Effect of methylglyoxal bis(guanylhydrazone) on polyamine metabolism in normal and regenerating rat liver and rat thymus

1. Injections of sublethal doses of methylglyoxal bis(guanylhydrazone), a potent inhibitor of putrescine-activated S-adenosylmethionine decarboxylase in vitro, resulted after a few days in an immense increase in the activity of S-adenosylmethionine decarboxylase in normal and regenerating rat liver and in rat thymus. The increase in the activity of S-adenosylmethionine decarboxylase was at least partly due to a marked lengthening of the half-life of the enzyme. 2. In regenerating liver and thymus there was also a moderate stimulation of the activity of ornithine decarboxylase (EC 4.1.1.17) and a marked accumulation of tissue putrescine. 3. Injection of methylglyoxal bis(guanylhydrazone) into the rat also markedly decreased the activity of diamine oxidase (EC 1.4.3.6) in thymus. 4. In no cases where doses of methylglyoxal bis(guanylhydrazone) close to the LD(50) dose for the rat were used was it possible to lower tissue spermidine content to any significant extent. 5. Methylglyoxal bis(guanylhydrazone) seemed to act as a competitive inhibitor for the substrate S-adenosylmethionine and as an uncompetitive inhibitor for the activator putrescine in the decarboxylation of S-adenosylmethionine in vitro. 6. In the diamine oxidase reaction, with putrescine as the substrate, methylglyoxal bis(guanylhydrazone) was a non-competitive inhibitor for putrescine.     

Ascaris suum and Onchocerca volvulus: S-adenosylmethionine decarboxylase

Putrescine-dependent S-adenosylmethionine decarboxylase (EC 4.1.1.50) was demonstrated in Ascaris suum and Onchocerca volvulus; activation was found to be about fourfold by putrescine. Mg2+ did not affect the enzyme activity. A. suum was taken as a model nematode and its S-adenosylmethionine decarboxylase was partially purified and characterized. The molecular weight was estimated to be 220,000. The apparent Km-value for adenosylmethionine was determined to be 17 microM. Methylglyoxal bis(guanylhydrazone) and berenil competitively inhibited the enzyme activity; the apparent Ki-values were found to be 0.24 microM and 0.11 microM, respectively. The dependence of filarial worms on uptake and interconversion of putrescine and polyamines as well as properties of the S-adenosylmethionine decarboxylase, different from the host enzyme, points to the polyamine metabolisms as a useful target for chemotherapy.     

Properties of S-adenosylmethionine decarboxylase from rabbit liver: effects of some hydrazone compounds on its activity

S-Adenosylmethionine decarboxylase (EC 4.1.1.50) has been partially purified from rabbit liver by ammonium sulphate fractionation and gel filtration and anion exchange chromatographies. Sodium dodecylsulphate-polyacrylamide disc gel electrophoresis analysis showed an approximate dimeric subunit mol. wt of 34,000. The enzyme showed a pH optimum at 7.5 (in phosphate buffer) and did not require bivalent cations for catalysis. The enzyme showed sigmoid kinetics to S-adenosylmethionine with a Hill coefficient of 1.7, which became michaelian with Km 70 microM in the presence of 2.5 mM putrescine. Methylglyoxal bis(guanylhydrazone) was an effective inhibitor of the enzyme, but phenylated derivatives of this compound as phenylglyoxal bis(guanylhydrazone) and diphenylglyoxal bis-(guanylhydrazone) inhibited less well.     

