Role of Pyridoxal Phosphate in Mammalian Polyamine Biosynthesis LACK of REQUIREMENT for MAMMALIAN S-ADENOSYLMETHIONINE DECARBOXYLASE ACTIVITY by ANTHONY E

Biochem. J. (1977) 166, 81-88 81 Printed in Great Britain

Role of Pyridoxal Phosphate in Mammalian Polyamine Biosynthesis LACK OF REQUIREMENT FOR MAMMALIAN S-ADENOSYLMETHIONINE DECARBOXYLASE ACTIVITY By ANTHONY E. PEGG Department ofPhysiology and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, U.S.A. (Received 31 December 1976)

1. Polyamine concentrations were decreased in rats fed on a diet deficient in vitamin B-6. 2. Ornithine decarboxylase activity was decreased by vitamin B-6 deficiency when assayed in tissue extracts without addition of pyridoxal phosphate, but was greater than in control extracts when pyridoxal phosphate was present in saturating amounts. 3. In contrast, the activity of S-adenosylmethionine decarboxylase was not enhanced by pyridoxal phosphate addition even when dialysed extracts were prepared from tissues of young rats suckled by mothers fed on the vitamin B-6-deficient diet. 4. S-Adenosylmethionine decarboxylase activities were increased by administration of methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidine)dinitrilo]diguanidine} to similar extents in both control and vitamin B-6-deficient animals. 5. The spectrum of highly purified liver S-adenosylmethionine decarboxylase did not indicate the presence of pyridoxal phosphate. After inactivation of the enzyme by reaction with NaB3H4, radioactivity was incorporated into the enzyme, but was not present as a reduced derivative of pyridoxal phosphate. 6. It is concluded that the decreased concentrations of polyamines in rats fed on a diet containing vitamin B-6 may be due to decreased activity of ornithine decarboxylase or may be caused by an unknown mechanism responding to growth retardation produced by the vitamin deficiency. In either case, measurements of S-adenosylmethionine decarboxylase and ornithine decarboxylase activity under optimum conditions in vitro do not correlate with the polyamine concentrations in vivo.

Polyamine biosynthesis in mammalian cells is DNA synthesis (Kay & Pegg, 1973; Otani et al., thought to be controlled by the activities of two key 1974; Fillingame et al., 1975; Relyea & Rando, 1975; enzymes, L-ornithine decarboxylase (EC 4.1.1.17) Mamont et al., 1976; Poso & Janne, 1976; Williams- and S-adenosyl-L-methionine decarboxylase (EC Ashman et al., 1976). It is possible that an important 4.1.1.50). Many growth-promoting stimuli have been facet of growth retardation by vitamin B-6 deficiency shown to increase cellular polyamine concentrations, might be due to inability to synthesize polyamines and this increase is brought about by increased because of decreased availability of an essential activities of these decarboxylases (Morris & Fillin- cofactor for L-ornithine decarboxylase and/or game, 1974; Raina & Janne, 1975; Tabor & Tabor, S-adenosyl-L-methionine decarboxylase. 1976). Evidence obtained by using specific antisera Mammalian L-ornithine decarboxylase has been and making direct measurements of the amount of purified in a number of laboratories (Raina & enzyme protein indicates that these increases in Janne, 1975; Tabor & Tabor, 1972, 1976), and there is activity are due to increased amounts of enzyme general agreement with the original observations protein (Pegg, 1974; Holtta, 1975; Theoharides & based on studies of the enzyme from rat prostate Canellakis, 1976; A. E. Pegg, unpublished work). (Pegg & Williams-Ashman, 1968; Pegg, 1970) that Although the exact function of polyamines within the this enzyme has a rather loosely bound pyridoxal cell is not yet well understood, there is compelling phosphate cofactor. There is controversy, however, evidence that these basic molecules play an essential about the prosthetic group of mammalian S- role in cellular physiology (Tabor & Tabor, 1972, adenosylmethionine decarboxylase. Early experi- 1976; Raina & Janne, 1975). Studies with inhibitors ments indicated that activity was lost on addition of of polyamine formation have indicated that the 4-bromo-3-hydroxybenzyloxyamine (NSD-1055) and enhanced concentrations of polyamines found in other reagents which react with carbonyl groups growing cells are essential for continued growth and (Pegg & Williams-Ashman, 1969; Pegg, 1970), but Vol. 166 82 A. E. PEGG

