Altered growth and polyamine catabolism following exposure of the chocolate spot pathogen Botrytis fabae to the essential oil of Ocimum basilicum.

Biomass of the fungal pathogen Botrytis fabae in liquid culture amended with two chemotypes of the essential oil of basil, Ocimum basilicum, was reduced significantly at concentrations of 50 ppm or less. The methyl chavicol chemotype oil increased the activity of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), but polyamine concentrations were not significantly altered. In contrast, the linalol chemotype oil decreased AdoMetDC activity in B. fabae, although again polyamine concentrations were not altered significantly. However activities of the polyamine catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO) were increased significantly in B. fabae grown in the presence of the essential oil of the two chemotypes. It is suggested that the elevated activities of DAO and PAO may be responsible, in part, for the antifungal effects of the basil oil, possibly via the generation of hydrogen peroxide and the subsequent triggering of programmed cell death.

Polyamine synthesis and interconversion by the Microsporidian Encephalitozoon cuniculi.

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.

Polyamines in the gastro-intestinal tract of goat kids and in the regenerating ruminal epithelium of sheep.

The aim of the present study was to investigate whether temporal changes in polyamine concentration and synthesis could be found in the luminal content and wall tissue of the rumen and abomasum, two organs which have entirely different growth patterns during the first month of life. In the abomasal mucosa there was a marked gradual decrease in the ornithine decarboxylase (ODC) activity during the first month of life, while the ODC activity in the ruminal mucosa was low during the whole experimental period. However, injury of the rumen wall was followed by increased ODC activity. The ODC activity in duodenal mucosa was about 10 times higher than in the ileal mucosa and the ruminal epithelium. In ruminal liquid a clear peak in ODC activity was observed during the period 51-70 days after birth. The polyamine concentration did not parallel the ODC activity, in either the ruminal epithelium or the ruminal liquid. Of the polyamines, the spermine concentration was always highest, and with the exception of duodenal mucosa, the putrescine concentration was lowest. In liver a clear decrease in spermidine concentration from day 1 to about day 60 after birth was observed. Otherwise no marked temporal changes in tissue polyamine concentrations were observed. Two and a half hours after oral administration of 14C-labelled spermine, nearly all of the radioactivity was found in the lumen of the gastrointestinal tract. On the other hand, 1 h after intravenous injection of polyamines the walls of the gastrointestinal tract were strongly labelled. In conclusion, the polyamines needed for ruminal epithelial development seem to come from sources other than the ruminal epithelium itself or the ruminal lumen.

Overexpression of arginine decarboxylase in transgenic plants.

The activity of arginine decarboxylase (ADC), a key enzyme in plant polyamine biosynthesis, was manipulated in two generations of transgenic tobacco plants. Second-generation transgenic plants overexpressing an oat ADC cDNA contained high levels of oat ADC transcript relative to tobacco ADC, possessed elevated ADC enzyme activity and accumulated 10-20-fold more agmatine, the direct product of ADC. In the presence of high levels of the precursor agmatine, no increase in the levels of the polyamines putrescine, spermidine and spermine was detected in the transgenic plants. Similarly, the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were unchanged. No diversion of polyamine metabolism into the hydroxycinnamic acid-polyamine conjugate pool or into the tobacco alkaloid nicotine was detected. Activity of the catabolic enzyme diamine oxidase was the same in transgenic and control plants. The elevated ADC activity and agmatine production were subjected to a metabolic/physical block preventing increased, i.e. deregulated, polyamine accumulation. Overaccumulation of agmatine in the transgenic plants did not affect morphological development.

Effect of feeding raw soybeans on polyamine metabolism in chicks and the therapeutic effect of exogenous putrescine.

