Relation of polyamine biosynthesis to the initiation of sprouting in potato tubers

The polyamines putrescine, spermidine, and spermine and their biosynthetic enzymes arginine decarboxylase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase are present in all parts of dormant potato (Solanum tuberosum L.) tubers. They are equally distributed among the buds of apical and lateral regions and in nonbud tissues. However, the breaking of dormancy and initiation of sprouting in the apical bud region are accompanied by a rapid increase in ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase activities, as well as by higher levels of putrescine, spermidine, and spermine in the apical buds. In contrast, the polyamine biosynthetic enzyme activities and titer remain practically unchanged in the dormant lateral buds and in the nonbud tissues. The rapid rise in ornithine decarboxylase, but not arginine decarboxylase activity, with initiation of sprouting suggests that ornithine decarboxylase is the rate-limiting enzyme in polyamine biosynthesis. The low level of polyamine synthesis during dormancy and its dramatic increase in buds in the apical region at break of dormancy suggest that polyamine synthesis is linked to sprouting, perhaps causally.     

Polyamine-deficient Neurospora crassa mutants and synthesis of cadaverine.

The polyamine path of Neurospora crassa originates with the decarboxylation of ornithine to form putrescine (1,4-diaminobutane). Putrescine acquires one or two aminopropyl groups to form spermidine or spermine, respectively. We isolated an ornithine decarboxylase-deficient mutant and showed the mutation to be allelic with two previously isolated polyamine-requiring mutants. We here name the locus spe-1. The three spe-1 mutants form little or no polyamines and grow well on medium supplemented with putrescine, spermidine, or spermine. Cadaverine (1,5-diaminopentane), a putrescine analog, supports very slow growth of spe-1 mutants. An arginase-deficient mutant (aga) can be deprived of ornithine by growth in the presence of arginine, because arginine feedback inhibits ornithine synthesis. Like spe-1 cultures, the ornithine-deprived aga culture failed to make the normal polyamines. However, unlike spe-1 cultures, it had highly derepressed ornithine decarboxylase activity and contained cadaverine and aminopropylcadaverine (a spermidine analog), especially when lysine was added to cells. Moreover, the ornithine-deprived aga culture was capable of indefinite growth. It is likely that the continued growth is due to the presence of cadaverine and its derivatives and that ornithine decarboxylase is responsible for cadaverine synthesis from lysine. In keeping with this, an inefficient lysine decarboxylase activity (Km greater than 20 mM) was detectable in N. crassa. It varied in constant ratio with ornithine decarboxylase activity and was wholly absent in the spe-1 mutants.     

Polyamine Metabolism in Acanthamoeba: Polyamine Content and Synthesis of Ornithine,Putrescine, and Diaminopropane1

Five polyamines which could be separated by high performance liquid chromatography were found in Acanthamoeba castellanii (strain Neff). These included in order of decreasing abundance: 1,3-diaminopropane, spermidine, spermine, norspermidine, and putrescine. Only diaminopropane and norspermidine had been found previously. Spermine was present in cultures grown in broth, but not in defined medium. Radioactive substrates were used to establish that putrescine was synthesized by decarboxylation of ornithine, ornithine was synthesized from arginine or citrulline, and diaminopropane was synthesized from spermidine. The presence of ornithine decarboxylase (EC 4.1.1.17), arginase (EC 3.5.3.1), and urease (EC 3.5.1.5) and the absence of arginine decarboxylase (EC 4.1.1.19) were established. A scheme for polyamine biosynthesis in A. castellanii is proposed.     