activity could not be restored by later addition of Highly purified S-adenosylmethionine decarboxyl- pyridoxal phosphate. Subsequently, two separate ase was purified from the livers of rats treated 24h groups have claimed that rat liver S-adenosylmethion- before death with 80mg of methylglyoxal bis(guanyl- ine decarboxylase could be stimulated by addition of hydrazone) {1,1'-[(methylethanediylidine)dinitrilo]- pyridoxal phosphate (Feldman et al., 1972; Sturman diguanidine}/kg body wt. The drug was dissolved in & Kremzner, 1974a), but others were unable to obtain 0.9 % (w/v) NaCI at a concentration of 40mg/ml and such a stimulation (Williams-Ashman et al., 1972; administered to male rats weighing between 300 and Schmidt & Cantoni, 1973; Pegg, 1974; Hannonen, 400g by intraperitoneal injection. This treatment 1976). The present paper provides data on the effects increases the concentration of the S-adenosylmeth- of vitamin B-6 deficiency on polyamine concen- ionine decarboxylase in the liver up to 20-fold (Pegg trations and the activities of these decarboxylases. et al., 1973; H6ltta et al., 1973; Pegg, 1974) and thus Even under conditions designed to maximize the provides an enriched starting material for the possibility of exposing S-adenosylmethionine de- purification. The enzyme was then purified by carboxylase in an apoenzyme form, it was not precipitation with (NH4)2SO4, affinity chromato- possible to demonstrate a requirement for pyridoxal graphy on columns of methylglyoxal bis(guanyl- phosphate. Further evidence that pyridoxal phos- hydrazone) linked to Sepharose and chromatography phate is not involved in the action of mammalian on DEAE-cellulose as previously described (Pegg, S-adenosylmethionine decarboxylase was obtained 1974). The final fraction was then passed through a by studies of the absorption spectrum of the purified column (40cmx2.5cm) of Bio-Gel A 1.5m pre- enzyme and its inactivation by reagents reacting with viously equilibrated with 2.5mM-putrescine/lOmM- carbonyl groups. These studies suggest that the sodium phosphate (pH7.0)/1 mM-dithiothreitol/ mammalian enzyme may resemble the bacterial O.1mM-disodium EDTA, and 5ml fractions were equivalent in having a covalently bound cofactor collected. The fractions having the highest activity which contains a carbonyl group. However, poly- from the column eluate were concentrated to about amine concentrations were significantly decreased by 1 ml by using a pressurized ultrafiltration cell giving a vitamin B-6-deficient diet. The possible (Amicon Corp., Lexington, MA, U.S.A.). The final mechanisms of this decrease are discussed. preparation had a specific activity of5800-6000 units/ mg and was stored at -20°C in small lots to avoid repeated freezing and thawing, which rapidly Methods inactivated the enzyme. In the absence of putrescine activity was lost very rapidly, but, under the con- S-Adenosylmethionine decarboxylase and orni- ditions described above, activity was lost only at the thine decarboxylase activities were assayed by rate of about 10% per week. Before use, the enzyme determination of the release of "4CO2 from carboxyl- was freed from putrescine by passage through a '4C-labelled substrates as previously described (Pegg column (1 cmx 20cm) of Sephadex G-10 equilibrated & Williams-Ashman, 1969). For the measurement with the buffer required for the particular experiment of S-adenosylmethionine decarboxylase activity, as described below. the assay medium contained 50mM-sodium phosphate The spectrum of the purified enzyme dissolved in buffer, pH7.2, 1 mM-dithiothreitol, 0.2mM-S-adeno- 50mM-potassium phosphate, pH7.0, was deter- syl[carboxyl-14C]methionine (2-5mCi/mmol) and up mined with a Unicam SP.8000 spectrophotometer. to 2 units of enzyme protein in a total volume of For reaction with NaB3H4, 0.2mg of enzyme protein 0.3 ml. For ornithine decarboxylase assay the medium was dissolved in 1 ml of0.5M-NH4HCO3 and 0.1 ml of contained 50mM-sodium phosphate buffer, pH7.2, 0.1 M-NaOH containing 0.1M-NaB3H4 (204.8mCi/ 2.5mM-dithiothreitol, 0.2mM-pyridoxal phosphate, mmol) added. After 4h the enzyme was dialysed 2mM-DL-[1-14C]ornithine (1-5mCi/mmol) and up to against 5 x 2 litres of 0.05M-NH4HCO3 over a 24h 4 units ofenzyme in a total volume of0.3 ml. A unit of period. A small sample was precipitated by the enzyme was defined as that catalysing the decarboxyl- addition of 5% (w/v) HC104 to assay for incor- ation of 1 nmol of substrate during a 30min incu- poration of radioactivity into the protein, and the bation at 37°C. remainder was freeze-dried. A portion of the 3H- Tissue extracts for the assay of enzyme activity labelled enzyme was mixed with unchanged enzyme were prepared by homogenization in 3 vol. of 1OmM- in 0.1 M-potassium phosphate/2.5mM-dithiothreitol sodium phosphate (pH7.2)/2.5 mM-dithiothreitol/ (pH6.9) and layered over a linear 5-20% (w/v) suc- 0.1 mM-disodium EDTA. The homogenate was rose gradient made up in the same buffer solution. centrifuged for h at 105000g and the supernatant The gradient was centrifuged for 48h at 40000rev./ fraction used for assay. The protein present min in the SW 40 rotor of a Beckman model L2-50 was determined by the method of Lowry et al. ultracentrifuge. Fractions (0.5 ml) were collected and (1951), with crystalline bovine serum albumin as a assayed for radioactivity and for S-adenosylmeth- standard. ionine decarboxylase activity. 1977 PYRIDOXAL PHOSPHATE AND MAMMALIAN POLYAMINE BIOSYNTHESIS 83