Differential effects of 2-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) on the testosterone-induced growth of ventral prostate and seminal vesicles of castrated rats

2-Difluoromethylornithine totally prevented any increases in putrescine and spermidine concentrations in the ventral prostate of castrated rats during a 6-day testosterone treatment. Prostatic ornithine decarboxylase activity was inhibited by 80%, whereas S-adenosylmethionine decarboxylase was stimulated by more than 9-fold. In seminal vesicle, the inhibition of putrescine and spermidine accumulation, as well as of ornithine decarboxylase activity, was only minimal, and no stimulation of S-adenosylmethionine decarboxylase was observed. Administration of methylglyoxal bis(guanylhydrazone) to castrated androgen-treated rats resulted in a marked increase in concentrations of all prostatic polyamines. Prostatic ornithine decarboxylase activity was nearly 2 times and adenosylmethionine decarboxylase activity 9 times higher than that of the testosterone-treated animals. In contrast with ventral prostate, methylglyoxal bis(guanylhydrazone) treatment inhibited moderately the accumulation of spermidine and spermine in seminal vesicle, although both ornithine decarboxylase and S-adenosylmethionine decarboxylase activities were stimulated. Difluoromethylornithine inhibited significantly the weight gain of ventral prostate, but methylglyoxal bis(guanylhydrazone) produced a substantial increase in prostatic weight. These changes were largely due to the fact that the volume of prostatic secretion was greatly decreased by difluoromethylornithine, whereas methylglyoxal bis(guanylhydrazone) increased the amount of secretion. Treatment with difluoromethylornithine strikingly increased the methylglyoxal bis(guanylhydrazone) content of both ventral prostate and seminal vesicle, but even under these conditions the drug concentration remained low in comparison with other tissues. The results indicate that a combined use of these two polyamine anti-metabolites does not necessarily result in a synergistic growth inhibition of the androgen-induced growth of male accessory sexual glands.     

Regulation of polyamine biosynthesis in tobacco. Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase

Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.     

Putrescine-sensitive (artifactual) and insensitive (biosynthetic) S-adenosyl-L-methionine decarboxylase activities of Lathyrus sativus seedlings.

The crude extracts of 3-day-old etiolated seedlings of Lathyrus sativus contained two S-adenosyl-L-methionine decarboxylase activities. The artifactual putrescine-dependent activity was due to the H2O2 generated by diamine oxidase (EC 1.4.3.6) of this plant system and was inhibited by catalase. This observation was confirmed by using an electrophoretically and immunologically homogeneous preparation of L. sativus diamine oxidase. In the presence of putrescine, diamine oxidase, in addition to S-adenosylmethionine, decarboxylated L-lysine, L-arginine, L-ornithine, L-methionine and L-glutamic acid to varying degrees. The decarboxylation was not metal-ion dependent. The biosynthetic S-adenosylmethionine decarboxylase (EC 4.1.1.21) was detected after removing diamine oxidase specifically from the crude extracts by employing an immunoaffinity column. This Mg2+-dependent decarboxylase was not stimulated by putrescine or inhibited by catalase. The enzyme activity was inhibited by semicarbazide, 4-bromo-3-hydroxybenzoylamine dihydrogen phosphate and methylglyoxal-bis (guanylhydrazone). It was largely localized in the shoots of the etiolated seedlings and was purified 40-fold by employing a p-hydroxymercuribenzoate/AH-Sepharose affinity column, which also separated the decarboxylase activity from spermidine synthase.     

Specific inhibition of the enzymic decarboxylation of S-adenosylmethionine by methylglyoxal bis(guanylhydrazone) and related substances

Methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidene)-dinitrilo]diguanidine} is a very potent inhibitor of putrescine-activated S-adenosylmethionine decarboxylases from many different mammalian tissues, including sublines of mouse L1210 leukaemia that are resistant to the drug as well as sublines that are sensitive. The inhibition of purified rat ventral prostate S-adenosylmethionine decarboxylase is competitive with respect to the S-adenosylmethionine substrate, and is much greater in the presence than in the absence of the activator putrescine. Inhibition by the drug depends, among other things, on the nature of the aliphatic amines that can serve as stimulators of rat prostate S-adenosylmethionine decarboxylase. Effects of some congeners of methylglyoxal bis(guanylhydrazone) on the enzyme are described.     

Insensitivity of cardiac phosphofructokinase to adrenergic activation in Zucker rats. A post-receptor defect.

