL, Naunyn-Schmiedeberg's 239, 507. Ehrismann, O., and Werle, E. (1948) Biochem. Z. 318, 560. Ekladius, L., and King, H. K. (1956) Biochem. J. 62, 7P. Ekladius, L., King, H. K., and Sutton, C. R. (1957) J. Gen. Microbiol. 17, 602. Elliott, K. A. C, and Jasper, H. H. (1959) Physiol. Revs. 39, 383. Epps, H. M. R. (1944) Biochem. J. 38, 242. Epps, H. M. R. (1945) Biochem. J. 39, 42. Erspamer, V., and Ciceri, C. (1957) Experientia 13, 87. 370 W. G. CLARK

Erspamer, V., Glàsser, Α., Pasini, C, and Stoppani, G. (1961) Nature 189, 483. Fellman, J. H. (1956) Proc. Soc. Exptl. Biol. Med. 93, 413. Fellman, J. H. (1959) Enzymologia 20, 366. Fellman, J. H., Fugita, T. S., and Robinson, E. (1960), personal communications. Ferrari, V., Ginoulhiac, E., and Tenconi, L. T. (1957) Boll. soc. ital. biol. sper. 33, 1418. Fowden, L. (1954) Exptl. Botany 5, 28. Fréter, K., Weissbach, H., Redfield, B., Udenfriend, S., and Witkop, B. (1958) Am. Chem. Soc. 80, 983. Frieden, E., Hsu, L. T., and Dittmer, K. (1951) /. Biol. Chem. 192, 425. Fromageot, C. (1953-1954) Harvey Lectures, Ser. 49, 1. Fromageot, C. (1956) Exposés ann. biochim. méd. 18, 111. Gâbor, M., Szorâdy, I., and Dirner, Z. (1952a) Acta Physiol. Acad. Sci. Hung. 3, 595. Gâbor, M., Szorâdy, I., and Dirner, Z. (1952b) Kisérletes Orvostudomâny 4, 100. Gaddum, J. H. (1957) Pharmacol. Revs. 9, 211. Gagnon, A. (1961) Rev. Can. Biol. 20, 7. Gale, E. F. (1940a) Biochem. J. 34, 392. Gale, E. F. (1940b) Bacteriol. Revs. 4, 135. Gale, E. F. (1941) Biochem. J. 35, 66. Gale, E. F. (1946) Advances in Enzymol. 6, 1. Gale, E. F. (1953) Brit. Med. Bull. 9, 135. Gale, E. F., and Epps, H. M. R. (1944) Biochem. J. 38, 232. Gangadharam, P. R. J., and Sirsi, M. (1956) J. Indian Inst. Sci. 38A, 33. Ganrot, P. O., Rosengren, A. M., and Rosengren, E. (1961) Experientia 17, 263. Geiger, E. (1944) Proc. Soc. Exptl. Biol. Med. 55, 11. Geschwind, I. I., and Li, L. H. (1953) Nature 172, 732. Gey, K. F., and Pletscher, A. (1960) Experientia 16, 372. Gey, K. F., Burkard, W. P., and Pletscher, A. (1961) Biochem. Pharmacol. 8, 383. Gey, K. F., and Petscher, A. (1962) Nature 194, 387. Gillespie, L., Jr. (1960) Ann. N. Y. Acad. Sci. 88, 1011. Gillespie, L., and Sjoerdsma, A. (1961) Med. Clin. N. Am. 45, 421. Ginsburg, Β. E. (1954) Research Pubis., Assoc. Research Nervous Mental Disease 33, 39. Ginsburg, Β. E., and Roberts, E. (1951) Anat. Record 111, 113. Goldberg, L. I., Da Costa, F. M., and Ozaki, M. (1960) Nature 188, 502. Gol'dshtein, V. D. (1960) Problemy Tuberk. 38, 46. Goldstein, M., Hagopian, M., and Dorfman, R. I. (1958) Abstr. Communs. I^th Intern. Congr. Biochem., Vienna, 1958 p. 105. Pergamon Press, New York. Gonnard, P. (1949) Bull. soc. chim. biol. 31, 194. Gonnard, P. (1951) Bull. soc. chim. biol. 33, 14. Gonnard, P. (1958) Compt. rend. acad. sci. 246, 3593. Gonnard, P., and Boigné, N. (1961) Bull. soc. chim. biol. 43, 609. Gonnard, P., and Chi, J. P. Ng. (1958) Bull. soc. chim. biol. 40, 485. Gonnard, P., and Chi, J. P. Ng. (1959a) Bull. soc. chim. biol. 41, 1455. Gonnard, P., and Chi, J. P. Ng. (1959b) Enzymologia 20, 237. Gonnard, P., and Nguyen-Philippon, C. (1959) Enzymologia 20, 231. Gonnard, P., and Nguyen-Philippon, C. (1961) Bull. soc. chim. biol. 43, 601. Gonnard, P., Pelou, Α., and Philippon, Mrs. C. Ng. (1958) Abstr. Communs. 4th Intern. Congr. Biochem., Vienna, 1958 p. 185. Pergamon Press, New York. Gonnard, P., Pelou, Α., and Philippon, Mrs. C. Ng. (1959) Bull. soc. chim. biol. 41,127. 9. INHIBITION OF AMINO ACID DECARBOXYLASES 371

Goodall, McC. (1950) Acta Chem. Scand. 4, 550. Goth, Α., Allman, R. M., Merritt, B. C., and Holman, J. (1951a) /. Pharmacol. Exptl. Therap. 103, 344P. Goth, Α., Allman, R. M., Merritt, B. C., and Holman, J. (1951b) Proc. Soc. Exptl. Biol Med. 78, 848. Greenberg, S. M., Masurat, T., Rice, E. G., Herndon, J. F., and Van Loon, E. J. (1959) Biochem. Pharmacol. 2, 308. Greig, M. E., Walk, R. Α., and Gibbons, A. J. (1959) J. Pharmacol. Exptl. Therap. 127, 110. Greig, M. E., Seay, P. H., and Freyburger, W. A. (1961) J. Neuropsychiat. 2 (Suppl. 1), 131. Griesemer, E. C., Fleming, R. M., Hartman, W. J., and Clark, W. G. (1961) Pharma cologist 3, 77. Grob, D. (1952) Bull. Johns Hopkins Hosp. 90, 341. Grunberger, D. J., and Sorm, F. (1954) Chem. listy 48, 1241. Grunberger, D. J., Skoda, J., and Sorm, F. (1948) Chem. listy 48, 1711. Grunberger, D. J., Skoda, J., and Sorm, F. (1955) Collection Czechoslov. Chem. Communs. 