Female Wistar-strain rats weighing 40-60 g at the a requirement for this cofactor for mammalian start of the experiment were fed on a pyridoxinc- S-adenosylmethionine decarboxylase, pregnant rats deficient diet (Nutritional Biochemicals Corp., were fed on a diet deficient in pyridoxine starting Cleveland, OH, U.S.A.) for 6 weeks. A similar from 1 week after conception and continuing until control group of rats received the same diet supple- 12 days after birth. The neonatal rats were therefore mented with pyridoxine hydrochloride supplied in deprived of vitamin B-6 both during embryonic the drinking water (50,cg/ml). Some of these rats development and during suckling. This procedure has were treated with methylglyoxal bis(guanylhydra- been used by others to study the role of pyridoxal zone) as described above. Developing rats were phosphate in the development of the nervous subjected to vitamin B-6 deficiency as described by system (Dakshinamurti & Stephens, 1969; Bayoumi Dakshinamurti & Stephens (1969). Female rats & Smith, 1972). Suckling rats from mothers fed on were placed on the vitamin B-6-deficient diet 1 week the vitamin B-6-deficient diet and from control after mating and the diet was continued until the mothers fed on the same diet supplemented with suckling rats were killed at 13 days of age. pyridoxine hydrochloride were killed 13 days after Pyridoxal phosphate concentrations were measured birth, and theactivities of ornithine decarboxylaseand by the ability to re-activate the apoenzyme of S-adenosylmethionine decarboxylase determined in bacterial tyrosine decarboxylase (Dakshinamurti & ultracentrifuged extracts from brain, liver and Stephens, 1969; Bayoumi & Smith, 1976). Spermine kidney. Results from these assays are shown in and spermidine were determined by butanol extrac- Table 1. When assayed in the presence of pyridoxal tion and separation by paper electrophoresis as phosphate, ornithine decarboxylase activity was previously described (Pegg et al., 1970). Putrescine significantly higher (up to threefold) in extracts from was determined by the ability to activate yeast the vitamin-deficient rats, but the reverse was the S-adenosylmethionine decarboxylase as described case when the assays were conducted in the absence by Harik et al. (1973). of pyridoxal phosphate (Table 1). Thus the stimu- DL-[1-14C]Ornithine (20-5OmCi/mmol) and S- lation by addition of pyridoxal phosphate was adenosyl-L-[carboxyl-'4C]methionine (25-56mCi/ 2-3-fold, depending on the tissue, for extracts from mmol) were purchased from New England Nuclear control rats, but was 12-20 fold for rats fed on the Corp., Boston, MA, U.S.A., and diluted to the vitamin-deficient diet. These results arc similar to appropriate specific radioactivity with unlabelled those found by others for other pyridoxal phosphate- material obtained from Sigma Chemical Co., St. dependent enzymes in extracts from vitamin B-6- Louis, MO, U.S.A. Methylglyoxal bis(guanylhydra- depleted rats and has been interpreted to indicate zone) was purchased from Aldrich Chemical Co., an increase in the amount of apoenzyme which may, Milwaukee, WI, U.S.A. at least in part, compensate for the decreased concentration of the cofactor (Dakshinamurti & Stephens, Results 1969; Bayoumi & Smith, 1972). The requirement for pyridoxal phosphate for To obtain a low tissue concentration of pyridoxal optimal activity of ornithine decarboxylase in these phosphate, which might result in the detection of crude tissue extracts is clear from the data in Table 1.

Table 1. S-Adenosylmethionine decarboxylase and ornithine decarboxylase activities in liver, kidney and brain extractsfrom pyridoxine-deficient developing rats Developing rats were exposed to pyridoxine deficiency by feeding a vitamin B-6-deficient diet to the mothers as described in the text. Enzyme assays were carried out as described in the Methods section. Pyridoxal phosphate, where present, was added at 0.2mM. Results are shown as means±s.E.M. for five separate determinations. Differences between the averages were tested for statistical significance by Student's t test: *P< 0.01 versus control; tP <0.05 versus control. S-Adenosylmethionine decarboxylase Ornithine decarboxylase activity activity (pmol of C02/30min per mg) (pmol of C02/30min per mg) Assayed-pyridoxal Assayed+pyridoxal Assayed-pyridoxal Assayed+pyridoxal Organ Diet phosphate phosphate phosphate phosphate Liver Vitamin B-6-deficient 108+9* 102 15t 6+1* 73+5* Control 160+12 155+21 16+3 45±4 Kidney Vitamin B-6-deficient 171±9 159+14 10±it 132+7* Control 172+12 164+ 8 18+4 45+3 Brain Vitamin B-6-deficient 127+9 115+9 7±2t 118+12* Control 134±18 114+12 15+2 42+9 Vol. 166 84 A. E. PEGG

