Proc. NatL Acad. Sci. USA Vol. 80, pp. 5144-5148, August 1983 Neurobiology

Identification in pituitary tissue of a peptide a-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid (secretory granules/endogenous inhibitors/a-melanotropin/post-translational processing/vitamin C) BETTY A. EIPPER*, RICHARD E. MAINS*, AND CHRISTOPHER C. GLEMBOTSKIt Department of Physiology, C-240, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262 Communicated by Curt P. Richter, May 20, 1983

ABSTRACT An enzymatic activity capable of producing an a- In this paper we present data showing that beef and rat pi- amidated peptide product from its glycine-extended precursor has tuitary secretory granules contain a copper-dependent enzy- been identified in secretory granules of rat anterior, intermedi- matic activity that requires the presence of molecular oxygen ate, and neural pituitary and bovine intermediate pituitary. High and is stimulated by ascorbic acid to produce an a-amidated levels of endogenous inhibitors of this a-amidation activity have peptide product from a glycine-extended precursor. also been found in tissue homogenates. The a-amidation activity is totally inhibited by addition of divalent metal ion chelators such as diethyldithiocarbamate, o-phenanthroline, and EDTA; a-ami- METHODS dation activity is restored to above control levels upon addition of Subceliular Fractionation. Pituitaries from adult male rats copper. The a-amidation reaction requires the presence of mo- were separated into pools of anterior, intermediate, and neural lecular oxygen. Of the various cofactors tested, ascorbic acid was pituitary; each pool of tissue was homogenized in buffered iso- the most potent stimulator of a-amidation. The a-amidation ac- tonic sucrose (0.25 M sucrose/20 mM Tris HCI, pH 7.4) and tivity has a neutral pH optimum and is primarily soluble following the anterior pituitary homogenates were incubated with DNase several cycles of freezing and thawing. Kinetic studies with the (20 ,g/ml) for 5 min at room temperature (4). Homogenates bovine intermediate pituitary granule-associated activity dem- were layered onto 10 ml of Percoll (diluted to a density of 1.08 onstrated a linear Lineweaver-Burk plot when D-Tyr-Val-Gly was g/ml with buffered isotonic sucrose) and centrifuged for 30 min the varied substrate; the apparent Km and Vm,, varied with the at 60,000 X g in a fixed-angle rotor (Beckman, type 40) (4). Per- concentration of ascorbic acid. The substrate specificity of the a- coll gradients were collected into approximately 0.3- to 0.4-ml amidation activity appears to be quite broad; the conversion of D- fractions, frozen and thawed several times, and assayed for a- Tyr-Val-Gly into D-Tyr-Val-NH2 is inhibited by the addition of a amidation activity. Bovine intermediate pituitary tissue was ho- variety of glycine-extended peptides. mogenized as described above and subjected to differential centrifugation before fractionation on a Percoll gradient. The Many of the bioactive peptides isolated from neural and en- homogenate was centrifuged for 10 min at 400 X g. The su- docrine tissues have an a-amide moiety at their carboxyl ter- pernatant was centrifuged for 30 min at 10,000 X g onto a 2- minus (1, 2). The presence of this a-amide group is so char- ml cushion of Percoll diluted to 1.08 g/ml with isotonic su- acteristic of bioactive peptides that Tatemoto et al (2) have crose; the resulting pellet, which contained secretory granules, succeeded in isolating several bioactive peptides by employing was layered onto 10 ml of Percoll and centrifuged as described a chemical assay for amino acid a-amides. In general, the pres- above. ence of the a-amide moiety is essential for full biological po- Amidation Assays. Synthetic D-Tyr-Val-Gly and D-Tyr-Val- tency of the