The inclusion of isolated soy protein in milk replacer diets for calves and neonatal pigs inhibits development of intestinal mucosal cells. Simultaneous administration of putrescine partially overcomes this effect. We therefore conducted experiments to determine the potential for dietary putrescine to overcome the toxicity of raw soybeans in chicks. In the first experiment, week-old chicks were fed either an isolated soy protein-based control diet or an isoenergetic and isonitrogenous diet containing 52% raw, ground soybeans for 14 d. The feeding of raw soybeans depressed (P < .001) growth and feed consumption, caused enlargement (P < .001) of the duodenum and pancreas, depressed (P < .001) activities of polyamine synthetic enzymes in the duodenum, and reduced (P < .01) duodenal tissue concentrations of putrescine. In the second experiment, the diet containing raw soybeans was fed with and without .2, .3, and .4% supplemental putrescine. The feeding of supplemental putrescine largely overcame the inhibition of growth due to the feeding of raw soybeans and increased intestinal putrescine concentrations. Putrescine supplementation had no effect, however, on pancreatic and intestinal enlargement in birds fed raw soybeans and tended to depress the activity of polyamine synthetic enzymes. The beneficial effects of putrescine supplementation were confirmed in the third experiment when up to 1.0% supplemental putrescine was fed. We conclude that the toxicity of raw soybeans to chicks can be overcome by feeding putrescine. These effects are likely due to improved nutrient uptake by overcoming the adverse effects of lectins in the intestinal tract and are not likely due to alleviation of the pancreatic enlargement caused by protease inhibitors.

Influence of sepsis and endotoxemia on polyamine metabolism in mucosa of small intestine in rats.

We examined the influence of sepsis and endotoxemia in rats on the biosynthesis of polyamines in small-intestinal mucosa. Sepsis was induced by cecal ligation and puncture (CLP); control rats were sham-operated. In other experiments, rats were treated with two subcutaneous injections of endotoxin (1 mg/kg) or corresponding injections of sterile saline. Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities and concentrations of putrescine, spermidine, and spermine were measured in jejunal mucosa at intervals during 16 hours. Sepsis stimulated ODC and SAMDC activities and increased putrescine and spermidine concentrations in jejunal mucosa. Injection of endotoxin resulted in metabolic changes similar to those observed following CLP. The results suggest that sepsis and endotoxemia stimulate polyamine biosynthesis in mucosa of small intestine. The role of polyamines in the regulation of cell proliferation and metabolic changes in the intestinal mucosa during sepsis remains to be determined.

Toxicity and growth-promoting potential of spermine when fed to chicks.

Previous studies have shown that the feeding of putrescine, a biogenic amine and the precursor of the mammalian polyamines, can promote whole-body growth of chicks. The current study was undertaken to determine the effect of spermine, also a biogenic amine and the most cationic of the polyamines, under similar conditions. In Exp. 1, 120 week-old chicks were fed purified crystalline amino acid-based diets containing 0, .2, .4, .6, .8, or 1.0% spermine for 14 d. Spermine proved highly toxic and growth rates were reduced compared with controls when even .2% was fed. In Exp. 2, chicks were fed 0, .0375, .0750, or .1000% spermine. These concentrations proved less toxic than those used in Exp. 1. Supplemental dietary cysteine was then provided at 0, .3, .6, and .9% together with 0, .025, .050, or .400% spermine (Exp. 3) because depletion of cellular glutathione has been suggested as contributing to spermine's toxicity. Even high levels of cysteine supplementation did not overcome spermine's toxicity. Subsequent dietary provision of L-2-oxothiazolidine-4-carboxylic acid (OTC, Exp. 4), a cysteine prodrug, showed that depletion of cellular glutathione was not likely a cause of spermine toxicosis. A trend toward increased weight gain and feed efficiency was observed when low concentrations of spermine were fed. It was concluded, however, that dietary spermine was more toxic to chicks than was previously seen for putrescine, that any growth-promoting effects of dietary spermine are small, and that supplements of dietary cysteine or OTC are unlikely to increase these effects by overcoming spermine toxicosis.

Localization of S-adenosylmethionine decarboxylase in murine tissues by immunohistochemistry.