In contrast, S-adenosylmethionine decarboxylase although there is a marked decrease in the activity was not enhanced by the addition of pyridoxal 5'-phosphate concentration in these pyridoxal phosphate to the assay medium with any of developing rats there is still a substantial amount theextracts from brain, liver or kidney tested, whether remaining, which cannot be decreased further, the tissues were obtained from control or vitamin because the rats do not survive beyond the second B-6-deprived rats (Table 1). Pyridoxal phosphate week (Bayoumi & Smith, 1972, 1976). Prolonged was added at 0.2mM in these experiments. Higher feeding of vitamin B-6-deficient diets to adult rats concentrations both inhibited the enzyme activity also does not produce decreases in tissue pyridoxal and increased the non-enzymic decarboxylation of phosphate concentrations much greater than this the substrate. No stimulation of enzymic decarb- (Sevigny et al., 1966; Lakshmi & Bamji, 1974). oxylation was achieved by addition of pyridoxal Thus an enzyme with a very high affinity for the phosphate even after dialysis of the tissue extracts pyridoxal phosphate cofactor might not be present for 24h against 2.5mM-putrescine/I mM-dithiothre- in the apoenzyme form even in the tissues of vitamin itol/50mM-potassium phosphate, pH7.0. Liver S- B-6-deprived animals. adenosylmethionine decarboxylase activity was some- To attempt to demonstrate the presence of such an what decreased in rats from the vitamin B-6-deficient apoenzyme of S-adenosylmethionine decarboxylase, group, but this effect was not abolished by the adult rats were fed on a vitamin B-6-deficient diet for addition of pyridoxal phosphate to the assay and 6 weeks and were then treated with the drug methyl- was not seen in extracts from kidney and brain. glyoxal bis(guanylhydrazone), which is known to (It should be emphasized that the results shown in cause a large increase in the amount of this enzyme Table 1 are expressed as enzyme activities per mg of present in mammalian cells (Pegg et al., 1973; protein present in the ultracentrifuged homogenates Fillingame & Morris, 1973; Holtta et al., 1973). and any changes observed are not due to changes It has been shown that the increased S-adenosyl- in the size of the organs from the vitamin-deficient methionine decarboxylase activity produced by this rats). drug is due to the stabilization of the enzyme against The data for ornithine decarboxylase given above normal intracellular degradation (Pegg et al., 1973; strongly suggest that pyridoxal phosphate concen- Pegg & Jefferson, 1974) and is associated with a trations were actually decreased in the extracts from marked increase in the enzyme protein present. In the rats exposed to the vitamin deficiency, and direct rat liver, a 10-20-fold increase in the amount of measurements of pyridoxal 5'-phosphate in these decarboxylase protein is produced within 24h (Pegg, organs indicated this was decreased by about 70%. 1974). The liver pyridoxal phosphate concentration (mean+ The effect ofgiving a diet lacking in vitamin B-6 on S.E.M. for the numbers of experiments in parentheses) the activity of liver S-adenosylmethionine decarb- was 9.3±1.9 (5)nmol/g wet wt. and that in the oxylase and on the degree of increase in hepatic and vitamin-deficient rats was 2.7 ±0.8 (4)nmol/g wet wt. renal decarboxylase produced by methylglyoxal These values are in reasonable agreement with those bis(guanylhydrazone) is shown in Table 2. Activity published by others (Sevigny et al., 1966; Lakshmi & was not decreased in rats fed on the vitamin B-6- Bamji, 1974; Bayoumi & Smith, 1976) and show that deficient diet, and on treatment with the drug,

Table 2. S-Adenosylmethionine decarboxylase activity in liver and kidney extracts from rats fed on a pyridoxine-deficient diet and treated with methylglyoxal bis(guanylhydraione) Rats were fed on a pyridoxine-deficient diet or the same diet supplemented with pyridoxine (control) as described in the Methods section. MGBG [methylglyoxal bis(guanylhydrazone); 80mg/kg], was given by intraperitoneal injection 24h before death. When added to the assay mixture, pyridoxal phosphate was present at 0.2nim. Results are shown as the mean±s.E.M. for five individual determinations in each group. S-Adenosylmethionine decarboxylase (nmol of C02/30min per mg) Tissue Diet Treatment Assayed-pyridoxal phosphate Assayed+pyridoxal phosphate Liver Control None 0.21 + 0.03 0.23 ± 0.04 MGBG 3.63+0.15 3.49±0.22 Vitamin B-6-deficient None 0.18+0.02 0.16+0.03 MGBG 3.71 + 0.27 3.65+0.09 Kidney Control None 0.24+ 0.02 0.22+ 0.05 MGBG 2.49+0.18 2.40+0.26 Vitamin B-6-deficient None 0.21+0.05 0.19+0.03 MGBG 2.63+0.21 2.39±0.30 1977 PYRIDOXAL PHOSPHATE AND MAMMALIAN POLYAMINE BIOSYNTHESIS 85