Glyoxal bis(guanylhydrazone), the parent compound of methylglyoxal bis(guanylhydrazone), was synthesized and tested for its ability to inhibit the biosynthesis of polyamines. It was found to be a powerful competitive inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), yet the lack of the methyl group at the glyoxal portion increased the apparent Ki value for the enzyme by about 30-fold in comparison with methylglyoxal bis(guanylhydrazone). Glyoxal bis(guanylhydrazone) inhibited diamine oxidase (EC 1.4.3.6) activity as effectively as did methylglyoxal bis(guanylhydrazone). The cellular accumulation curves of glyoxal bis(guanylhydrazone) in L1210 cells were practically superimposable with those of methylglyoxal bis(guanylhydrazone), and the uptake of both compounds was distinctly stimulated by a prior treatment with 2-difluoromethylornithine. The drug decreased the concentration of spermidine in a dose-dependent manner and, in contrast with methylglyoxal bis(guanylhydrazone), without a concomitant accumulation of putrescine. The fact that putrescine concentrations were decreased in cells exposed to glyoxal bis(guanylhydrazone) was, at least in part, attributable to an inhibition of ornithine decarboxylase (EC 4.1.1.17) activity in cells treated with the compound. Under these experimental conditions equivalent concentrations of methylglyoxal bis(guanylhydrazone) [1,1'-[(methylethanediylidine)dinitrilo]diguanidine] elicited large increases in the enzyme activity. When combined with difluoromethylornithine, glyoxal bis(guanylhydrazone) potentiated the growth-inhibitory effect of that drug. Taking into consideration the proven anti-leukaemic activity of glyoxal bis(guanylhydrazone), its effectiveness to inhibit spermidine biosynthesis (without raising the concentration of putrescine) as well as its suitability for combined use with inhibitors of ornithine decarboxylase, this drug is apparently worthy of further testing in tumour-bearing animals, especially in combination with difluoromethylornithine or related inhibitors of ornithine decarboxylase.     

Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylmethionine decarboxylase activity in vitro. The enzyme from both sources was most powerfully inhibited by ethylglyoxal bis(guanylhydrazone). All the diguanidines likewise inhibited diamine oxidase activity in vitro. The maximum intracellular concentrations of the ethyl and dimethylated analogues achieved in activated lymphocytes were only about one-fifth of that of the parent compound. However, both derivatives appeared to utilize the polyamine-carrier system, as indicated by competition experiments with spermidine.     

Biochemical characteristics ofS-adenosylmethionine decarboxylase from carnation (Dianthus caryophyllus L.) petals

We have partially purified S-adenosylmethionine decarboxylase (EC 4.1.1.50, SAMDC) from carnation (Dianthus caryophyllus L.) petals and generated polyclonal antibodies against CSDC 16 protein (Leeet al., 1996) overexpressed inE. coli. The protein has been purified approximately 126.8 fold through the steps involving ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephacryl S-300 gel filtration. Its molecular mass was 42 kDa in native form and we could also detect a band of 32 kDa molecular mass on SDS-PAGE in western blot analysis using the polyclonal antibodies. The Km value of this enzyme forS-adenosylmethionine was 26.3 μM. The optimum temperature and pH forS-adenosylmethionine decarboxylase activity were 35°C and pH 8.0, respectively. Putrescine and Mg²⁺ had no effects on the activation of the enzyme activity. Mg²⁺ did not have any significant effects on the enzyme activity. SAMDC activity was inhibited by putrescine, spermidine and spermine. Methylglyoxal bis-(guanylhydrazone) (MGBG), carbonyl reagents such as hydroxylamine and phenylhydrazine, and sulfhydryl reagent such as 5,5′dithio-bis (2-nitrobenzoic acid) (DTNB) were effective inhibitors of the enzyme. However, isonicotinic acid hydrazide known as an inhibitor of 5′-pyridoxal phosphate (PLP) dependent enzyme activity had no significant effect on the enzyme activity. These results and our previously reported results (Leeet al., 1997b) suggest thatS-adenosylmethionine decarboxylase is a heterodimer, αβ, and some carbonyl group and sulfhydryl group are involved in the catalytic activity.     