S-adenosylmethionine decarboxylase (AdoMetDC), one of three key enzymes in polyamine biosynthesis, is present in minute concentrations in adult tissues, whereas increased AdoMetDC activity occurs in conjunction with differentiation and growth. The occurrence of AdoMetDC in tissues has earlier been studied by biochemical technique only. In this work, an antiserum against recombinant human AdoMetDC was used to investigate the localization of the enzyme in different mouse tissues. In all tissues studied, immunolabeling was seen in cytoplasm, while cell nuclei were negative. In the kidney, AdoMetDC immunoreactivity was confined to the inner part of the cortex; the outer part of the cortex and the medulla displayed only a weak AdoMetDC immunoreaction. In the cortex, renal tubules were strongly reactive, while renal corpuscules were weakly immunolabeled. In developing teeth, AdoMetDC immunoreactivity displayed a gradient of distribution, the immunolabeling intensity being increased from the cervical region to the tip of the cusps. In the incisor, post-secretory ameloblasts were strongly labeled. In the oral mucosa, epithelial cells demonstrated stronger immunolabeling than did cells of the subjacent connective tissue. As for muscle cells, immunoreactive material was confined to the periphery of the fibers. In vitro, treatment with DL-alpha-difluoromethylornithine (DFMO) or methylglyoxal bis-(guanylhydrazone) (MGBG) led to an increase in AdoMetDC. It can be concluded that antibodies to recombinant human AdoMetDC provide a tool for the immunohistochemical localization of AdoMetDC, and that the distribution of the enzyme in the tissues studied gives further support to the importance of polyamines in the development and functions of these organs.

Comparison of androgen regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase gene expression in rodent kidney and accessory sex organs.

Androgen regulation of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities and accumulation of the mRNAs encoding these two enzymes in rodent kidney and accessory sex organs were studied. The ODC mRNA concentration and enzyme activity were increased by androgens in kidney, prostate, and seminal vesicle of 3-day castrated mice and rats, whereas AdoMetDC mRNA and protein levels were androgen inducible only in rodent accessory sex organs. ODC and AdoMetDC mRNAs were regulated in prostate and seminal vesicles in a coordinate fashion, with the maximal levels reached within 24-48 h of steroid exposure. The extent of induction was similar for the two gene products, and ODC and AdoMetDC mRNA accumulation occurred primarily in epithelial cells of accessory sex organs, as shown by in situ hybridization studies. The murine ODC promoter contains an androgen-responsive element (ARE)-like sequence at about -910 nucleotides from the cap site; this element binds to androgen receptor in vitro, albeit with much lower affinity than some other AREs. In transient expression studies with CV-1 cells, an ODC promoter construct (pODCCAT) conferred androgen responsiveness upon the reporter gene. ODC mRNA accumulation is androgen regulated in epithelial cells of proximal tubules in both murine and rat kidney; however, different subtypes of the proximal tubular cells respond in the two species, as revealed by in situ hybridization studies. Expression of the ODC gene was induced more markedly and for a longer duration in the murine than in the rat kidney, but the initial response occurred faster in the rat kidney. The relatively slow kinetics of ODC mRNA accumulation in mouse kidney were not due to recruitment of new cells to respond; rather, in situ hybridization studies indicated that there was progressive accumulation of the mRNA in the responding cells. Collectively, these data indicate that the genes for two key enzymes in polyamine biosynthesis are not regulated in an identical fashion in different androgen target tissues, such as rodent kidney and accessory sex organs.

Protection from chlordecone-amplified carbon tetrachloride toxicity by cyanidanol: regeneration studies.