activity of the liver enzyme was increased some 20- under conditions giving a maximal rate of reaction, fold and that of the kidney enzyme was increased there was agreater amount ofornithine decarboxylase about 13-fold. There was no difference in the amount activity in hepatic extracts from the vitamin-deprived of increase produced by the drug in the control or animals (Table 3). Since it is difficult to evaluate how vitamin B-6-deficient animals. Also, addition of active the ornithine decarboxylase enzyme is within pyridoxal phosphate in amounts up to 0.2mm did not the cell, because of the ready dissociation of the produce any increase in activity of S-adenosyl- pyridoxal phosphate cofactor, it is not possible to methionine decarboxylase in any of these extracts determine to what extent the increased amount ofthe (Table 2). Pyridoxal phosphate in the livers of these apoenzyme compensates for the diminution in rats fell by 55% from 24.1±3.2 (4) to 13.3±1.8 (4) cofactor concentration. nmol/g wet wt. (means+s.E.M. for the numbers of Table 4 shows the concentrations ofputrescine and experiments in parentheses). It is therefore apparent polyamines in tissues from control and vitamin from these results that normal S-adenosylmethionine B-6-deficient rats. There are significant decreases in decarboxylase activity can be maintained and that the concentrations of putrescine and spermidine, greatly enhanced amounts of this enzyme can be but not of spermine, in liver, kidney and brain of induced even in the presence of significantly rats fed on the vitamin-deficient diet for 6 weeks. decreased pyridoxal phosphate. A decreased activity of ornithine decarboxylase As found in the newborns, the activity of ornithine could be responsible for the decrease in putrescine decarboxylase in the liver extracts from the adult and spermidine, since the putrescine produced by this rats fed on a vitamin B-6-deficient diet was stimulated enzyme is both the precursor of spermidine and also by addition of pyridoxal phosphate to a greater an activator of S-adenosylmethionine decarboxylase extent than that from control extracts. When assayed (Pegg & Williams-Ashman, 1969). As discussed below, the data given above are in conflict with reports of others that S-adenosylmethionine decarboxylase activity could be en- Table 3. Ornithine decarboxylase activity in hepatic hanced by the presence of pyridoxal phosphate. extracts from vitamin B-6-deficient rats Assays were carried out as described in Table 1. However, the following observations based on studies Results are shown as means±s.E.M. for five separate of the highly purified enzyme from rat liver support determinations. Pyridoxal phosphate was present at the suggestion that this decarboxylase does not have a 0.2mM when added. Statistical significance of the pyridoxal phosphate cofactor. The decarboxylase differences between the means was determined by was purified from the livers of rats treated with Student's t test. *P <0.01 versus control; tP<0.05 methylglyoxal bis(guanylhydrazone) by affinity versus control. chromatography as previously described (Pegg, Ornithine decarboxylase activity 1974). The final preparation had a specific activity (pmol of C02/30min per mg) of about 0.2,umol of CO2 liberated from S-adenosylmethionine/min per mg when assayed at pH7.2 in Assayed-pyridoxal Assayed+pyridoxal the presence of 2.5mM-putrescine and 0.2mM-S- Diet phosphate phosphate adenosylmethionine. This activity is considerably less Control 15+3 22+2 than that of homogeneous S-adenosylmethionine Vitamin B-6- 7+2t 65+8* decarboxylase from Escherichia coli, which has deficient diet Vmax. 1.4,umol of CO2 released/min per mg (Wickner

Table 4. Polyamine concentrations in liver, brain and kidney ofvitamin B-6-deficient rats Putrescine and polyamine concentrations were determined as described in the Methods section. The rats were treated as described in Table 2. Results are given in the mean+s.E.M. for five separate determinations. Statistical significance of the differences between the means were determined by Student's t test: *P <0.01 versus control; tP<0.05 versus control. Polyamine concentrations (nmol/g wet wt.) Tissue Diet Putrescine Spermidine Spermine Liver Control 21+3 817+ 58 708 + 15 Vitamin B-6-deficient 9+ 1* 565 + 34* 732 + 21 Kidney Control 44±5 335+ 23 675+ 37 Vitamin B-6-deficient 19+2* 198 +44t 585 + 21 Brain Control 15+3 496+ 37 385+19 Vitamin B-6-deficient 8± 2t 382+28t 395 + 23 Vol. 166 86 A. E. PEGG