20, 507. Gubarev, Ε. M. (1960) Mikrobiol. Zhur., Akad. Nauk. Ukr. R.S.R., Inst. Mikrobiol. im. D. K. Zabolotnogo 22, 54. Guirard, Β. M., and Snell, Ε. E. (1954) J. Am. Chem. Soc. 76, 4745. Gunsalus, I. C. (1951) Harvey Lectures, Ser. 40, 40. Gupta, S. P., Gupta, N. P., and Agarwal, W. S. (1960) Indian J. Med. Research 48, 287. Guroff, G., and Udenfriend, S. (1960) J. Biol. Chem. 235, 3518. Hado, T. (1959) Nagoya Med. J. 5, 203. Hagen, P., and Welch, A. D. (1956) Recent Progr. Hormone Research 12, 27. Hagihira, H., Ogata, M., Takedatsu, N., and Suda, M. (1960) J. Biochem. (Tokyo) 47, 139. Hagihira, H., Lin, E. C. C, Samiy, A. H., and Wilson, T. H. (1961) Biochem. Bio phys. Research Communs. 4, 478. Halpern, Β. N. (1957) Practitioner 178, 659. Halpern, Β. N., and Briot, M. (1956) Rev. franc études clin, et biol. 1, 151. Halpern, Β. N., Neveu, T., and Branellec, A. (1957) Compt. rend. soc. biol. 151, 858. Hanson, A. (1958) Naturwissenschaften 45, 423. Hanson, A. (1959) Acta Chem. Scand. 13, 1366. Happold, F. C. (1956) Proc. 3rd Intern. Congr. Biochem., Brussels, 1955 p. 32. Happold, F. C, and Ryden, R. (1952) Nature 169, 115. Hartman, W. J., Akawie, R. L, and Clark, W. G. (1955) J. Biol. Chem. 216, 507. Hartman, W. J., Clark, W. G., and Cyr, S. D. (1961) Proc. Soc. Exptl. Biol. Med. 107, 123. Haughton, B. G., and King, H. K. (1957) Biochem. J. 65, 36P. Haverback, B. J., Sjoeidsma, Α., and Terry, L. L. (1956) New Engl. J. Med. 255, 270. Hawkins, J., and Walker, J. M. (1952) Brit. J. Pharmacol. 7, 152. Hayashi, M. (1957) Vitamins (Kyoto) 13, 148. Heilbronn, E. (1960) Biochem. J. 76, 45P. Hempling, H. G. (1961) Federation Proc. 20, 139. Hess, S. M., Connamacher, R. H., Ozaki, M., and Udenfriend, S. (1961) J. Pharmacol. Exptl. Therap. 134, 129. Heyl, D., Harris, S. Α., and Folkers, K. (1948) J. Am. Chem. Soc. 70, 3429. 372 W. G. CLARK

Heyl, D., Luz, E., Harris, S. Α., and Folkers, K. (1951) Proc. Intern. Congr. Pure and Appt. Chem., 12th Congr., p. 75. Heyl, D., Luz, E., Harris, S. Α., and Folkers, K. (1952) Am. Chem. Soc. 74, 414. Hicks, R., and West, G. B. (1958a) Nature 181, 1342. Hicks, R., and West, G. B. (1958b) Nature 182, 401. Hicks, R. M., and Clarke, P. H. (1959) Enzymologia 21, 169. Hoare, D. S. (1956) Biochim. et Biophys. Acta 19, 141. Holtz, P. (1941) Ergeb. Physiol., biol. Chem. u. exptl. PharmakoL 44, 230. Holtz, P. (1959) Pharmacol. Revs. 11, 317. Holtz, P. (1960a) In "Fermente, Hormone-und Vitamine" (R. Ammon and W. Dirscherl, eds.), 3rd éd., Vol. 2, p. 396. Georg Thiem, Stuttgart. Holtz, P. (1960b) Monatsschr. Psychiat. u. Neurol. 140, 175. Holtz, P., and Heise, R. (1937) Arch, exptl. Pathol, u. PharmakoL, Naunyn-Schmiede- berg's 186, 377. Holtz, P., and Heise, R. (1938) Arch, exptl. Pathol, u. PharmakoL, Naunyn-Schmiede- berg's 191, 87. Holtz, P., and Westermann, E. (1956a) Naturwissenschaften 43, 38. Holtz, P., and Westermann, E. (1956b) Biochem. Z. 327, 502. Holtz, P., and Westermann, E. (1957) Arch, exptl. Pathol, u. PharmakoL, Naunyn- Schmiedeberg's 231, 311. Holtz, P., Reinhold, Α., and Credner, K. (1939) Z. physioL Chem., Hoppe-Seyler's 261, 278. Holtz, P., Stock, K., and Westermann, E. (1956) Arch, exptl. Pathol, u. PharmakoL, Naunyn-Schmiedeberg's 228, 322. Hope, D. B. (1955) Biochem. J. 59, 497. Hope, D. B. (1957) Neurochem. 1, 364. Hope, D. B. (1959) Proc. 4th Intern. Congr. Biochem., Vienna, 1958 13, 63. Horvath, A. (1957) Nature 179, 968. Horvath, A. (1958) Enzymologia 19, 297. Horvath, A. (1959) Abstr. Communs. 21st Intern. Congr. PhysioL Sci., Buenos Aires p. 128. Horvath, Α., Orrego, F., and McKennis, H., Jr. (1961) Federation Proc. 20, 5. Howe, A. F., and Treffers, H. P. (1952) Nature 170, 123. Huang, L, Tannenbaum, S., Blume, L., and Hsia, D. Y. (1961) Proc. Soc. Exptl. Biol. Med. 106, 533. Hughes, D. E. (1949) Biochem. J. 45, 325. Hughes, D. E. (1950) Biochem. J. 46, 231. Hurwitz, J. (1952) Biochim. et Biophys. Acta 9, 496. Hurwitz, J. (1953) J. Biol. Chem. 205, 935. Hurwitz, J. (1955) ^ /. Biol. Chem. 217, 513. Imiya, T. (1941) Osaka Shiritsu Daigaku Igaku Zasshi 40, 1494. Ishikawa, Y., and Obata, Y. (1955) Nippon Nogei-kagaku Kaishi 29, 689. Ito, F., Aoki, R., Hayashi, Α., Yuasa, M., Tone, K., and Nishio, K. (1958) J. Chest. Diseases (Osaka) 2, 106. Iwamoto, F., and Nukada, T. (1961) J. Biochem. (Japan) 49, 177. Jacobs, F. Α., Flaa, R. C, and Belk, W. F. (1960) J. Biol. Chem. 235, 3224. Jacquez, J. A. (1961) Am. J. PhysioL 200, 1063. Janke, A. (1951) Arch. Mikrobiol. 15, 472. Jollès-Bergeret, B., Labouesse, J., and Chatagner, F. (1960) Bull. soc. chim. biol. 42, 51. 9. INHIBITION OF AMINO ACID DECARBOXYLASES 373