Previous work has shown that chlordecone (CD)-amplified CCl4 hepatotoxicity and lethality can be mitigated by pretreatment with cyanidanol. These studies also revealed that stimulated hepatocellular regeneration might play an important role in the cyanidanol protection of CD-amplified CCl4 toxicity. The present studies conducted over a time course of 0 to 120 hr after CCl4 challenge describe sequential changes in hepatic [3H]thymidine incorporation into hepatocellular nuclear DNA, polyamines and related enzymes, and histomorphometry of liver sections from variously treated rats. Male Sprague-Dawley rats (125-150 g) were maintained on a control diet or on a diet contaminated with CD (10 ppm) for 15 days and/or pretreated with cyanidanol (250 mg/kg, ip) at 48, 24, and 2 hr before a single ip injection of either a standard protocol dose (100 microliters/kg) or a low dose (50 microliters/kg, L) of CCl4 on Day 16 of the dietary protocol. Cyanidanol pretreatment significantly stimulated the hepatic [3H]thymidine incorporation into hepatocellular nuclear DNA of control rats irrespective of CD pretreatment. Similarly, polyamine metabolism was altered favorably for cell division, although mitotic index (metaphase) was not increased. Cyanidanol-stimulated [3H]thymidine incorporation was highly suppressed in rats receiving the CD + CCl4 standard dose combination treatment up to 36 hr, but after this time point a marked increase was observed. Hepatocellular regeneration, quantified histomorphometrically as volume density of cells in metaphase, was progressively increased in rats protected from CD + CCl4 interaction by cyanidanol, starting at 36 hr and lasting until 72 hr. Favorably altered polyamine metabolism was evident from the stimulated ornithine decarboxylase, as well as from the stimulated interconversion of the higher polyamines to maintain increased concentration of putrescine. Challenge by the same dose of CCl4 (100 microliters/kg) to CD-pretreated rats not protected by cyanidanol failed to cause any increase in [3H]thymidine incorporation up to 36 hr and resulted in animal death starting at 36 hr. In the surviving rats, [3H]thymidine incorporation at 48 hr was increased, but was less than 50% of the increase observed in the cyanidanol group. In these rats, attenuation in the stimulation of cell division and insufficiently increased putrescine levels were observed, which are consistent with the inadequate level of hepatocellular regeneration. With rats receiving CD + CCl4(L) combination, the [3H]thymidine incorporation at 48 hr was less than 50% of the increase of cyanidanol-protected rats. Cyanidanol pretreatment to the CD + CCl4 group of rats prevented the decrease in the hepatic DNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Ornithine and S-adenosylmethionine decarboxylase activities and polyamine contents in developing muscle tissues and primary cultures of normal and polymyopathic hamsters.

Polyamine (putrescine, spermidine, and spermine) contents and ornithine (ODC) and S-adenosylmethionine (SAMDC) decarboxylase activities have been assessed in an age-dependent manner, in normal and polymyopathic (dystrophic) hamster skeletal muscle, heart, and tongue extract and in primary tongue myoblast and skin fibroblast cultures. At 2 weeks of age, polyamine contents were significantly elevated in all of the dystrophic hamster tissues studied when compared with their age-matched controls. The degree of this elevation decreased with the age of the animals, generally, to a level where no significant difference in polyamine contents could be noted between normal and dystrophic hamster tissues. ODC and SAMDC activities in whole tissue extracts were consistently highest in 2-week-old muscle extracts and also declined with age. However, no significant changes in ODC or SAMDC activities were evident in any of the dystrophic muscle tissues studied when compared with their age-matched controls. Polyamine contents in dystrophic hamster myoblast and fibroblast primary cultures were also during proliferation (1 and 2 days after the initial seeding) compared with cultures prepared from normal hamsters. ODC and SAMDC activities in primary myoblast and fibroblast cultures clearly reflected the rate of cell proliferation, with highest activities found in subconfluent cell cultures. However, in general, no significant dystrophic-related abnormality in ODC or SAMDC activity was evident in proliferating myoblast or fibroblast cultures. These results suggest that the elevated polyamine contents of dystrophic hamster tissues and primary cultures may be due to a deficiency in polyamine catabolism or transport.

Antitumor effect of a new multienzyme inhibitor of polyamine synthetic pathway, methylglyoxal-bis(cyclopentylamidinohydrazone), against human and mouse leukemia cells.