0. A B L II 0 II .0 0.3 F 0i I8 0 1.8 A 0.2 06 -I 0. 1- .5.I

x A o. F 4 - 0 t Ov -.=

I 240 280 320 360 400 440 2 0

Wavelength (nm) 0 5 10 15 20 2!5 04 Fig. 1. Spectrum of S-adenosylmethionine decarboxylase Fraction no. Spectra were determined on a Unicam SP. 8000 Fig. 2. Sucrose gradient sedimentation of 3H- recording spectrophotometer at a protein concen- labelled S-adenosylmethionine decarboxylase tration of about 0.25mg/mil in 0.1 M-potassium Inactivated 3H-labelled S-adenosylmethionine de- phosphate buffer, pH7.0. carboxylase (25000d.p.m., about Igg) prepared by reaction with NaB3H4, was mixed with active enzyme (20 units, about 4pg) and layered on a 5-20% sucrose gradient as described in the Methods section. et al., 1970), but the purified mammalian enzyme After centrifugation for 48h at 40000rev./min in appeared to be homogeneous on electrophoresis on the SW40 rotor of a Beckman L-2 ultracentrifuge, polyacrylamide gels and on sedimentation-equili- fractions (0.5ml) were collected and assayed for brium analysis in the ultracentrifuge, with a mol.wt. radioactivity (s) and S-adenosylmethionine decarb- of about 69000 (A. E. Pegg, unpublished work). oxylase activity (0). The ratio of radioactivity to 1 activity is also shown (-) and the positions of marker Fig. shows the spectrum of the purified enzyme. proteins [E. coli alkaline phosphatase (A), bovine The enzyme has some at absorption wavelengths serum albumin (B) and lysozyme (L)] are indicated. greater than 310nm, but there is no sign of peaks of absorption characteristic of pyridoxal phosphate. As reported by several groups working with less- pure preparations (Pegg & Williams-Ashman, 1969; The relative proportions of these products differed Schmidt & Cantoni, 1973; Hannonen, 1975; Pegg & with the time of hydrolysis, but we were unable to Conover, 1976), the activity of the purified S-adeno- find conditions such that only a single product was sylmethionine decarboxylase was lost on exposure to obtained. However, none of the labelled products reagents reacting with carbonyl groups. After reac- corresponded to reduced derivatives of pyridoxal tion with NaB3H4 followed by extensive dialysis phosphate prepared and treated with acid in the same the inactivated enzyme contained radioactivity, way. indicating that a closely associated chemical residue had been reduced. To prove that this radioactivity Discussion was associated with the enzyme rather than with a The present experiments are in marked disagree- contaminating protein or with a non-diffusible ment with those published by others, who claimed impurity in the NaB3H4 (Wickner et al., 1971) the that S-adenosylmethionine decarboxylase activity labelled, inactivated enzyme was mixed with could be stimulated by the addition of pyridoxal unchanged S-adenosylmethionine decarboxylase and phosphate to the assay medium (Feldman et al., separated by centrifugation on a sucrose gradient. 1972; Sturman & Kremzner, 1974a). In the present As shown in Fig. 2 the fractions containing radio- experiments, activity of S-adenosylmethionine de- activity corresponded exactly to those having enzyme carboxylase was not stimulated by addition of activity. The amount of 3H incorporated into the pyridoxal phosphate even when extracts were enzyme corresponded to the reduction of one prepared from vitamin B-6-deficient animals. Also, carbonyl group per 60000 daltons of enzyme protein. extensive dialysis of these extracts for 24h against The inactivated enzyme could not be re-activated two changes of lOOvol. of buffer [2.5mM-putrescine/ by addition of pyridoxal phosphate, and the 2.5 mM-dithiothreitol / 10mma-sodium phosphate radioactivity associated with it could be released (pH 7.2)/0.1 mM-disodium EDTA] did not lead to only after acid hydrolysis of the enzyme protein. any significant loss of activity provided that We have not yet been able to identify the labelled putrescine was present throughout dialysis. The material; after heating the labelled protein in explanation for this discrepancy is unclear, but one constant-boiling HCI under reduced pressure at possible explanation is that, as discussed by 100C, several radioactive products were found. Williams-Ashman et al. (1972), pyridoxal phosphate 1977 PYRIDOXAL PHOSPHATE AND MAMMALIAN POLYAMINE BIOSYNTHESIS 87 can lead to decarboxylation ofS-adenosylmethionine the activity of this enzyme measured in the absence in a non-enzymic reaction. In the present studies this of additional pyridoxal phosphate was observed, non-enzymic decarboxylation (which was deducted whereas Sturman & Kremzner (1974a,b) found an fromthe measured activities) was small and represen- increase. This difference could result from the ted less than 5 % of the activity found in the presence different homogenization conditions or from a of the enzyme, but in the presence of certain metal different degree of pyridoxal phosphate depletion. ions much greater rates ofnon-enzymic reaction have A similar increase in amount of apoenzyme during been noted (Coppoc et al., 