Kahlson, G. (1960) Lancet , i 67. Kahlson, G., and Rosengren, E. (1959a) Nature 184, 1238. Kahlson, G., and Rosengren, E. (1959b) J. Physiol. {London) 149, 66. Kahlson, G., Rosengren, E., Westling, H., and White, T. (1958) Physiol. (London) 144, 337. Kahlson, G., Rosengren, E., and White, T. (1959) ./ Physiol. (London) 145, 30. Kahlson, G., Nilsson, K., Rosengren, E., and Zederfeldt, B. (1960a) Lancet ,i 230. Kahlson, G., Rosengren, E., and White, T. (1960b) J. Physiol. (London) 151, 131. Kamrin, R. P., and Kamrin, A. A. (1961) J. Neurochem. ,6 219. Karrer, P. (1947) Schweiz. Z. Pathol, u. Bakteriol. 10, 351. Kating, H. (1954) Naturwissenschaften 41, 188. Kato, R. (1959) Experientia 15, 424. Kato, R. (I960) J. Neurochem. ,5 202. Kato, F. R., and Valzelli, L. (1959) Boll. soc. ital. biol. sper. 35, 1238. Kauffmann, F., and Miller, V. (1955) Acta Pathol. Microbiol. Scand. 36, 173. Kawashima, M. (1957) Yakugaku Zasshi 77, 758. Kiliam, K. F., and Bain, .J A. (1957a) J. Pharmacol. Exptl. Therap. 119, 255. Killam, K. F., and Bain, .J A. (1957b) J. Pharmacol. Exptl. Therap. 119, 263. Kimura, K., Kuwano, S., and Hikino, H. (1958) Yakugaki Zasshi 78, 236. Kizer, D. E., and Chan, S. (1961) Cancer Research 21, 489. Knivett, V. A. (1954) Biochem. J. 58, 480. Knox, W. E., Auerbach, V. H., and Lin, E. C. C. (1956) Physiol. Revs. 36, 164. Kobayashi, S. (1958) Vitamins (Kyoto) 14, 99. Kochetkov, N. K., Khomutov, R. M., Karpeïskiï, M. Ya., Budovskiï, Ε. I., and Severin, E. S. (1959) Doklady Akad. Nauk. S.S.S.R. 126, 1132. Koike, M. (1954) Seikagaku 26, 528. Koizumi, K., Inoue, F., and Yoshimatsu, H. (1958) Osaka Daigaku Igaku Zasshi 10, 141. Konishi, S. (1957) Vitamins (Kyoto) 13, 426. Kooka, T. (1957) Vitamins (Kyoto) 13, 128. Kostyo, J. L., and Engel, F. L. (1960) Endocrinology 67, 708. Kostyo, J. L., and Knobil, E. (1959a) Endocrinology 65, 395. Kostyo, J. L., and Knobil, E. (1959b) Endocrinology 65, 525. Kostyo, J. L., and Schmidt, J. E. (1962) Endocrinology 70, 381. Kostyo, J. L., Hotchkiss, J., and Knobil, E. (1959) Science 130, 1653. Kraychy, S., and Gallagher, T. F. (1957) J. Am. Chem. Soc. 79, 754. Krebs, H. A. (1948) Biochem. J. 43, 51. Krebs, H. A. (1950) Biochem. J. 47, 605. Krebs, Η. Α., Eggleston, L. V., and Knivett, V. A. (1955) Biochem. J. 59, 185. Kfecek, J., Sterzl, J., Kfeckova, J., and Vaicenbacher, V. (1950) Gasopis lékâru ceskych 89, 1. Krishnaswamy, P. R., and Giri, Κ. V. (1956) Biochem. «/..62, 301. Kuchinskas, E. J. (1961) Abstr. lS9lh Meeting Am. Chem. Soc, St. Louis p. 10C. Kuchinskas, E. J., and Du Vigneaud, V. (1957) Arch. Biochem. et Biophys. 66, 1. Kuchinskas, E. J., Horvath, Α., and Du Vigneaud, V. (1957) Arch. Biochem. Biophys. 68, 69. Kurotsu, T., Ito, Α., Tane, T., and Ban, T. (1955). Med. J. Osaka Univ. (Eng. Ed.) 6, 347. Labouesse, J., Chatagner, F., and Bergeret, B. (1959) Biochim. et Biophys. Acta 35, 226. 374 W. G. CLARK

Labouesse-Mercouroff, J., Chatagner, F., and Jollès-Bergeret, B. (1960) Biochim. et Biophys. Acta 39, 372. Lagerborg, Α., and Clapper, W. E. (1952) J. Bacteriol. 63, 393. La Grutta, G., and Monaco, P. (1958) Boll. soc. ital. biol. sper. 34, 1677. Langemann, H. (1951) Brit. J. Pharmacol. 6, 318. Lestrovaya, Ν. N., and Mardashev, S. R. (1960) Biokhimiya (Biochemistry) (Eng. ed.) 25, 168. Lévy, J., and Michel-Ber, E. (1960a) Compt. rend. acad. sci. 250, 4061. Levy, J., and Michel-Ber, E. (1960b) Compt. rend. acad. sci. 250, 415. Lichtenstein, N., Ross, H. E., and Cohen, P. P. (1953a) Nature 171, 45. Lichtenstein, N., Ross, H. E., and Cohen, P. P. (1953b) J. Biol. Chem. 201, 117. Lin, E. C. C, and Hastings, T. (1960) Am. J. PhysioL 199, 127. Lin, E. C. C, Civen, M., and Knox, W. E. (1958) J. Biol. Chem. 233, 1183. Lin, E. C. C, Hagihira, H., and Wilson, T. H. (1961) Federation Proc. 20, 243. Litwin, J., and Kordecki, R. (1958) Polski Tygod. Lekar. i Wiadomoêci Lekar. 13, 1542. Lovenberg, W., Weissbach, H., and Udenfriend, S. (1962) J. Biol. Chem. 237, 89. Ludwig, G. D. (1960) J. Clin. Invest. 