peptide. NH2 (3) were purchased from Bachem and were further pu- The amino acid sequences of many precursors to a-amidated rified by reversed-phase HPLC on a Waters C18 ,Bondapak peptides have been deduced over the last several years; in every column; the column was equilibrated with 4% CH3CN in 0.1% case the amino acid residue (X) that is a-amidated in the prod- trifluoroacetic acid and eluted with a linear gradient to 20% uct peptide (-X-NH2) is followed in the precursor by a glycyl CH3CN in 0.1% trifluoroacetic acid over 60 min (5). Monoio- residue (-X-Gly-). The combined proteolysis/amidation signal dinated D-Tyr-Val-Gly and monoiodinated D-Tyr-Val-NH2 were has generally been found to consist of the sequence -X-Gly-basic- prepared by reaction of 2.0 Ag of peptide with 1.0 mCi (1 Ci basic-, where the basic residues can be Lys or Arg (1). Bradbury = 3.7 x 1010 Bq) of Na'"I by using the Iodobead procedure et al (3) used a synthetic glycine-extended peptide (D-Tyr-Val- (unpublished data; ref. 6); iodinated peptide was separated from Gly) resembling the carboxyl terminus of the presumed pre- free iodine by adsorption to an octadecasilyl cartridge (5). For cursor to a-melanocyte-stimulating hormone (a-MSH) to de- use in amidation assays, iodinated D-Tyr-Val-Gly was first passed tect a peptide a-amidation activity in pig pituitary secretory through a 0.5-ml column of SP-Sephadex-C25-120 (40- to 120- granules; the corresponding peptide amide (D-Tyr-Val-NH2) and ,m beads) equilibrated with 10 mM Na phosphate (pH 5.0). In glyoxylate were produced and the nitrogen of the glycyl residue some early experiments a mixture of mono- and diiodinated D- was shown to be the source of the amide nitrogen (3). The re- Tyr-Val-Gly was produced by reaction of 2 ,g of peptide with action mechanism proposed involved removal of hydrogen from 1 mCi of Na'"I by using the hypochlorite procedure (4); where the carboxyl-.terminal glycine and spontaneous hydrolysis of the imino there was no redox cofactor for the re- resulting linkage; Abbreviations: MSH, melanocyte-stimulating hormone; ACTH, corti- action specified (3). cotropin; TES, N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid. * Present address: Dept. of Neuroscience, Johns Hopkins University The publication costs of this article were defrayed in part by page charge School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. payment. This article must therefore be hereby marked "advertise- t Present address: Dept. of Pharmacology/G3, School of Medicine, ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. University of Pennsylvania, Philadelphia, PA 19104. 5144 Downloaded by guest on September 27, 2021 Neurobiology: Eipper et al. Proc. Natl. Acad. Sci. USA 80 (1983) 5145 indicated, this material was used for amidation assays. The glycine substrates had met with failure; the activity studied by monoiodinated D-Tyr-Val-Gly is referred to as 125I-D-Tyr-Val- Bradbury et al. was more readily detected after gel filtration of Gly. a secretory granule lysate than in crude subcellular fractions (3). Assay conditions are described in the figure legends; unless Therefore, we decided to use the synthetic 125I-D-Tyr-Val-Gly otherwise noted, assays were carried out in duplicate at 370C substrate to search for a-amidation activity in pituitary homoge- in a final volume of 40 pil with =20,000 cpm of l25I-D-Tyr-Val- nate fractionated on Percoll gradients (Fig. 1). Rat anterior, in- Gly. The standard stock assay buffer was 150 mM TES [N- termediate, and neural pituitary were analyzed separately. The tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid] ti- fractions were assayed for P-endorphin immunoactivity to lo- trated to pH 7.0 with NaOH; in the final assay solution it was cate secretory granules derived from corticotropin (ACTH)/en- generally diluted to about 100 mM. Assays were quenched by dorphin cells and to provide a comparison to our previous sub- the addition of 1.3 ml of 10 mM Na phosphate (pH 5.0) and D- cellular fractionations of pituitary tissue on Percoll gradients Tyr-Val-Gly and D-Tyr-Val-NH2 were separated by application (4). As shown previously, the peak of f3-endorphin immunoac- of the sample to a 1.0-ml column of SP-Sephadex-C25-120 tivity nearest the bottom of each gradient (fractions 2-9 in Fig. equilibrated with 10 mM Na phosphate (pH 5.0); after six washes 1) represents secretory granules; the peaks of immunoactivity (1.3 ml each) with the starting buffer, to remove unreacted sub- around fractions 19-21 are in the region of the gradient con- strate, 125I-D-Tyr-Val-NH2 was eluted from the column with taining rough endoplasmic reticulum and Golgi apparatus marker three 1.3-ml washes of 0.5 M NaCl in 50 mM Na phosphate enzymes and material at the top of the gradient is soluble (4). (pH 5.0). The efficacy of this separation system was established For each of the three tissues, a-amidation activity was found with synthetic 1251-D-Tyr-Val-Gly and 1251-D-Tyr-Val-NH2. almost exclusively in the secretory granule region. The rat an- Samples were assayed for radioactivity at '-75% efficiency in terior pituitary homogenate contained an unexpectedly large a Micromedic 4/200 gamma scintillation counter. Results of amount of a-amidation activity given that the major anterior some early experiments are expressed as % conversion. For most pituitary hormones are not a-amidated. In contrast, a-MSH (a assays a known amount of synthetic D-Tyr-Val-Gly (generally major product of the intermediate pituitary) and oxytocin and between 0.2 and 25 ,uM) was added to the reaction mix and vasopressin (major products of the neural lobe) are a-amidated. results are expressed as pmol of product formed per ,g of pro- Reversed-phase HPLC analysis of the reaction products formed tein per hr. Preliminary studies demonstrated that the kinetic by the granule-associated activity from each of the three tissues constants (Km and Vmax) for 127I-D-Tyr-Val-Gly and D-Tyr-Val- indicated that >97% of the labeled reaction product comi- Gly in the a-amidation assay were indistinguishable (unpub- grated with synthetic 125I-D-Tyr-Val-NH2. All further analyses lished data). Protein concentrations were determined as de- of a-amidation activity utilized pools of secretory granule-as- scribed (4). sociated a-amidation activity from Percoll gradients similar to those shown in Fig. 1. The same assay was used to detect a- RESULTS amidation activity associated with bovine intermediate pituitary Detection of a-Amidation Activity. Until the report by secretory granules. Bradbury et aL (3), attempts to detect a-amidation of peptidyl- As can be seen in Fig. 1, a greater proportion of the a-ami-

40 20 30 Cq ,, , , 10I 15- 20 10 "0 CSQ

10 Fraction FIG. 1. a-Amidation activity in pituitary homogenates fractionated on Percoll gradients. Pituitaries from 25 adult male rats (average weight, 392 g) were separated into pools of anterior (Left), intermediate (Center), and neural (Right) pituitary, homogenized, and fractionated on Percoll gradients. Amidation assays contained 125I-D-Tyr-Val-Gly (a mixture of mono- and diiodinated substrate) and an aliquot of each Percoll fraction (5 ,ul for anterior pituitary; 10 ,ul for intermediate and neural pituitary); assays were carried out for 5 hr; results are all expressed as % conversion per 10-,l aliquot. Radioimmunoassays for /3-endorphin utilized antiserum Danielle (4); 3endorphin immunoactivity per pituitary was: anterior lobe, 88 pmol; intermediate lobe, 254 pmol; neural lobe, 99 pmol. As expected, the anterior pituitary homogenate contained about one-third as much 3-endorphin immunoactivity as the intermediate pituitary homogenate; the 3-endorphin immunoactivity in the neural pituitary homogenate was due to the difficulty ofremoving adhering intermediate pituitary cells from the neural lobe. For further analyses of a-amidation activity, fractions withthehighest a-amidation activity were pooled: anteriorpituitary, fractions 1-5; intermediate pituitary, fractions 3-5; neural pituitary, fractions 4-6. Downloaded by guest on September 27, 2021 5146 Neurobiology: Eipper et al. Proc. Natl. Acad. Sci.,USA 80 (1983)