1971; Williams-Ashman pyridoxine deficiency has been noted for glutamate et al., 1972). The conclusion that S-adenosylmethion- decarboxylase (Bayoumi & Smith, 1976; Morris & ine decarboxylase can be fully active in rats fed on a Fillingame, 1974) and for tyrosine transaminase diet deficient in vitamin B-6, although directly (Tryfiates & Morris, 1974). This suggests that the cell contradictory to the results of Sturman & Kremzner may try to compensate for the decreased activity of (1974a), has been independently reported by these key enzymes in pyridoxal phosphate deficiency Hannonen (1976) and by Eloranta et al. (1976). by increasing the concentrations of the enzyme pro- The findings reported here, (a) that S-adenosyl- tein so that they would compete more effectively for methionine decarboxylase activity is not decreased the available cofactor. It has been proposed that in developing or adult rats fed with a vitamin the very rapid turnover of ornithine decarboxylase B-6-deficient diet, (I) that S-adenosylmethionine is due to the fact that its pyridoxal phosphate cofactor decarboxylase activity can be greatly increased by is very loosely bound and that it is degraded in the treatment with methylglyoxal bis(guanylhydrazone) apoenzyme form (Litwack & Rosenfield, 1973). in these vitamin B-6-deprived animals, (c) that activity However, if this was the only factor controlling was not lost on extensive dialysis or increased by ornithine decarboxylase concentrations in the vitamin the addition of pyridoxal phosphate, and (d) that B-6-deficient rats, the ornithine decarboxylase acti- the spectrum of the purified enzyme does not vity would be expected to decrease rather than indicate the presence ofpyridoxal phosphate, provide increase as found in the present study. convincing evidence that pyridoxal phosphate is Finally, thefindingsthatspermidineandputresceine not a cofactor of this enzyme. It appears that this is concentrations are diminished in the tissues of rats the first mammalian amino acid decarboxylase that fed on a pyridoxine-deficient diet [which also con- does not have this cofactor. A number of bacterial firms the data of Sturman & Kremzner 1974a,b) enzymes concerned with amino acid metabolism and of Eloranta et al. (1976)] may indicate that the have bound carbonyl groups as part of cofactors effective ornithine decarboxylase activity in vivo is which are not pyridoxal phosphate. These include decreased owing to the decreased amounts of the histidine decarboxylase (Riley & Snell, 1968), cofactor. This would imply that ornithine decarb- S-adenosylmethionine decarboxylase (Wickner et al., oxylase activity in vivo under these conditions forms 1970) and proline reductase (Hodgkins & Abeles, the rate-limiting step in polyamine synthesis. This 1967), which have covalently bound pyruvate, and assumption has frequently been made (see Raina & serine/threonine dehydratase and urocanase (George Janne, 1975; Tabor & Tabor, 1976), but it should be & Phillips, 1970), which have a-oxobutyrate. emphasized that polyamine concentrations may be The L-serine/threonine dehydratase from sheep liver regulated by modifications both of the rate of has also been shown to contain a-oxobutyrate synthesis and of breakdown. Further, it is clear that rather than pyridoxal phosphate (Kapke & Davis, putrescine concentrations play a key role both in 1975). In the present experiments a small proportion regulating the rate of spermidine synthesis through of the radioactivity derived from the mammalian precursor availability and activation of S-adenosyl- S-adenosylmethionine decarboxylase after reduction methionine decarboxylase and incontrolling ornithine with NaB3H4 and acid hydrolysis co-chromato- decarboxylase activity by a mechanism which is graphed with lactate in several solvent systems not yet well understood (Raina & Janne, 1975; (Hodgkins & Abeles, 1967; Wickner et al., 1970), Fong et al., 1976). It is possible therefore that a suggesting that the mammalian enzyme may resemble decrease in the polyamine concentrations is brought that from E. coli in having a bound pyruvate group. about by the growth retardation produced by the However, until the other labelled products are pyridoxine-deficient diet rather than by a specific identified this question cannot be settled. decrease in the rate of synthesis of polyamines owing In contrast with the findings on S-adenosylmeth- to a deficiency of pyridoxal phosphate. However, ionine decarboxylase, the reports of Sturman & whatever the mechanism by which putrescine and Kremzner (1974a,b) that ornithine decarboxylase spermidine concentrations are decreased, it is clear apoenzymes appeared to be present in increased that the ornithine decarboxylase and S-adenosyl- amounts in pyridoxal phosphate-depleted rats were methionine decarboxylase activities measured under confirmed both in young adult rats and in newborns. optimal conditions in vitro do not correlate with However, in the present experiments a decrease in polyamine concentrations in vivo, Vol. 166 88 A. E. PEGG