39, 44. McCormick, D. B., and Snell, Ε. E. (1959) Proc. Natl. Acad. Sci. U.S. 45, 1371. McCormick, D. B., and Snell, Ε. E. (1961) J. Biol. Chem. 236, 2085. McCormick, D. B., Guirard, B. M., and Snell, Ε. E. (1960) Proc. Soc. Exptl. Biol. Med. 104, 554. McCormick, D. B., Gregory, M. E., and Snell, Ε. E. (1961) Biol. Chem. 236, 2076. McGilvery, R. W., and Cohen, P. P. (1948) J. Biol. Chem. 174, 813. MacLagan, N. F., and Wilkinson, J. H. (1951) Nature 168, 251. Makino, K., and Kinoshita, T. (1955) Vitaminol. (Osaka) 1, 98. Makino, K., and Koike, M. (1954a) Nature 174, 1056. Makino, K., and Koike, M. (1954b) Enzymologia 17, 157. Makino, K., Kinoshita, T., Aramaki, Y., and Shintani, S. (1954) Nature 174 , 275. Malkiel, S., and Werle, M. D. (1951) Science 114, 98. Manchester, K. L., and Young, F. G. (1960) Biochem. J. 75, 487. Mandelstam, J. (1956) Proc. 3rd Intern. Biochem. Congr., Brussels, 1955 p. 98. Mandokoro, Y. (1957) Vitamins (Kyoto) 13, 161. Marco, C. de. (1957) Biochim. et Biophys. Acta 25, 634. Mardashew, S. R. (1949) Uspekhi Sovremennoï Biol. 28, 354 and 365. Mardashew, S. R., and Chao, T. (1960) Doklady Akad. Nauk. S.S.S.R. 133, 230. Mardashew, S. R., and Etingof, R. N. (1948) Biokhimiya 13, 469. Mardashew, S. R., and Semina, L. A. (1961) Biokhimiya 26, 31. Marrack, .J R. (1951) In "The Enzymes" (J. B. Sumner and K. Myrbâck, eds.), Vol. ,1 p. 343. Academic Press, New York. Marshall, P. B. (1961) Brit. J. Pharmacol. 16, 50. Martin, G. .J (1946) J. Biol. Chem. 166, 389. Martin, G. .J (1951) "Biological Antagonism. The Theory fo Biological Relativity." Blakiston, Philadelphia and Toronto. Martin, G. J., and Beiler, .J M. (1947) Arch. Biochem. 15, 201. Martin, G. J., Ichniowski, O. T., Wisansky, W. Α., and Ansbacher, S. (1942) Am. J. PhysioL 136, 66. Martin, G. J., Tolman, L., and Brendel, R. (1947) Arch. Biochem. 15, 323. Martin, G. J., Brendel, R., Beiler, J. M., Moss, J., Avakian, S., Urist, H., Tolman, L., and Alpert, S. (1948) Am. J. Pharm. 120, 189. 9. INHIBITION OF AMINO ACID DECARBOXYLASES 375

Martin, G. J., Graff, M., Brendel, R., and Beiler, J. M. (1949) Arch. Biochem. 21,177. Mascitelli-Coriandoli, E., and Boldrini, R. (1959) Experientia 15, 229. Mason, M. (1957) J. Biol. Chem. 227, 61. Mason, M., and Gullekson, E. (1959) J. Am. Chem. Soc. 81, 1517. Mason, M., and Gullekson, E. (1960) J. Biol. Chem. 235, 1312. Mathews, R. E. F. (1958) Pharmacol. Revs. 10, 359. Matsuda, M., and Makino, K. (1961a) Biochim. et Biophys. Acta 48, 192. Matsuda, M., and Makino, K. (1961b) Biochim. et Biophys. Acta 48, 194. Matsuda, G., Nishioka, Y., Okamura, Y., Kiya, I., and Iwasaki, A. (1955) Nagasaki Igakkai Zassi 30, 142. Mauron, J., and Bujard, E. (1960) Proc. IVth Intern. Congr. Biochem., Vienna, 1958 15 (Sect. 4), 94. Pergamon Press, New York. Mazelis, M. (1959) Biochem. Biophys. Research Communs. 1, 59. Meadow, P., and Work, E. (1958) Biochim. et Biophys. Acta 29, 180. Meister, A. (1955) In "A Symposium on Amino Acid Metabolism" (W. D. McElroy and H. B. Glass, eds.), p. 3. Johns Hopkins Press, Baltimore, Maryland. Meister, A. (1957) In "Biochemistry of the Amino Acids." Academic Press, New York. Melnykovych, J., and Johansson, K. R. (1955) J. Exptl. Med. 101, 507. Melnykovych, J., and Johansson, K. R. (1958) Proc. Soc. Exptl. Biol. Med. 98, 323. Melnykovych, J., and Johansson, K. R. (1959) J. Bacteriol. 77, 638. Melnykovych, G., and Snell, Ε. E. (1958) J. Bacteriol. 76, 518. Metzler, D. E., Ikawa, M., and Snell, Ε. E. (1954) /. Am. Chem. Soc. 76, 648. Michel, M., and Francois, A. C. (1956) Compt. rend. acad. sci. 242, 1770. Miyake, M. (1957) Vitamins {Kyoto) 13, 139. Miyaki, K., Hayashi, M., Wada, T., and Matsumato, Y. (1959) Chem. & Pharm. Bull. {Tokyo) 7, 62. Mitchell, R. G., Logan, G. B., Peters, G. Α., and Henderson, L. L. (1954) J. Allergy 25, 504. Moldave, Κ. (1958) Arch. Biochem. Biophys. 75, 418. Miller, V. (1954) Acta Pathol. Microbiol. Scand. 35, 259. Miller, V. (1956) "Bidrag til den biokemiske inddeling af Enterobacteriaceae," Dissertation. Univ. Copenhagen. Monaco, P. (1958a) Boll. soc. ital. biol. sper. 34, 900. Monaco, P. (1958b) Boll. soc. ital. biol. sper. 34, 1464. Monaco, P., and Cascio, G. (1959) Farmaco {Pavia) Ed. sci 14, 352. Monaco, P., and La Grutta, G. (1959) Arch. ital. sci. farmacol. 9, 1. Monaco, P., and La Grutta, G. (1961) Arch, intern, pharmacodynamic 131, 32. Morii, S. (1941) Biochem. Z. 309, 354. Moriya, T. (1958) Vitamins (Kyoto) 14, 90. Morrison, J. F. (1950) Australian J. Exptl. Biol. Med. Sci. 28, 311. Murphy, G. F., and Sourkes, T. L. (1961) Arch. Biochem. Biophys. 93, 338. Nadkarni, G. B., and Sreenivasan, A. (1957) Proc. Indian Acad. Sci. B46, 138. Namba, S. (1957) Vitamins (Kyoto) 13, 322. Nash, J. B. (1952) Texas Repts. Biol. and Med. 10, 639. Neame, K. D. (1961) J. Neurochem. 6, 358. Neveu, Th. (1960) /. Physiol. (Paris) 52, 1. Nickerson, M. (1959) Pharmacol. Revs. 11, 443. Nishizawa, Y. (1958) Vitaminol. (Osaka) 4, 63. Nishizawa, Y., Kodama, T., and Kooka, T. (1957) J. Vitaminol. (Osaka) 3, 309. 376 W. G. CLARK