Regulation of S-adenosylmethionine decarboxylase activity in rat liver and prostate

Two methods were used for the quantitation of S-adenosylmethionine decarboxylase protein. The first involved titrating the active site of the enzyme by reduction of the Schiff base between 3H-decarboxylated S-adenosylmethionine and the pyruvate prosthetic group with sodium cyanoborohydride. The second method was radioimmunoassay with rabbit antiserum which was used to determine the total immunoreactive enzyme protein. It was found that the increased S-adenosylmethionine decarboxylase activity produced in rat prostate by treatment with alpha-difluoromethylornithine and in both prostate and liver by methylglyoxal bis(guanylhydrazone) were due entirely to increases in the amount of enzyme protein. The ratio of enzyme activity to protein (measured by either method) remained constant in rats treated with the drugs. Treatment with 2% alpha-difluoromethylornithine in the drinking water for 3 days increased prostatic S-adenosylmethionine decarboxylase protein by 5-fold. A substantial part, but not all, of this increase could be accounted for by a slowing of the rate of degradation of the enzyme. The half-life for loss of activity and titratable protein after inhibition of protein synthesis by cycloheximide was increased from 35 to 108 min by treatment with alpha-difluoromethylornithine. However, the half-life for loss of immunoreactive protein which was considerably longer was only increased from 139 to 213 min. The molecular weight of the S-adenosylmethionine decarboxylase subunit determined by immunoblotting was 32,000, and no smaller immunoreactive fragments were detected. These results indicate that spermidine depletion produced by alpha-difluoromethylornithine affects the degradation of S-adenosylmethionine decarboxylase at an early step involving the loss of the active site without substantial breakdown of the protein.     

Regulation of DNA synthesis and cell division by polyamines in Catharanthus roseus suspension cultures

Summary Various inhibitors of polyamine biosynthesis were used to study the role of polyamines in DNA synthesis and cell division in suspension cultures of Catharanthus roseus (L.) G. Don. Arginine decarboxylase (ADC; EC 4.1.1.19) was the major enzyme responsible for putrescine production. DL -difluoromethylarginine inhibited ADC activity, cellular putrescine content, DNA synthesis, and cell division. The effect was reversible by exogenous putrescine. Ornithine decarboxylase (ODC; EC 4.1.1.17) activity was always less than 10% of the ADC activity. Addition of DL -difluoromethylornithine had no effect on ODC activity, cellular polyamine levels, DNA synthesis, and cell division within the first 24 h but by 48 to 72 h it did inhibit these activities. Methylglyoxal bis(guanyl-hydrazone) inhibited S-adenosylmethionine decarboxylase (EC 4.1.1.50) activity without affecting DNA synthesis and cell division.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - SAMDC S-adenosylmethionine decarboxylase - DFMA DL -difluoro-methylarginine - DFMO DL -difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone)     

Putrescine activated S-adenosylmethionine decarboxylase from Trypanosoma brucei brucei

Trypanosoma brucei brucei contained a S-adenosyl-L-methionine decarboxylase (AdoMetDC) strongly activated by putrescine. The enzyme was also activated to a lesser extent by cadaverine and 1,3-diaminopropane. Spermidine and spermine had no effect on basal activity of the enzyme. However, they interfered with putrescine activation of trypanosomal AdoMetDC. The trypanosomal enzyme could not be precipitated with antiserum against human AdoMetDC. The trypanosomal AdoMetDC enzyme subunit was labeled by reaction with ³⁵S-decarboxylated AdoMet in the presence of NaCNBH₄, and found to have a molecular weight of 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit was readily degraded on storage to a form with a molecular weight of 26 kDa. The specificity of labeling of AdoMetDC by this procedure was confirmed by the prevention of ³⁵S-decarboxylated S-adenosylmethionine (AdoMet) binding in the presence of specific AdoMetDC inhibitors [either methylglyoxal bis(guanylhydrazone (MGBG), a reversible inhibitor, or 5-deoxy-5-[(2-hydrazinoethyl)methylamino]adenosine (MHZEA), an irreversible inactivator]. As compared to human AdoMetDC, the trypanosomal enzyme showed weaker binding to a column of MGBG-Sepharose and also was significantly less sensitive to inhibition by MGBG and its congener ethylglyoxal bis(guanylhydrazone) (EGBG). Thus, the trypanosomal AdoMetDC differs significantly from its mammalian and bacterial counterparts and may therefore be exploited as a specific target for chemotherapy of trypanosomiasis.     