dation activity was associated with particulate fractions than was Table 1. Ability of various divalent cations to decrease the P3-endorphin immunoactivity. When fractions from the rough inhibitory effect of diethyldithiocarbamate on endoplasmic reticulum/Golgi apparatus or from the soluble re- a-amidation activity gion of any of the tissues shown in Fig. 1 were mixed with the Pituitary enzymatically active granule fractions from anterior pituitary, a-amidation activity could be totally inhibited. For the gradient Treatment Anterior Intermediate Neural shown in Fig. 1 Left, addition of 1 pl of fraction 19 or fraction Control 100 100 100 26 completely inhibited the a-amidation activity in 2.5 /1 of the 2 AM DDC 1.4 3.3 1.2 secretory granule pool. The occurrence of potent inhibitors of + 10 AM CaCl2 0.7 6.1 1.3 a-amidation activity may explain why previous studies con- + 10AWMgCl2 0.9 2.0 1.2 sistently failed to detect a-amidation activity (1). The inhibitory + 10AM.ZnSO4 0.7 1.8 0.7 activity is stable to boiling; when insoluble material is sedi- + 10 pM CdCH3COOH 2.2 L.a 0.3 + 2.3 4.3 1.8 mented from these boiled samples the supernatant is largely 10 PM CoC12 + 10 MM FeSO4 0.5 1.0 0.3. depleted of inhibitory activity. The profile of inhibitory activity + 10 pM MnCl2 0.6 1.0 1.0 on the Percoll gradient tends to follow protein levels and is highest + 10,uM NiSO4 0.3 2.8 2.2 in the rough endoplasmic reticulum/Golgi apparatus and in the + 10 AM CuSO4 120 169. 147 soluble regions. Inhibitory activity is also present in the secre- + 2 AM CuSO4 224 221 143 tory granule pool and can be revealed by the addition of a boiled enzyme blank to an aliquot of active enzyme. Assays contained 0.2 AM D-Tyr-Val-Gly, 0.25 mM ascorbic acid, 100 Preliminary Characterization of a-Amidation Activity. The Agofcatalaseperml, 1251-D-Tyr-Val-Gly, and 97.5 mM NaTES(pH 7.0) as well as the additives listed. The anterior pituitary sample (2.3 ,g of pH optimum for the granule-associated a-amidation activity in protein per assay) was incubated for 4 hr. control reaction velocity was each of the rat tissues and in bovine intermediate pituitary was 0.085 pmol of product per ag per hr (9.7% conversion). The interme- found to be about pH 7.0, with a rapid decline in activity below diate pituitary sample (3.0 ug of protein per assay) was incubated for pH 6 or above pH 7.5 (unpublished data). For all of the granule 5 hr; control reaction velocity was 0.014 pmol/,g per hr (2.7% con- pools, the reaction was shown to be linear in amount of granule- version). The neural pituitary sample (3.3 ,ug of protein per assay) was up to 6 ug of protein per assay tube. incubated for 5 hr; control reaction velocity was 0.022 pmol/,ug per hr associated protein, about (4.6% conversion). Data are expressedas % control. DDC, diethyldithio- The reaction was essentially linear in time up to about 5 hr. A carbamate. boiled enzyme blank showed no detectable activity (<0.1% conversion). Centrifugation of the frozen and thawed granule copper was capable of reversing the inhibitory effect of die- pools (85,000 X g for 30 min) demonstrated that over two-thirds thyldithiocarbamate. The concentration of copper resulting in of the a-amidation activity was soluble. maximal stimulation