Nishizawa, Y., Kodama, T., and Miyake, M. (1958a) /. Vitaminol. (Osaka) 4, 1. Nishizawa, Y., Kodama, T., and Hayashi, M. (1958b) J. Vitaminol. (Osaka) 4, 132. Nishizawa, Y., Kodama, T., and Kumagai, T. (1958c) J. Vitaminol. (Osaka) 4, 138. Nishizawa, Y., Kodama, T., and Namba, S. (1958d) Vitaminol. (Osaka) 4, 264. Nishizawa, Y., Kodama, T., and Kobayashi, S. (1959a) Vitaminol. (Osaka) 5, 241. Nishizawa, Y., Kodama, T., and Yamanaka, T. (1959b) J. Vitaminol. (Osaka) 5, 111. Nishizawa, Y., Kodama, T., and Konishi, S. (1959c) J. Vitaminol. (Osaka) 5, 117. Nishizawa, Y., Kodama, T., and Moriya, T. (1959d) J. Vitaminol. (Osaka) 5, 229. Nishizawa, Y., Kodama, T., and Sugahara, S. (1960) J. Vitaminol. (Osaka) 6, 236. Noall, M. W. (1960) Biochim. et Biophys. Acta 40, 180. Noall, M. W., Riggs, T. R., Walker, L. M., and Christensen, H. N. (1957) Science 126, 1002. Nossal, P. M. (1952) Biochem. J. 50, 349. Nozaki, J. (1958) Vitamins (Kyoto) 14, 310. Oates, J. Α., Jr., Gillespie, L., Jr., Crout, J. R., and Sjoerdsma, A. (1960a) J. Clin. Invest. 39, 51P. Oates, J. Α., Gillespie, L., Udenfriend, S., and Sjoerdsma, A. (1960b) Science 131, 1890. Okunuki, K. (1937) Botan. Mag. (Tokyo) 51, 270. Okunuki, K. (1942) Kagaku (Tokyo) 12, 221. Okunuki, K. (1943) Acta Phytochim. (Japan) 13, 155. Olivard, J., and Snell, Ε. E. (1955) /. Biol. Chem. 213, 215. Oliver, W. H. (1952) Gen. Microbiol. 7, 329. Oxender, D. L., and Royer, M. (1961) Federation Proc. 20, 140. Ozaki, M. (1959) Nippon Yakurigaku Zasshi 55, 1467. Page, E. W., and Reed, R. (1945) Am. J. Physiol. 143, 122. Pal, P. R., and Christensen, H. N. (1958) J. Biol. Chem. 234, 613. Palm, D. (1958) Arch, exptl. Pathol, u. Pharmakol. Naunyn-Schmiedeberg's 234, 206. Pare, C. M. B., Sandler, M., and Stacey, R. S. (1957) Lancet i, 551. Pare, C. M. B., Sandler, M., Stacey, R. S. (1958a) Abstr. Proc. Intern. Meeting Neuropsychopharmacol., Rome, p. 96. Produtti Roche, S.P.A., Milano. Pare, C. M. B., Sandler, M., and Stacey, R. S. (1958b) Lancet ii, 1099. Parrot, J., and Laborde, C. (1955) /. Physiol. (Paris) 47, 263. Parrot, J. L., and Laborde, C. (1956) In "Histamine" (G. E. W. Wolstenholme, éd.), Ciba Foundation Symposium, p. 57. Little, Brown, Boston, Massachusetts. Parrot, J., and Laborde, C. (1959) J. Physiol. (Paris) 51, 546P. Parrot, J., and Reuse, J. (1954) J. Physiol. (Paris) 46, 99. Perry, H. M., Jr., Teitlebaum, S., and Schwartz, P. L. (1955) Federation Proc. 14,113. Pfister, K., 3rd, Leanza, W. J., Conbere, J. P., Becker, H. J., Matzuk, A. R., and Rogers, E. F. (1955) J. Am. Chem. Soc. 77, 697. Pietra, G. D., Procaccini, S., Capano, V., and De Lorenzo, F. (1959) Boll. soc. ital. sper. 35, 1875. Pletscher, Α., and Gey, K. F. (1960) Med. Exptl. 2, 259. Pletscher, Α., and Gey, K. F. (1961) Nature 190, 918. Pogrund, R. S., and Clark, W. G. (1953) Abstr. Commun. XIX Intern. Physiol. Congr., Montreal p. 682. Pogrund, R. S., Drell, W., and Clark, W. G. (1955) Federation Proc. 14,116. Pogrund, R. S., Drell, W., and Clark, W. G. (1961) J. Pharmacol. Exptl. Therap. 131, 294. Pollock, M. R. (1959) In "The Enzymes" (P. D. Boyer, H. Lardy, and K. Myrbàck, eds.), 2nd éd., Vol. 1, p. 619. Academic Press, New York. 9. INHIBITION OF AMINO ACID DECARBOXYLASES 377

Polonovski, M., Gonnard, P., and Schapira, G. (1946) Compt. rend. soc. biol. 140,280. Porter, C. C., Totaro, J. Α., and Leiby, C. M. (1961) J. Pharmacol. Exptl. Therap. 134, 139. Price, S. A. P., and West, G. B. (1960) J. Pharm. and Pharmacol. 2, 617. Rajewsky, B., Bûcker, H., and Pauly, H. (1959) Arch. Biochem. Biophys. 82, 229. Riggs, T. R., and Walker, L. M. (1958) J. Biol. Chem. 233, 132. Riggs, T. R., and Walker, L. M. (1960) J. Biol. Chem. 235, 3603. Rindi, G., and Ferrari, G. (1959a) Nature 183, 608. Rindi, G., Perri, V., and Ventura, U. (1959b) Nature 183, 1126. Rindi, G., Perri, V., and Ventura, U. (1959c) Giorn. biochim. (Engl, ed.) ,8 149. Roberts, E. (1952a) Federation Proc. 11, 275. Roberts, E. (1952b) J. Biol. Chem. 198, 495. Roberts, E. (1953) J. Biol. Chem. 202, 359. Roberts, E. (I960) Am. J. Orthopsychiat. 