Methylglyoxal-bis(cyclopentylamidinohydrazone) (MGBCP) has been synthesized as a multienzyme inhibitor for the polyamine-synthesizing pathway. This drug inhibited S-adenosylmethionine decarboxylase (EC 4.1.1.50), spermine synthase and spermidine synthase activities, competitively with S-adenosylmethionine, spermidine, and putrescine, respectively. MGBCP inhibited the growth of human leukemia Molt 4B and K 562 cells at 10 to 100 microM concentrations. Spermidine and spermine levels were markedly depressed in these MGBCP-treated leukemic cells, and the synthesis of protein, but not of DNA or RNA, was significantly diminished. In in vivo experiments, MGBCP depleted spermidine and spermine in the P388 leukemic ascites cells, and prolonged the survival time of mice bearing P388 leukemia. The S-adenosylmethionine decarboxylase-stabilizing effect of MGBCP in mouse liver, Molt 4B and K 562 cells was much less than that of the parent inhibitor methylglyoxal-bis(guanylhydrazone). Induction of ornithine decarboxylase activity by MGBCP in the cultured leukemic cells was also much less than that by methylglyoxal-bis(guanylhydrazone).

Reduced amounts of S-adenosylmethionine decarboxylase in the adrenal glands of rats following administration of piribedil or 2-deoxyglucose.

The activity of S-adenosylmethionine decarboxylase (SAM-DC) is decreased in the adrenal gland of the rat following physical stress, metabolic stress or administration of dopamine agonists [M. Ekker and T. L. Sourkes, Endocrinology 120, 1299 (1987)]. Immunotitration studies with a serum directed against purified rat liver SAM-DC show that the reduction in activity of the enzyme following administration of 2-deoxyglucose or piribedil was paralleled by a decrease in the amount of immuno-reactive protein. There was no difference in the half-life of SAM-DC activity between piribedil-treated rats and controls. The properties of an extensively purified preparation of the adrenal enzyme resembled those of SAM-DC obtained from rat liver. It is suggested that the reduction in adrenal SAM-DC activity and protein content caused by stress is due to a reduction in the rate of synthesis of the enzyme.

Structural and mechanistic properties of E. coli adenosylmethionine decarboxylase.

Adenosylmethionine decarboxylase catalyzes one of the first committed steps in polyamine biosynthesis. It is a member of a small class of decarboxylases that use a pyruvovyl prosthetic group rather than the more common pyridoxal cofactor. We have recently shown that AdoMet decarboxylase from E. coli is composed of stoichiometric amounts of two types of subunits; alpha (Mr = 19,000), and beta (Mr = 14,000). The NH2-terminal of the alpha subunit is blocked by the pyruvoyl group and can be sequenced only after reductive amination, which converts this to an alanine residue. The beta subunit, on the other hand, has an unblocked NH2-terminal and sequences normally. The molecular weight of the holoenzyme, estimated by gel filtration, is 136,000 suggesting that the enzyme is an alpha 4 beta 4 octamer. AdoMet decarboxylase undergoes a time dependent inactivation during turnover. The mechanism of this inactivation involves a transamination from the product, decarboxylated AdoMet, and the pyruvoyl group generating an NH2-terminal alanine. The nascent product aldehyde then eliminates methylthioadenosine, resulting in the formation of acrolein, which covalently labels the alpha subunit. How this mechanism may explain AdoMet decarboxylase turned over, and how AdoMet decarboxylase inhibitors can affect its half life will be discussed.

Ornithine decarboxylase and S-adenosylmethionine decarboxylase activity in the accessory sex organs of intact, castrated, and androgen-stimulated castrated rats.

We have studied the activities of ornithine decarboxylase and adenosylmethionine decarboxylase in the 10(6)-m/s2 supernatants of the different lobes of the prostate and the seminal vesicles of castrates, androgen-stimulated castrates, and intact controls. After castration L-ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (AMDC) activities fell in all tissues examined. The induction of kinetics was followed for 72 h after administration of testosterone propionate to castrated rats. AMDC activities increased more rapidly than ODC activities in every tissue studied. Peak activities were reached more rapidly in the dorsal lobe than in the other tissues. ODC activity of the ventral lobe increased linearly for 48 h after stimulation. In the other tissues studied, ODC activity reached a maximum after 24 h and thereafter leveled off or decreased. In conclusion we have found distinct differences in ODC and AMDC activity in various tissues and major differences between treatment groups, with near extinction of activity at castration. In castrates stimulated with testosterone, the between-group differences prevailed but with different patterns of ODC versus AMDC activity. AMDC is seemingly rate-limiting in polyamine synthesis in stimulated tissues, while ODC controls synthesis in tissues from castrated rats.