of a-amidation activity in each pool of pi- In preliminary studies of the pH optimum for a-amidation tuitary enzyme activity was determined in separate experi- activity it was observed that the use of citrate or phosphate buffers ments and was usually found to be about 2 ,uM; this result is resulted in diminished activity (to approximately one-fourth) variable,, suggesting that the optimal copper concentration needs compared to use of sulfonic acid buffers. This observation sug- to be determined for each preparation of granules. The inhib- gested that divalent metals might affect the a-amidation activ- itory activity of the rough endoplasmic reticulum/Golgi ap- ity; therefore, the effect of various metal chelators on a-ami- paratus and soluble fractions was completely eliminated by ad- dation activity was determined (Fig. 2). Addition of dieth- dition of the appropriate level of copper. yldithiocarbamate (1 ,uM), o-phenanthroline (20 ,uM), or EDTA Because glyoxylate was identified as one of the products of (20 ,uM) resulted in total inhibition of a-amidation activity in the a-amidation of glycine-extended peptides by Bradbury et all tissues tested. The ability of a variety of divalent cations to al.. (3), the overall reaction must involve oxidation. -Copper-re- reverse the inhibitory effect of diethyldithiocarbamate on rat quiring enzymes are often involved in interactions with mo- anterior, intermediate, and neural pituitary a-amidation activ- lecular oxygen (7, 8); therefore, the effect of removal of mo- ity was determined (Table 1). Of all of the metals tested, only lecular oxygen on a-amidation activity was determined, (Table 2). For all of the tissues tested, replacement of molecular ox- ygen with argon (or nitrogen) resulted in almost total inhibition 5 of a-amidation activity. The above results indicate that there are two possible re- 4 Table 2. Dependence of a-amidation reaction on

,-i 3- molecular oxygen x Reaction velocity, % activity 2 Source of pmol/,ug per hr remaining pituitary enzyme Air Argon in argon Rat Anterior 10.4 0.65 6.2 0 Intermediate 2.27 0.078 3.4 30 10 3.0 1.0 0.3 0.1 0.03 0.01 Neural 1.94 0.15 7.7 Chelator, gM Bovine intermediate 3.14 0.42 13.3

FIG. 2. Effect of chelators on a-amidation. activity. All assay tubes Assays were incubated for 2 hr and contained 1"I-D-Tyr-Val-Gly, 25 contained 0.2 AM D-Tyr-Val-Gly, '25I-D-Tyr-Val-Gly, 101 mM Na TES pM D-Tyr-Val-Gly, 2 PM CuS04, 0.25 mM ascorbic acid, 100 pg of cat- (pH 7.0), 2.8 ug of rat anterior pituitary granule-associated protein, alase per ml, and 114 mM Na TES (pH 7.0) under an atmosphere of air and the indicated concentration of chelator; assays were incubated for or a humidified stream of argon. Amount of protein added per assay 4 hr and data are presented as fmol of product formed in that time. 0, tube was: rat anterior pituitary, 12 ug; rat intermediate pituitary, 3.0 Diethyldithiocarbamate; a, o-phenanthroline; A, EDTA; -*, control. ug; rat neural pituitary, 3.3 Ag; bovine intermediate pituitary, 7.0 jig. Downloaded by guest on September 27, 2021 Neurobiology: Eipper et al. Proc. Natl. Acad. Sci. USA 80 (1983) 5147 action mechanisms. The reaction could require a reducing agent and produce glyoxylate and water, using two atoms of oxygen 3.5 from molecular oxygen; this is the pattern observed with mono- oxygenases such as dopamine /3-hydroxylase (9-11). Alterna- 3.0 F tively, the reaction might involve no reducing agent and pro- duce H202 and an unstable imino intermediate, which would 2.51- spontaneously hydrolyze to yield glyoxylate and the amidated 2.0 F peptide; this reaction scheme is like that suggested by Brad- 0. Km = 2.5 juM bury et al. (3) and is similar to the mechanism followed by amine T - Y Vmax = 1.2 pmol//ug per hr oxidases (11). 