30, 15. Roberts, E., and Eidelberg, E. (1959) Intern. Rev. Neurobiol. ,2 279. Roberts, E., and Frankel, S. (1951) J. Biol. Chem. 188, 789. Roberts, E., Younger, F., and Frankel, S. (1951) J. Biol. Chem. 191, 277. Roberts, E., Rothstein, M., and Baxter, C. F. (1958) Proc. Soc. Exptl. Biol. Med. 97, 796. Roberts, E., Baxter, C. F., Van Harreveld; Α., Wiersma, C. A. G., Adey, W. R., and Killam, K. F., (eds.) (1960) "Inhibition in the Nervous System and Gamma- Aminobutyric acid." Pergamon Press, New York. Roche, J., Michel, R., and Gregorio, P. de (1961) Biochim. et Biophys. Acta 47, 398. Rohrlich, M. (1957) Z. Lebensm.-Untersuch. u. -Forsch. 105, 189. Rohrlich, M., and Rasmus, R. (1956) Naturwissenschaften 43, 88. Rosen, F., Milholland, R. J., and Nichol, C. A. (1959) Federation Proc. 18, 543. Rosengren, E. (1960) Acta Physiol. Scand. 49, 364. Rothschild, A. M., and Schayer, R. W. (1959) Biochim. et Biophys. Acta 34, 392. Ruiz, A. S., and Zaragoza, F. M. (1959) /. Bioquim. Latinoam. 5, 1. Sagawara, S. (1958) Vitamins (Kyoto) 14, 117. Saito, T. (1957) Seikagaku 29, 346. Sakuragi, T., and Kummerow, F. (1957) Arch. Biochem. Biophys. 71, 303. Samiy, A. E. (1952) Federation Proc. 11, 136. Sandler, M. (1959a) Lancet ii, 318. Sandler, M. (1959b) Lancet ii, 516. Sandler, M., and Close, H. G. (1959) Lancet ii, 316. Sankar, D. V. S., and Bender, L. (1960) Recent Advances in Biol. Psychiatry, Proc. Ann. Conv. Soc. Biol. Psychiatry, 14th, Atlantic City, 1959 2, 363. Sano, I., Taniguchi, K., Gano, T., Takesada, M., and Kakimoto, Y. (1960) Klin. Wochschr. 38, 57. Scardi, V., and Bonavita, V. (1957a) Acta Vitaminol. 11, 117. Scardi, V., and Bonavita, V. (1957b) Boll. soc. biol. sper. 33, 770. Scardi, V., and Bonavita, V. (1958) Experientia 14, 7. Schales, O. (1951) In "The Enzymes" (J. B. Sumner and K. Myrbâck, eds.), Vol. 2, p. 216. Academic Press, New York. Schales, O., and Schales, S. S. (1946a) Arch. Biochem. 11, 155. Schales, O., and Schales, S. S. (1946b) Arch. Biochem. 11, 445. , Schales, O., and Schales, S. S. (1949) Arch. Biochem. 24, 83. Schanberg, S. M., and Giarman, N. J. (1960a) Pharmacologist 2, 67. Schanberg, S. M., and Giarman, N. J. (1960b) Biochim. et Biophys. Acta 41, 556. Schanberg, S. M., Mcllroy, C. Α., and Giarman, N. J. (1961) Federation Proc. 20, 1 378 W. G. CLARK

Schayer, R. W. (1956a) Federation Proc. 15, 347. Schayer, R. W. (1956b) Am. J. PhysioL 186, 199. Schayer, R. W. (1956c) Am. J. PhysioL 187, 63. Schayer, R. W. (1956d) In "Histamine" (G. E. W. Wolstenholme, éd.), Ciba Foun dation Symposium, p. 298. Little, Brown, Boston, Massachusetts. Schayer, R. W. (1957) Am. J. PhysioL 189, 533. Schayer, R. W. (1959) Federation Proc. 18, 317. Schayer, R. W. (1960) Science 131, 226. Schayer, R. W. (1961) Chemotherapia 3, 128. Schayer, R. W., and Ganley, O. H. (1959a) Am. J. PhysioL 197, 721. Schayer, R. W., and Ganley, O. H. (1959b) Federation Proc. 18, 137. Schayer, R. W., and Ganley, O. H. (1960) Federation Proc. 19, 210. Schayer, R. W., and Ganley, O. H. (1961) Allergy 32, 204. Schayer, R. W. (1962) Am. J. PhysioL 202, 66. Schayer, R. W., and Kobayashi, Y. (1956) Proc. Soc. Exptl. Biol. Med. 92, 653. Schayer, R. W., Wu, K. Y. T., and Smiley, R. L. (1954a) Am. J. PhysioL 179, 481. Schayer, R. W., Smiley, R. L., and Davis, K. J. (1954b) Proc. Soc. Exptl. Biol. Med. 87, 590. Schayer, R. W., Davis, K. J., and Smiley, R. L. (1955) A m. J. PhysioL 182, 54. Schayer, R. W., Rothschild, Z., and Bizony, P. (1959) Am. J. PhysioL 196, 295. Schayer, R. W., Houlihan, P., Sestokas, E., and Chapin, L. (1960) Am. J. PhysioL 198, 1187. Scheunert, Α., Haenel, H., and Meyer, I. (1957) Intern. Z. Vitaminforsch. 27, 464. Schlenk, F., and Fischer, A. (1947) Arch. Biochem. 12, 69. Schormuller, J., and Leichter, L. (1955a) Z. Lebensm.-Untersuch. u. -Forsch. 102, 13. Schormuller, J., and Leichter, L. (1955b) Z. Lebensm.-Untersuch. u. -Forsch. 102, 97. Schreus, T., and Stuttgen, G. (1950) Z. ges. exptl. Med. 115, 547. Schrodt, G. R., Dizon, F. Y., Peskoe, L. Y., Howell, R. S., Robie, C. H., and Stevenson, T. D. (1960) Am. Practitioner and Dig. Treatment 11, 750. Schuler, W., and Wyss, E. (1960) Arch, intern, pharmacodynamic 128, 431. Schumann, E. L., Greig, M. E., Heinzelman, R. V., and Seay, P. H. (1961) J. Med. Pharm. Chem. 3, 567. Seaman, G. R. (1960) J. Bacteriol. 80, 830. Senez, J. C., Cattaneo-Lacombe, J., and Beaumond, P. (1959) Proc. 4th Intern. Congr. Biochem., Vienna, 1958 15 (Sect. 4), p. 96. Pergamon Press, New York. Sher, I. H., and Mallette, M. F. (1954) Arch. Biochem. Biophys. 