Note Added in Proof (Received 16 May 1977) Mamont, P. S., Bohlen, P., McCann, P. P., Bey, P., Schuber, F. & Tardie, C. (1976) Proc. Natl. Acad. Sci. After this paper was submitted for publication, it U.S.A. 73, 1626-1630 was reported that yeast S-adenosylmethionine de- Morris, D. R. & Fillingame, R. H. (1974) Annu. Rev. carboxylase lacks pyridoxal phosphate and contains Biochem. 43, 303-325 a bound pyruvate group (Cohn et al., 1977). Otani, S., Mizogucki, Y., Matsui, I. & Morisawa, S. (1974) Mol. Biol. Rep. 1, 431-436 Pegg, A. E. (1970) Ann. N.Y. Acad. Sci. 171, 977-987 This work was supported by grant CA18138 from the Pegg, A. E. (1974) Biochem. J. 141, 581-583 National Cancer Institute, DHEW, and by an Established Pegg, A. E. & Conover, C. (1976) Biochem. Biophys. Res. Investigatorship from the American Heart Association. Commun. 69, 766-774 Pegg, A. E. & Jefferson, L. S. (1974) FEBS Lett. 40, References 321-324 Pegg, A. E. & Williams-Ashman, H. G. (1968) Biochem. J. Bayoumi, R. A. & Smith, W. R. 0. (1972) J. Neurochem. 108, 533-539 19, 1883-1897 Pegg, A. E. & Williams-Ashman, H. G. (1969) J. Biol. Bayoumi, R. A. & Smnith, W. R. 0. (1976) J. Neurochem. Chem. 244, 682-693 26, 405-407 Pegg, A. E., Lockwood, D. H. & Williams-Ashman, Cohn, M. S., Tabor, C. W. & Tabor, H. (1977) Fed. H. G. (1970) Biochem. J. 117, 17-31 Proc. Fed. Am. Soc. Exp. Biol. 36, 714 Pegg, A. E., Corti, A. & Williams-Ashman, H. G. (1973) Coppoc, G. L., Kallio, P. & Williams-Ashman, H. G. Biochem. Biophys. Res. Commun. 52, 696-701 (1971) Int. J. Biochem. 2, 673-681 Poso, H. & Janne, J. (1976) Biochem. J. 158,485-488 Dakshinamurti, K. & Stephens, M. C. (1969) J. Raina, A. & Janne, J. (1975) Med. Biol. 53, 121-147 Neurochem. 16, 1515-1522 Relyea, N. & Rando, R. R. (1975) Biochem. Biophys. Res. Eloranta, T. O., Kajander, E. 0. & Raina, A. M. (1976) Commun. 67, 392-402 Biochem. J. 160, 287-294 Riley, W. D. & Snell, E. E. (1968) Biochemistry 7, Feldman, M. J., Levy, C. C. & Russell, D. H. (1972) 3520-3528 Biochemistry 11, 671-677 Schmidt, G. L. & Cantoni, G. L. (1973) J. Neurochem. Fillingame, R. H. & Morris, D. R. (1973) Biochem. 20, 1373-1385 Biophys. Res. Commun. 52, 1020-1025 Sevigny, S. J., White, S. L., Halsey, M. L. & Johnston, Fillingame, R. H., Jorstad, C. M. & Morris, D. R. (1975) F. A. (1966) J. Nutr. 88, 50-65 Proc. Natl. Acad. Sci. U.S.A. 72, 4042-4045 Sturman, J. A. & Kremzner, L. T. (1974a) Biochim. Fong, W. F., Heller, J. S. & Canellakis, E. S. (1976) Biophys. Acta 372, 162-170 Biochim. Biophys. Acta 428, 456-465 Sturman, J. & Kremzner, L. T. (1974b) Life Sci. 14, George, D. J. & Phillips, A. T. (1970) J. Biol. Chem. 245, 977-982 528-537 Tabor, C. W. & Tabor, H. (1976) Annu. Rev. Biochem. 45, Hannonen, P. (1975) Acta Chem. Scand. Ser. B29, 295-299 285-306 Hannonen, P. (1976) Acta Chem. Scand. Ser. B30,121-124 Tabor, H. & Tabor, C. W. (1972) Adv. Enzymol. Relat. Harik, S. I., Pasternak, G. W. & Snyder, S. M. (1973) Areas Mol. Biol. 36, 203-268 Biochim. Biophys. Acta 304, 753-764 Hodgkins, D. & Abeles, R. H. (1967) J. Biol. Chem. 242, Theoharides, T. C. & Canellakis, Z. N. (1976) J. Biol. 5158-5159 Chem. 251, 1781-1784 Hoitta, E. (1975) Biochim. Biophys. Acta 399, 420-427 Tryfiates, G. P. & Morris, H. P. (1974) J. Natl. Cancer Holtta, E., Hannonen, P., Pispa, J. & Janne, J. (1973) Inst. 52, 1259-1263 Biochem. J. 136, 669-676 Wickner, R. B., Tabor, C. W. & Tabor, H. (1970) J. Biol. Kapke, G. & Davis, L. (1975) Biochemistry 14, 4273-4276 Chem. 245, 2132-2139 Kay, J. E. & Pegg, A. E. (1973) FEBSLett. 29, 301-304 Wickner, R. B., Tabor, C. W. & Tabor, H. (1971) Lakshmi, A. V. & Bamji, M. S. (1974) Br. J. Nutr. 32, Methods Enzymol. 17, 647-651 249-256 Williams-Ashman, H. G., Janne, J., Coppoc, G. L., Litwack, G. & Rosenfield, S. (1973) Biochem. Biophys. Geroch, M. E. & Schenone, A. (1972) Adv. Enzyme Res. Commun. 52, 181-188 Regul. 10, 225-245 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, Williams-Ashman, H. G., Corti, A. & Tadolini, B. (1976) R. J. (1951) J. Biol. Chem. 193, 265-275 Ital. J. Biochem. 25, 5-32

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