Effect of estradiol on the activities of ornithine decarboxylase and S-adenosyl-methionine decarboxylase in tissues of ovariectomized rats.

Polyamines play an important role in cellular growth and differentiation. Ornithine decarboxylase (ODC) and S-adenosyl-methionine decarboxylase (SAMD) catalyze the rate-limiting steps of polyamine formation. Many hormones have been known to affect these decarboxylase activities in their target tissues. This study investigated the effect of estradiol (E2) on the activities of ODC and SAMD and on the growth of three estrogen receptor-containing organs: uterus, kidney and liver. Two weeks after ovariectomy, Sprague-Dawley rats received a subcutaneous injection of E2 (10 micrograms/rat). The ODC activity and organ weight were determined at various intervals after E2 administration. The E2 treatment increased the weight of uterus but not that of liver and kidney. In the uterus, ODC activity showed biphasic increases at 6 h and 16-24 h respectively after E2 injection. Renal ODC activity increased gradually and peaked at 18-24 h whereas hepatic ODC activity remained unchanged. Daily injection of E2 over a 3-day period provoked an increase of uterine weight, uterine ODC and SAMD activity. The same treatment also increased renal ODC activity, however it had no effect on the renal SAMD activity and the kidney Our results indicate that E2 has different effects on the activities of ODC and SAMD in three organs tested, and the increase of ODC activity is not necessarily correlated with the tissue growth.

Escherichia coli S-adenosylmethionine decarboxylase. Subunit structure, reductive amination, and NH2-terminal sequences.

S-Adenosylmethionine decarboxylase is one of a small group of enzymes that use a pyruvoyl residue as a cofactor. Histidine decarboxylase from Lactobacillus 30a, the best studied pyruvoyl-containing enzyme, has an (alpha beta)6 subunit structure with the pyruvoyl moiety linked through an amide bond to the NH2-terminal of the larger alpha subunit (Recsei, P. A., Huynh, Q. K., and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 973-977). To examine potential structural analogies between the two enzymes, we have isolated and partially characterized S-adenosylmethionine decarboxylase. The purified enzyme comprises equimolar amounts of two subunits of Mr = 14,000 and 19,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and has a native molecular weight of 136,000 (by gel filtration). Approximately 4 mol of [methyl-3H] adenosylmethionine are incorporated per mol of enzyme (Mr = 136,000) when the enzyme is inactivated with this substrate and NaCNBH3. These data suggest an (alpha beta)4 structure with 1 pyruvoyl residue for each alpha beta pair. The two subunits have been separated by reversed-phase high performance liquid chromatography after reduction and carboxymethylation. The smaller subunit (beta) has a free amino terminus. The amino terminus of the larger subunit (alpha) appears to be blocked by a pyruvoyl group; this subunit can be sequenced only after this group is converted to an alanyl residue by reduction with sodium cyanoborohydride in the presence of ammonium acetate. This work suggests that S-adenosylmethionine decarboxylase is structurally much more similar to histidine decarboxylase than previously thought.

Differential turnover of the subunits of ribonucleotide reductase in synchronized leukemia L1210 cells.

Ribonucleotide reductase catalyzes the rate-limiting step in the de novo synthesis of 2'-deoxyribonucleoside 5'-triphosphates that is required for DNA replication. The mammalian enzyme consists of two nonidentical protein subunits that are both required for enzyme activity. In leukemia L1210 cells, enriched in G1-phase cells by centrifugal elutriation, it was found that ribonucleotide reductase activity increased as the cells progressed to S-phase. The two subunits making up the holoenzyme did not increase coordinately. The nonheme iron subunit increased much more rapidly than the effector-binding (EB) subunit. The activity of the holoenzyme paralleled the level of the EB subunit, which was limiting. The half-lives of the holoenzyme and its subunits were determined in S-phase cells by treatment with cycloheximide. The half-lives of the holoenzyme and the nonheme iron and EB subunits, as determined by enzyme activity, were 3.5, 7.6, and 4 h, respectively. These half-lives are consistent with the data that indicate that the EB subunit is the limiting component in L1210 cells.