52, 331. Sherman, H. (1954) In "The Vitamins" (W. H. Sebrell, Jr. and R. S. Harris, eds.), Vol. 3, p. 265. Academic Press, New York. Shimada, K., Imaizumi, Y., and Okui, S. (1954) Osaka Daigaku Igaku Zassi 6, 265 Shimura, K., Nagayama, H., and Kikuchi, A. (1956) Nature 177, 935. Shintani, S. (1956) J. Vitaminol. (Osaka) 2, 185. Shintani, S. (1957a) Yakugaku Zasshi 77, 736. Shintani, S. (1957b) Yakugaku Zasshi 77, 993. Shintani, S. (1957c) Yakugaku Zasshi 77, 781. Shishova, O. A. (1956) Biokhimiya 21, 105. Shishova, Ο. Α., and Gorozhankina, L. A. (1959) Voprosy Pitaniya 18, 44. Shukuya, R., and Schwert, G. W. (1960a) J. Biol. Chem. 235, 1649. Shukuya, R., and Schwert, G. W. (1960b) J. Biol. Chem. 235, 1653. Shukuya, R., and Schwert, G. W. (1960c) Biol. Chem. 235, 1658. Siliprandi, N. (1960) Rev. espan. fisiol. 16, 1. Simmonet, G., Chapeville, F., and Fromageot, P. (1960) Bull. soc. chim. biol. 42, 891. 9. INHIBITION OF AMINO ACID DECARBOXYLASES 379

Sjoerdsma, A. (1960) Ann. Ν. Y. Acad. Sci. 88, 933. Sjoerdsma, A. (1961) Circulation Research 9, 734. Sjoerdsma, Α., Oates, J. Α., Zaltzman, P., and Udenfriend, S. (1960) New Engl. J. Med. 263, 585. Sloane-Stanley, G. H. (1949a) Biochem. J. 44, 567. Sloane-Stanley, G. H. (1949b) Biochem. J. 45, 556. Smith, S. E. (1960) Brit. J. Pharmacol. 15, 319. Snell, Ε. E. (1958) Vitamins and Hormones 16, 77. Snell, Ε. E. (1959) Proc. 4th Intern. Congr. Biochem., Vienna, 1958 9, 250. Pergamon Press, New York. Snell, Ε. E., and Jenkins, W. T. (1959) J. Cellular Corny. Physiol. 54, 161. Sôrbo, B., and Heyman, T. (1957) Biochim. et Biophys. Acta 23, 624. Sorm, F., and Grunberger, D. (1953) Chem. listy 47, 1657. Sorm, F., and Tursk^, T. (1954) Chèm. listy 48, 1403. Sorm, F., Grunberger, D., and Skoda, .J (1955) Chem. listy 49, 121. Sourkes, T. L., Drujan, B. D., and Woodford, U. R. (1958) Federation Proc. 17, 153. Sourkes, T. L., Murphy, G. F., and Woodford, V. R., Jr. (1960) ./ Nutrition 72, 145. Sourkes, T. L., Murphy, G. F., Chavez, B., and Zielinska, M. (1961) J. Neurochem. 8, 109. Spinks, Α., and Burn, J. H. (1952) Brit. J. Pharmacol. 7, 93. Steensholt, G., Flikke, M., and Joner, P. E. (1956) Proc. 3rd Intern. Biochem. Congr., Brussels, 1955 p. 38. Stein, G. Α., Bronner, Η. Α., and Pfister, K. (1955) J. Am. Chem. Soc. 77, 700. Sterzl, J., and Kfecek, J. (1949) Nature 164, 700. Sterzl, J., and Kfecek, J. (1950) Gasopis lekaru ceskych 89, 1. Storck, R. (1951) Rev. fermentations et inds. aliment. 6, 185. Sutton, C. R., and King, H. K. (1960) Biochem. J. 73, 43P. Tashian, R. E. (1960) Proc. Soc. Exptl. Biol. Med. 103, 407. Tashian, R. E. (1961) Metabolism, Clin, and Exptl. 10, 393. Taylor, E. S., and Gale, E. F. (1945) Biochem. J. 39, 52. Telford, J. M., and West, G. B. (1960) Brit. J. Pharmacol. 15, 532. Tenenhouse, Α., and Quastel, J. H. (1960) Can. J. Biochem. and Physiol. 39, 1311. Testa, E., Bonati, Α., and Pagani, G. (1961) Chimia (Switz.) 15, 314. Thoai, Ng.-V., and Chevillard, L. (1951a) Compt. rend. acad. sci. 232, 1444. Thoai, Ng.-V., and Chevillard, L. (1951b) Compt. rend. soc. biol. 145, 485. Thibault, Ο. (1950) Ann. endocrinol {Paris) 11, 219. Thibault, Ο. (1952a) Ann. endocrinol (Paris) 13, 949. Thibault, Ο. (1952b) Arch. sci. physiol. 6, 349. Tower, D. B. (1956) Am. J. Clin. Nutrition 4, 329. Tower, D. B. (1959) Proc. 4th Intern. Congr. Biochem., Vienna, 1958 3, 213. Perga mon Press, New York. Tower, D. B. (1960) "Neurochemistry of Epilepsy. Seizure Mechanisms and their Management." C. C Thomas, Springfield, Illinois. Trendelenburg, J. (1953) Brit. J. Pharmacol. 8, 454. Tsukada, Y., Nagata, Y., and Hirano, S. (1960) Seitai no Kagaku 11, 281. Turner, J. M., and Happold, F. C. (1961) Biochem. J. 78, 364. Tursk£, T. (1960) Nature 187, 322. Uchida, T. (1954) Igaku to Seibutsugaku 32, 107. Udenfriend, S., Weissbach, H., and Bogdanski, D. F. (1957) Ann. Ν. Y. Acad. Sci. 66, 602. Ueda, K., Akedo, H., and Suda, M. (1960) J. Biochem. (Japan) 48, 584. 380 W. G. CLARK