S-adenosylmethionine decarboxylase from baker''s yeast.

1. S-Adenosyl-L-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) was purified more than 1100-fold from extracts of Saccharomyces cerevisiae by affinity chromatography on columns of Sepharose containing covalently bound methylglyoxal bis(guanylhydrazone) (1,1'[(methylethanediylidene)dinitrilo]diguanidine) [Pegg, (1974) Biochem J. 141, 581-583]. The final preparation appeared to be homogeneous on polyacrylamide-gel electrophoresis at pH 8.4. 2. S-Adenosylmethionine decarboxylase activity was completely separated from spermidine synthase activity [5'-deoxyadenosyl-(5'),3-aminopropyl-(1),methylsulphonium-salt-putrescine 3-aminopropyltransferase, EC 2.5.1.16] during the purification procedure. 3. Adenosylmethionine decarboxylase activity from crude extracts of baker's yeast was stimulated by putrescine, 1,3-diamino-propane, cadaverine (1,5-diaminopentane) and spermidine; however, the purified enzyme, although still stimulated by the diamines, was completely insensitive to spermidine. 4. Adenosylmethionine decarboxylase has an apparent Km value of 0.09 mM for adenosylmethionine in the presence of saturating concentrations of putrescine. The omission of putrescine resulted in a five-fold increase in the apparent Km value for adenosylmethionine. 5. The apparent Ka value for putrescine, as the activator of the reaction, was 0.012 mM. 6. Methylglyoxal bis(guanylhydrazone) and S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine) were powerful inhibitors of the enzyme. 7. Adenosylmethionine decarboxylase from baker's yeast was inhibited by a number of conventional carbonyl reagents, but in no case could the inhibition be reversed with exogenous pyridoxal 5'-phosphate.     

Detection of proenzyme form of S-adenosylmethionine decarboxylase in extracts from rat prostate

Previous work in which the synthesis of S-adenosylmethionine decarboxylase was studied by translation of its mRNA indicated that it was formed as a proenzyme having a M.W. of about 37,000 that was cleaved to form the enzyme sub-unit of M.W. 32,000 in a putrescine-stimulated reaction. The extent to which the proenzyme accumulates in vivo and is affected by the putrescine concentration was studied by subjecting prostate extracts to Western immunoblotting procedures. The proenzyme form was readily detectable in control prostates (about 4% of the total) and this proportion was increased to 25% when the rats were pretreated for 3 days with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. Conversely, it was decreased to almost undetectable levels after treatment with methylglyoxal bis(guanylhydrazone). These results indicate that the processing of the proenzyme form of S-adenosylmethionine decarboxylase is regulated by the cellular putrescine concentration. This conversion provides another step at which polyamine biosynthesis may be controlled.     

The influence of nerve section on the metabolism of polyamines in rat diaphragm muscle.

Concentrations of spermidine, spermine and putrescine have been measured in rat diaphragm muscle after unilateral nerve section. The concentration of putrescine increased approx. 10-fold 2 days after nerve section, that of spermidine about 3-fold by day 3, whereas an increase in the concentration of spermine was only observed after 7-10 days. It was not possible to show enhanced uptake of either exogenous putrescine or spermidine by the isolated tissue during the hypertrophy. Consistent with the accumulation of putrescine, activity of ornithine decarboxylase increased within 1 day of nerve section, was maximally elevated by the second day and then declined. Synthesis of spermidine from [14C]putrescine and either methionine or S-adenosylmethionine bt diaphragm cytosol rose within 1 day of nerve section, but by day 3 had returned to normal or below normal values. Activity of adenosylmethionine decarboxylase similarly increased within 1 day of nerve section, but by day 3 had declined to below normal values. Activity of methionine adenosyltransferase was elevated throughout the period studied. The concentration of S-adenosylmethionine was likewise enhanced during hypertrophy. Administration of methylglyoxal bis(guanylhydrazone) produced a marked increase in adenosylmethionine decarboxylase activity and a large increase in putrescine concentration, but did not prevent the rise in spermidine concentration produced by denervation. Possible regulatory mechanisms of polyamine metabolism consistent with the observations are discussed.     