Polyamine metabolism in Acanthamoeba: polyamine content and synthesis of ornithine, putrescine, and diaminopropane

Five polyamines which could be separated by high performance liquid chromatography were found in Acanthamoeba castellanii (strain Neff). These included in order of decreasing abundance: 1,3-diaminopropane, spermidine, spermine, norspermidine, and putrescine. Only diaminopropane and norspermidine had been found previously. Spermine was present in cultures grown in broth, but not in defined medium. Radioactive substrates were used to establish that putrescine was synthesized by decarboxylation of ornithine, ornithine was synthesized from arginine or citrulline, and diaminopropane was synthesized from spermidine. The presence of ornithine decarboxylase (EC 4.1.1.17), arginase (EC 3.5.3.1), and urease (EC 3.5.1.5) and the absence of arginine decarboxylase (EC 4.1.1.19) were established. A scheme for polyamine biosynthesis in A. castellanii is proposed.     

Polyamine metabolism in potassium-deficient bacteria

The metabolism of polyamines was studied in K(+)-dependent strains of Escherichia coli. When these stringent organisms were in a medium containing Na(+) instead of K(+), protein synthesis was arrested, but synthesis of ribonucleic acid continued as it would in a relaxed organism. The Na(+) medium inhibited synthesis of spermidine and S-adenosylmethionine. However, the synthesis of putrescine was accelerated at least five- to eightfold. Exogenous ornithine doubled even this rate of putrescine synthesis but did not increase the low level of putrescine synthesis in the K(+) medium. In K(+) or Na(+) media, with or without 0.3 mm arginine, putrescine was derived almost entirely from ornithine via ornithine decarboxylase. Addition of spermidine (5 mm) to a Na(+) culture markedly inhibited putrescine synthesis. The ornithine decarboxylase of an extract of a K(-)-dependent strain prepared at low ionic strength was separated from ribosomes, deoxyribonucleic acid, and associated polyamines by centrifugation, and from many ions by ultrafiltration and fractionation on Sephadex G-100. Addition of Na(+) and K(+) salts to 200 mm was markedly inhibitory. The combined reductions both in synthesis of the inhibitor spermidine and in intracellular ionic strength may explain the in vivo activation of this enzyme.     

Polyamine metabolism in Ancylostoma ceylanicum and Nippostrongylus brasiliensis

Spermidine was detected as the major polyamine of Ancylostoma ceylanicum as well as Nippostrongylus brasiliensis. Spermine was present in lower amounts whereas the level of putrescine was even less. S-Adenosylmethionine decarboxylase, a rate-limiting enzyme in the biosynthetic pathway of polyamines, was demonstrated at low levels in both parasites. Decarboxylation of lysine and arginine was absent or negligible and that of ornithine questionable, as the enzyme activity was not inhibited by alpha-difluoromethylornithine while RMI 71,645, an irreversible inhibitor of ornithine aminotransferase, strongly inhibited the liberation of CO2 from ornithine. High activity of ornithine aminotransferase was observed in both the parasites and may interfere with the assay for ornithine decarboxylase. Adults of A. ceylanicum were found to rapidly take up spermidine and spermine from incubation medium while uptake of putrescine was very low. These results indicate that hookworms depend on uptake and interconversion rather than de novo synthesis for their polyamine requirement.     

Utilization of putrescine in tobacco cell lines resistant to inhibitors of polyamine synthesis

Three tobacco cell lines have been analyzed which are resistant to lethal inhibitors of either putrescine production or conversion of putrescine into polyamines. Free and conjugated putrescine pools, the enzymic activities (arginine, ornithine, and S-adenosylmethionine decarboxylases), and the growth characteristics during acidic stress were measured in suspension cultures of each cell line. One cell line, resistant to difluoromethylornithine (Dfr1) had a very low level of ornithine decarboxylase activity which was half insensitive to the inhibitor in vitro. Intracellular free putrescine in Dfr1 was elevated 10-fold which was apparently due to a 20-fold increase in the arginine decarboxylase activity. The increased free putrescine titer was not reflected in an increased level of spermidine, spermine, or putrescine conjugation. Dfr1 cultures survived acidic stress at molarities which were lethal to wild type cultures. Two other mutants, resistant to methylglyoxal bis(guanylhydrazone) (Mgr3, Mgr12), had near normal levels of the three decarboxylases and normal titers of free putrescine, spermidine, and spermine. Both mutants however had elevated levels of conjugated putrescine. Mgr12 had an increased sensitivity to acidic medium. These results suggest that increased levels of free putrescine production may enhance the ability of tobacco cells to survive acid stress. This was supported by the observation that cytotoxic effects of inhibiting arginine decarboxylase in wild type cell lines were dependent on the acidity of the medium.     

Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings

A purified preparation of arginine decarboxylase fromCucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine andPi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase,viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3–4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine andvice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.     

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.     

Acquisition of polyamines by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

Both the polyamine content and the route of acquisition of polyamines by Rickettsia prowazekii, an obligate intracellular parasitic bacterium, were determined. The rickettsiae grew normally in an ornithine decarboxylase mutant of the Chinese hamster ovary (C55.7) cell line whether or not putrescine, which this host cell required in order to grow, was present. The rickettsiae contained approximately 6 mM putrescine, 5 mM spermidine, and 3 mM spermine when cultured in the presence or absence of putrescine. Neither the transport of putrescine and spermidine by the rickettsiae nor a measurable rickettsial ornithine decarboxylase activity could be demonstrated. However, we demonstrated the de novo synthesis of polyamines from arginine by the rickettsiae. Arginine decarboxylase activity (29 pmol of 14CO2 released per h per 10(8) rickettsiae) was measured in the rickettsiae growing within their host cell. A markedly lower level of this enzymatic activity was observed in cell extracts of R. prowazekii and could be completely inhibited with 1 mM difluoromethylarginine, an irreversible inhibitor of the enzyme. R. prowazekii failed to grow in C55.7 cells that had been cultured in the presence of 1 mM difluoromethylarginine. After rickettsiae were grown in C55.7 in the presence of labeled arginine, the specific activities of arginine in the host cell cytoplasm and polyamines in the rickettsiae were measured; these measurements indicated that 100% of the total polyamine content of R. prowazekii was derived from arginine.     

The effect of salt stress on polyamine biosynthesis and content in mung bean plants and in halophytes

The activity of L-arginine decarboxylase (EC 4.1.1.19) and L-ornithine decarboxylase (EC 4.1.1.17), polyamine content, and incorporation of arginine and ornithine into polyamines, were determined in mung bean [Vigna radiata (L.) Wilczek] plants subjected to salt (hypertonic) stress (NaCl at 0.51–2.27 MPa). Changes in enzyme activity in response to hypotonic stress were determined as well in several halophytes [Pulicaria undulata (L.), Kostei, Salsola rosmarinus (Ehr.) Solms-Laub, Mesembryanthemum forskahlei Hochst, and Atriplex halimus L.]. NaCl stress, possibly combined with other types of stress that accompanied the experimental conditions, resulted in organ-specific changes in polyamine biosynthesis and content in mung bean plants. The activity of both enzymes was inhibited in salt-stressed leaves. In roots, however, NaCl induced a 2 to 8-fold increase in ornithine decarboxylase activity. Promotion of ornithine decarboxylase in roots could be detected already 2 h after exposure of excised roots to NaCl, and iso-osmotic concentrations of NaCl and KCl resulted in similar changes in the activity of both enzymes. Putrescine level in shoots of salt-stressed mung bean plants increased considerably, but its level in roots decreased. The effect of NaCl stress on spermidine content was similar, but generally more moderate, resulting in an increased putrescine/spermidine ratio in salt-stressed plants. Exposure of plants to NaCl resulted also in organ-specific changes in the incorporation of both arginine and ornithine into putrescine: incorporation was inhibited in leaf discs but promoted in excised roots of salt-stressed mung bean plants. In contrast to mung bean (and several other glycophytes), ornithine and arginine decarboxylase activity in roots of halophytes increased when plants were exposed to tap water or grown in a pre-washed soil—i.e. a hypotonic stress with respect to their natural habitat. NaCl, when present in the enzymatic assay mixture, inhibited arginine and ornithine decarboxylase in curde extracts of mung bean roots, but did not affect the activity of enzymes extracted from roots of the halophyte Pulicaria. Although no distinct separation between NaCl stress and osmotic stress could be made in the present study, the data suggest that changes in polyamines in response to NaCl stress in mung bean plants are coordinated at the organ level: activation of biosynthetic enzymes concomitant with increased putrescine biosynthesis from its precursors in the root system, and accumulation of putrescine in leaves of salt-stressed plants. In addition, hypertonic stress applied to glycophytes and hypotonic stress applied to halophytes both resulted in an increase in the activity of polyamine biosynthetic enzymes in roots.     