Umbreit, W. W. (1954) In "The Vitamins" (W. H. Sebrell, Jr. and R. S. Harris, eds.), Vol. 3, p. 236. Academic Press, New York. Umbreit, W. W. (1955) In "Amino Acid Metabolism" (W. D. McElroy and B. Glass, eds.), p. 45. Johns Hopkins Press, Baltimore, Maryland. Umbreit, W. W., and Waddell, J. G. (1949) Proc. Soc. Exptl. Biol. Med. 70, 293. Van Meter, W. G., Avala, G. F., Costa, E., and Himwich, H. E. (1960) Federation Proc. 19, 265. Villee, C. A. (1960) In "Enzymes in Health and Disease" (D. M. Greenberg and H. A. Harper, eds.), p. 73. C. C Thomas, Springfield, Illinois. Vyshepan, E. D., Ivanova, K. L, and Chernukh, A. M. (1959) Byull. Eksptl. Biol. Med. 8, 52. Wachi, T., Matsumoto, T., and Kita, N. (1959) Kekkaku 34, 659. Wachstein, M., and Lobel, S. (1956) Am. J. Clin. Pathol. 26, 910. Wachstein, M., and Moore, C. (1958) Proc. Soc. Exptl. Biol. Med. 97, 905. Wada, H., and Snell, Ε. E. (1961) Biol. Chem. 236, 2089. Wada, H., Morisue, T., Morino, Y., Sakamoto, Y., and Ichihara, K. (1961) Kôso Kagaku Shimpojiumu 14, 116. Waksman, A. (1957) Arch, intern, physiol. et biochim. 65, 171. Waton, N. G. (1956a) Biochem. J. 64, 318. Waton, N. G. (1956b) Brit. J. Pharmacol. 11, 119. Weil-Malherbe, H. (1955) In "Biochemistry of the Developing Nervous System" (H. Waelsch, ed.), p. 458. Academic Press, New York. Weil-Malherbe, H. (1956) Lancet ii, 282. Weissbach, H., and Redfield, B. G. (1960) J. Biol. Chem. 235, 3287. Weissbach, H., Bogdanski, D. F., Redfield, B. G., and Udenfriend, S. (1957) /. Biol. Chem. 227, 617. Weissbach, H., Lqvenberg, W., and Udenfriend, S. (1960a) Biochem. Biophys. Re search Communs. 3, 225. Weissbach, H., Redfield, B. G., and Titus, E. (1960b) Nature 185, 99. Weissbach, H., Lovenberg, W., and Udenfriend, S. (1961) Biochim. et Biophys. Acta 50, 177. Wenzel, D. G., and Beckloff, G. N. (1958) J. Am. Pharm. Assoc. 47, 844. Werle, E. (1936) Biochem. Z. 287, 292. Werle, E. (1940) Biochem. Z. 304, 201. Werle, E. (1940-1941) Biochem. Z. 309, 61. Werle, E. (1941) Biochem. Z. 311, 270. Werle, E. (1943a) Die Chemie 56, 141. Werle, E. (1943b) Fermentforschung 17, 103. Werle, E. (1947) Z. Vitamin-, Hormon- u. Fermentforsch. 1, 504. Werle, E. (1951) Angew. Chem. 63, 550. Werle, E. (1961) Naturwissenschaften 48, 54. Werle, E., and Aures, D. (1959) Z. physiol. Chem., Hoppe-Seyler's 316, 45. Werle, E., and Aures, D. (I960) Psychiatria (Basel) 140, 226. Werle, E., and Bruninghaus, S. (1951) Biochem. Z. 321, 492. Werle, E., and Gleissner, R. (1951) Z. Vitamin-, Hormon- u. Fermentforsch. 4, 450. Werle, E., and Heitzer, K. (1938) Biochem. Z. 299, 420. Werle, E., and Hermann, H. (1937) Biochem. Z. 291, 105. Werle, E., and Koch, W. (1949) Biochem. Z. 319, 305. Werle, E., and Krautzun, K. (1938) Biochem. Z. 295, 315. Werle, E., and Peschel, W. (1949) Biochem. Z. 320, 1. 9. INHIBITION OF AMINO ACID DECARBOXYLASES 381

Werle, E., and Raub, A. (1948) Biochem. Z. 318, 538. Werle, E., Schauer, Α., and Hartung, G. (1955) Klin. Wochschr. 33, 562. West, G. B. (1953) /. Pharm. and Pharmacol. 5, 311. West, G. B. (1958) J. Pharm. and Pharmacol. 10, 92. Westermann, E. (1956) Naunyn-Schmiedeberg's Arch, exptl. Pathol, u. Pharmakol. 228, 159. Westermann, E., Balzer, H.} , and Knell, J. (1958) Arch, exptl. Pathol, u. Pharmakol. Naunyn-Schmiedeberg s 234, 194. Willett, H. P. (1958) Proc. Soc. Exptl. Biol. Med. 99, 177. Williams, R. J., Eakin, R. E., Beerstecher, E., Jr., and Shive, W. (1950) In "The Biochemistry of the Β Vitamins." Reinhold, New York. Wilson, J. E., and Du Vigneaud, V. (1950) J. Biol. Chem. 184, 63. Wilson, W. R., Fisher, F. D., and Kirkendall, W. M. (1961) /. Clin. Invest. 40,1089. Wingo, W. J., and Awapara, J. J. (1950) Biol. Chem. 187, 267. Wiseman, G., and Ghadially, F. N. (1955) Brit. J. Cancer 9, 480. Wiss, O., and Weber, F. (1958) Med. J. Osaka Univ. 8 (Suppl.), 41. Wohl, M. G., Levy, Η. Α., Szutka, Α., and Maldia, G. (1960) Proc. Soc. Exptl. Biol. Med. 105, 523. Yabe, Y., Akita, Y., and Akita, K. (1957) Okayama-Igakkai-Zasshi 69, 2135. Yamada, K., Sawaki, S., and Hayami, S. (1957) Vitaminol. (Osaka) 3, 68. Yamada, K., Sawaki, S., and Yazaki, C. (1959) /. Vitaminol. (Osaka) 5, 249. Yamagami, A. (1958) Osaka Daigaku Igaku Zassi 10, 417. Yamanaka, T. (1957) Vitamins (Kyoto) 13, 422. Yeh, S. D. J., Solomon, J. D., and Chow, B. F. (1959) Federation Proc. 18, 357. Yoneda, M., and Asano, N. (1953) Science 117, 277. Yoshimura, T. (1958) Vitamins (Kyoto) 14, 107. Youatt, J. (1958) Biochem. J. 68, 193. Yuwiler, Α., Geller, E., and Eiduson, S. (1959) Arch. Biochem. 80, 162. Yuwiler, Α., Geller, E., and Eiduson, S. (1960) Arch. Biochem. 89, 143. Zile, M. H. (1959) "Monoamine Oxidase in Relation to Thyroid Hormones," Dis sertation, Univ. Wisconsin, Madison, Dissertation Abstr. 19, 103. Zile, M. H. (1960) Endocrinology 66, 311. Zile, M., and Lardy, H. A. (1959) Arch. Biochem. Biophys. 82, 411.