1.5 F Given the striking similarities of this a-amidation activity to dopamine P-hydroxylase, ascorbic acid was tested as a possible 1.0 cofactor (Fig. 3). The ai-amidation activity in rat anterior pi- 0.5 tuitary was stimulated 33-fold, whereas the activity in beef in- [ Km = 42 AMM termediate pituitary was stimulated 15-fold by ascorbic acid. Vmax = 39 pmol/,ug per hr The effect of several other reduced and oxidized cofactors on 0 I I a-amidation activity was compared to that of ascorbic acid. For 0 0.2 0.4 0.6 0.8 1.0 the granule-associated a-amidation activity from rat anterior pi- 1/S, JM tuitary and beef intermediate pituitary, ascorbic acid was found to be the most potent stimulator of a-amidation activity (un- FIG. 4. Determination ofKm and V.., for bovine intermediate pi- tuitary granule-associated a-ariidation activity. Assays were incu- published data). Consistent with the requirement for molecular bated for 4 hr in a reaction mix containing the indicated amount of D- oxygen, none of the oxidized cofactors stimulated the a-ami- Tyr-Val-Gly, a constant amount of'251-D-Tyr-Val-Gly, 2 AuM CUSO4, 4.2 dation reaction. Ag of bovine intermediate pituitary granule-associated protein, and 136 The optimal ascorbic acid concentration rises as the concen- mM Na TES (pH 7.0); one set of assays was carried out with no added tration of substrate D-Tyr-Val-Gly rises-e.g., the optimal as- ascorbic acid and catalase (e) and one set included 0.5 mM ascorbic acid and 100 .g ofcatalase per ml (o). Kinetic constants were calculated by corbic acid concentration is 3 mM with 25 /iM D-Tyr-Val-Gly an least and 0.3 mM with 0.25 p.M D-Tyr-Val-Gly. As is the case for unweighted squares method. dopamine ,-hydroxylase, an appropriate amount of catalase must be added along with ascorbic acid or inhibition of the a-ami- tivities were quite similar at this level of analysis (unpublished dation reaction will occur (9-11); catalase alone has no signif- data). Several potential physiologically relevant substrates (gly- icant effect on a-amidation activity and appears to act by sta- cine-extended peptides), such as a-N-acetyl-ACTH-(1-14) and bilizing the added ascorbic acid (10, 11). y2-MSH, were potent inhibitors of the a-amidation of D-Tvr- Substrate Specificity of a-Amidation Activity. To get an idea Val-Gly. Several other glycine-extended peptides, not expected of the specificity of the a-amidation activity, the ability of var- to serve as physiological substrates for the activity, also inhib- ious potential substrate and product peptides (each added at ited a-amidation [ACTH-(1-10), which terminates -Trp-Gly 100 /iM final concentration) to inhibit conversion of D-Tyr-Val- (positions 9 and 10, respectively); Tyr-Gly-Gly]. The impor- Gly into D-Tyr-Val-NH2 was determined. The specificities of tance of the carboxyl-terminal glycyl residue is illustrated by the rat anterior pituitary and beef intermediate pituitary ac- the fact that neither Tyr-Gly-Gly-Phe nor ACTH-(1-13) was a potent inhibitor of a-amidation. Several a-amidated peptides, including a-MSH, had no significant effect on activity at the 100 E concentration tested. Other pro-ACTH/endorphin-related peptides not containing potential a-amidation sites [f-endor- phin-(1-27); CLIP or ACTH-(18-39)] also had no effect on the reaction. Kinetic studies were carried out to determine the effect of CZ varying the concentration of D-Tyr-Val-Gly on reaction veloc- in every case 50 ity; examined, reaction velocity was found to in- crease to a saturation level. The granule-associated activities behaved in a Michaelis-Menten manner to D-Tyr-Val-Gly as the varied substrate; data for the bovine intermediate pituitary granule-associated activity are shown in Fig. 4. The Km and Vma. 0 for D-Tyr-Val-Gly showed a striking variation with the amount of