Prolactin stimulation of Nb 2 node lymphoma cell division is inhibited by polyamine biosynthesis inhibitors

The mitogenic action of prolactin in Nb 2 node lymphoma cells was inhibited by two drugs which interfere with polyamine biosynthesis. At concentrations of 0.5 mM and above alpha-difluoromethyl ornithine (DFMO), which inhibits ornithine decarboxylase and the conversion of ornithine to putrescine, significantly attenuated the mitogenic effect of prolactin. This inhibition was prevented by the addition of putrescine, spermidine, or spermine to the culture medium. At concentrations of 1 microM and above methylglyoxal bis(guanylhydrazone) (MGBG), which inhibits S-adenosylmethionine decarboxylase and hence the conversion of putrescine to spermidine and spermine, abolished the mitogenic action of prolactin. This inhibition was prevented by the addition of spermidine or spermine, but not putrescine, to the culture medium. These studies show that ongoing polyamine biosynthesis is essential for prolactin to express its mitogenic effect in this lymphoma cell line.     

Paradoxical enhancement of S-adenosylmethionine decarboxylase in rat tissues following administration of the specific inhibitor methyl glyoxal bis(guanylhydrazone)

Treatment of rats with large but sublethal doses of methyl glyoxal bis(guanylhydrazone), a potent $\text{in vitro}$ inhibitor of animal S-adenosylmethionine decarboxylases, causes marked increases in the enzyme activity of extracts of kidney, ventral prostate, and testis which had been extensively dialyzed to remove any remaining drug. One day after administration of the inhibitor to female rats, the renal S-adenosylmethionine decarboxylase activity was 12 times the normal level and remained greatly enhanced for a further 24 hr. As indicated by decline in decarboxylase activity following depression of protein biosynthesis by injection of cycloheximide, the apparent half-life of the kidney enzyme in normal female rats is roughly 2 hr; in contrast, the apparent half-life of the enzyme is elevated to a value of more than 20 hr in animals that were previously treated with methyl glyoxal bis(guanylhydrazone). The increased renal S-adenosylmethionine decarboxylase activity following administration of the specific enzyme inhibitor $\text{in vivo}$ may thus be due, at least in part, to stabilization of the enzyme against intracellular inactivation as a result either of direct combination of the enzyme protein with the inhibitor, or with substance(s) in the tissue whose levels are influenced by treatment with methyl glyoxal bis(guanylhydrazone).     

Purification,properties and regulation of the level of bovine S-adenosylmethionine decarboxylase during lymphocyte mitogenesis

S-Adenosylmethionine decarboxylase was purified from the livers of calves treated with methylglyoxal bis (guanylhydrazone) to elevate the level of the enzyme. Purified bovine S-adenosylmethionine decarboxylase was similar in specific activity and subunit molecular weight (32 000) to the enzymes previously isolated from rat and mouse. The bovine liver enzyme immunologically crossreacted with S-adenosylmethionine decarboxylase from resting and mitogenically activated bovine lymphocytes. The rate of enzyme synthesis in activated lymphocytes was determined by labeling the cells with [³H]leucine and isolating the radioactive decarboxylase by affinity chromatography and sodium dodecyl sulfate gel electrophoresis. The rate of enzyme syntheis was increased 10-fold by 9 h after mitogen treatment, which accounts for the initial increase in cellular enzymatic. There was no further incraese in the rate of S-adenosylmethionine decarboxylase synthesis that correlated with a second elevation of activity occuring at approx. 24 h after mitogenic activation. It was concluded that the second increase in enzyme activity was due to lengthening the intracellular half-life of the enzyme by 2-fold.