ascorbic acid added to the reaction mixture. In the absence 0 l {i of added ascorbic acid, the Km was 2.5 u.M and the Vma? was 10 3 1.0 0.3 0.1 0.03 0.01 0 1.2 pmol/ttg of protein per hr; when ascorbic acid (0.5 mM) Ascorbate, mM was added, the Km increased 17-fold and the Vmax increased 33- fold. Similar effects of ascorbic acid on the apparent Km and FIG. 3. Effect of ascorbic acid on a-amidation activity. Assays con- Vm. were seen for the rat anterior, intermediate, and neural tained the indicated concentration of ascorbic acid, 100 ,ug of catalase pituitary granule-associated a-amidation activities (unpub- perml, 25 ,uM D-Tyr-Val-Gly, 125 D--Tyr-Val-Gly, 2 ,uM CUS04, and 135 lished data). mM Na TES (pH 7.0). Assays with the beef intermediate pituitary ac- tivity (0) were for 3 hr with 2.8 jig of protein per assay; data are pre- sented as % ofmaximal reaction velocity (14 pmol/,ug ofprotein per hr DISCUSSION at 3.0mM ascorbic acid). Assays with the ratanterior pituitary activity (@) were carried out for 3 hr with 3.4 ,g of protein per assay; maximal Taking into account the effects of molecular oxygen and ascor- reaction velocity was 28 pmol/ftg of protein per hr at 1.0 mM ascorbic bic acid on the a-amidation activity reported here and the iden- acid. tification of glyoxylate as a product of peptide amidation (3), Downloaded by guest on September 27, 2021 5148 Neurobiology: Eipper et al. Proc. Natl. Acad. Sci. USA 80 (1983) the following reaction seems the most likely: perhaps the depolarization-related efflux of ascorbate from ascorbate dehydro- or semidehydroascorbate central nervous system tissue that has been observed in vitro 00 and in vivo (22) is related to ascorbate associated with granules \ / ~~~~~~~~~~~1111 of peptidergic as well as adrenergic neurons. Consideration of D-Tyr-Val-Gly + r-Val-NH2+ H-C-C-OH + H20 the involvement of ascorbate and copper in peptidylglycine a- glyoxylate amidation offers an important approach toward understanding -Bradbury et aL (3) showed that the amide nitrogen is derived the multitude of effects associated with ascorbate and copper from the glycyl residue. Studies utilizing 1802 and measuring deficiencies. ascorbic acid consumption will be required to substantiate this We thank Shawn Mulvihill and Diane Honnecke for expert technical mechanism. assistance and George Tarver for the drawings.-,.This research was sup- The a-amidation activity shares many characteristics with ported by National Institutes of Health Grants AM-18929 and AM-19859 another secretory granule-associated enzyme, dopamine 13-hy- and by the McKnight Foundation. droxylase (8-11); both use copper, molecular oxygen, and ascorbate. Both enzymes are also. affected by heat-stable en- 1. Mains, R. E., Eipper, B. A., Glembotski, C. C. & Dores, R. M. (1983) Trends NeuroSci. 6, 229-235. dogenous inhibitory activity that can be overcome by addition 2. Tatemoto, K., Carlquist, M. & Mutt, V. (1982) Nature (London) of copper (11, 12). The presence of inhibitory activity in crude 296, 659-660. cell-free extracts may account for previous failures to detect a- 3. Bradbury, A. F., Finnie, M. D. A. & Smyth, D. G. (1982) Nature amidation activity. (London) 298, 686-688. We have observed a similar a-amidation activity in rat and 4. Glembotski, C. C. (1982) J. Biol. Chem. 257, 10501-10509. bovine anterior and intermediate pituitary, rat neural pituitary, 5. Bennett, H. P. J., Browne, C. A. & Solomon, S. (1981) Biochem- istry 20, 4530-4538. and mouse AtT-20 corticotropic tumor cells (unpublished data). 6. Pierce Chemical Company (1982) Pierce Chemical Company Pro- Although the physiological substrates for the a-amidation ac- tein Digest (Pierce Chemical, Rockville, IL), Vol. 6, pp. 2-3. tivity from some of these tissues (e.g., anterior pituitary) are 7. Cass, A. E. G. & Hill, H. A. 0. (1980) in CIBA Foundation Sym- not known, the inhibition of this activity by a variety of glycine- posium 79 (new series) Biological Roles of Copper, eds. Evered, extended peptides suggests that many a-amidated peptides may D. & Lawrenson, G. (Excerpta Medica, New York), pp. 71-84. be produced by the same a-amidating activity. There is a large 8. Ullrich, V. & Duppel, W. (1975) in The Enzymes, ed. Boyer, P. D. (Academic, New York), Vol. 12B, pp. 253-297. amount of a-amidation activity in the intermediate pituitary. 9. May, S. W, Phillips, R. S., Mueller, P. W. & Herman, H. H. (1981) Considering the kinetic constants observed for D-Tyr-Val-Gly J. Biol. Chem. 256, 2258-2261. and the concentration of pro-ACTH/endorphin-derived pep- 10. Levin, E. Y., Levenberg, G. & Kaufman, S. (1960)J. Biol. Chem. tides in the secretory granule pool, the steady-state concentra- 235, 2080-2086. tion of a substrate like ACTH-(1-14) should be negligible. At- 11. Hunt, D. M. (1980) in CIBA Foundation Symposium 79 (new se- tempts to identify ACTH-(1-14) in intermediate pituitary are ries) Biological Roles of Copper, eds. Evered, D. & Lawrenson, G. (Excerpta Medica, New York), pp. 247-260. in agreement with this prediction (13, 14). 12. Orcutt, J. C. & Molinoff, P. B. (1976) Biochem. Pharmacol. 25, It is interesting to note that a-amidation of pancreatic poly- 1167-1174. peptide and a-MSH decreases rapidly when cells normally pro- 13. Eipper,.B. A., Glembotski, C. C. .& Mains, R. E. (1983)J. Biol. ducing these peptides are placed in tissue culture (13-15). In- Chem. 258, 7292-7298. stead, cultured rat intermediate pituitary cells synthesize and 14. Glembotski, C. C., Eipper, B. A. & Mains, R. E. (1983) J. Biol. secrete ACTH-(1-14) peptides (13, Chem. 258, 7299-7304. glycine-extended acetylated 15. Paquette, T. L., Gingerich, R. & Sharp, D. (1981) Biochemistry 14). Knowledge of the requirements of the a-amidation activity 20, 7403-7408. in vitro may make it possible to manipulate culture conditions 16. Owen, C. A., Jr. (1981) Copper Deficiency and Toxicity (Noyes, until the ability to synthesize a-amidated peptides is regained. Park Ridge, NJ). Neurological dysfunction is invariably present in copper-de- 17. Sourkes, T. L. (1980) in CIBA Foundation Symposium 79 (new se- ficient mammals (11, 16). Addition of this peptidylglycine a- ries) Biological Roles of Copper, eds. Evered, D. & Lawrenson, the short list of G. (Excerpta Medica, New York), pp. 143-154. amidation activity to 'rather copper-requiring 18. Harris, E. D., Rayton, J. K., Balthrop, J. E., DiSilvestro, R. A. enzymes opens up many new areas to-be considered when trying & Garcia-de-Quevdo, M. (1980) in CIBA Foundation Symposium to explain the changes associated with copper deficiency and 79 (new series) Biological Roles of Copper, eds. Evered, D. & toxicity. The activity of other copper-requiring enzymes such Lawrenson, G. (Excerpta Medica, New York), pp. 163-177. as dopamine /3-hydroxylase and lysyl oxidase is decreased in 19. Basu, T. K. & Schorah, C. J. (1982) Vitamin C in Health and Dis- dietary copper deficiency (11, 16-18). Similarly, ascorbate de- ease (Avi, Westport, CT). 20. Noble, S. & Woodhill, J. M. (1982) Vitamin C (MTP, Lancaster, ficiency has been shown to affect dopamine ,3-hydroxylase, pro- England). line and lysine hydroxylation, and carnitine biosynthesis (19). 21. Ingebretsen, O. C., Terland, 0. & Flatmark, T. (1980) Biochim. Interestingly, pituitary levels of ascorbate are higher than lev- Biophys. Acta 628, 182-189. els of any other tissue (19, 20). High levels of ascorbate are as- 22. Milby, K. H., Mefford, I. N., Chey, W. & Adams, R. N. (1981) sociated with adrenal medullary chromaffin granules (21) and Brain Res. BulL 7, 237-242. Downloaded by guest on September 27, 2021