Streptomycin Resistance (rpsL) Produces an Absolute Requirement for Polyamines for Growth of an Escherichia coli Strain Unable to Synthesize Putrescine and Spermidine [Δ(speA-speB) ΔspecC]

The presence of certain rpsL (strA) mutations in a strain of Escherichia coli that cannot synthesize putrescine or spermidine because of deletions in ornithine decarboxylase, arginine decarboxylase, and agmatine ureohydrolase, converts a partial requirement for polyamines for growth into an absolute requirement.     

Activities of arginine and ornithine decarboxylases in various plant species

In extracts from the youngest leaves of Avena sativa, Hordeum vulgare, Zea Mays, Pisum sativum, Phaseolus vulgaris, Lactuca sativa, and four pyrrolizidine alkaloid-bearing species of Heliotropium, the activities of ornithine decarboxylase, close to V_max, ranged between traces and 1.5 nanomoles per hour per gram fresh weight when based on putrescine formed during incubation with labeled ornithine. The arginine decarboxylase activities in the same extracts ranged between 8 and 8000 nanomoles per hour per gram fresh weight being lowest in the borages and highest in oat and barley. α-Difluoromethylornithine and α-difluoromethylarginine inhibited ornithine and arginine decarboxylases, respectively, in all species. Agmatine, putrescine, spermidine, and spermine were found in all, diaminopropane in eight, and cadaverine in three species.

No correlation was observed between arginine or ornithine decarboxylase level and the levels of total polyamines. The in vitro decarboxylase activities found in the borages cannot explain the high accumulation of putrescine-derived pyrrolizidines in their youngest leaves if the pyrrolizidines are produced in situ from arginine and/or ornithine as precursors; other possibilities are discussed.

In assays of ornithine decarboxylase, an interference of decarboxylation not due to this enzyme was observed in extracts from all species. In arginine decarboxylase assays, the interfering decarboxylation as well as the interference of arginase were apparent in two species. Addition of aminoguanidine was needed to suppress oxidative degradation of putrescine and agmatine during incubation of extracts from pea, bean, lettuce, Heliotropium angiospermum, and Heliotropium indicum.

     

Polyamine metabolism in some helminth parasites

Polyamine levels of some helminth parasites were analyzed by reverse phase HPLC of benzoyl derivatives. Setaria cervi, Acanthocheilonema viteae, Hymenolepis nana, H. diminuta, and Ascaridia galli contained higher levels of spermine than spermidine while in Ancylostoma ceylanicum and Nippostrongylus brasiliensis the spermidine levels were higher than spermine; putrescine was either absent or present in minor quantities. The enzymes of polyamine biosynthesis viz., ornithine decarboxylase, S-adenosyl methionine (SAM)-decarboxylase, and arginine decarboxylase were present in very low to negligible amounts in all the parasites examined. A. ceylanicum exhibited high activity of ornithine amino transferase (OAT) and catalyzed appreciable decarboxylation of ornithine. The ornithine decarboxylating activity of A. ceylanicum was localized in the particulate fraction containing mitochondria, not inhibited by alpha-difluoromethyl ornithine, the specific inhibitor of ornithine decarboxylase (ODC), but inhibited in the presence of glutamate, suggesting the involvement of mitochondrial OAT rather than a true ODC in ornithine decarboxylation in this parasite. Significant activity of polyamine oxidase was also detected in helminth parasites. The absence of polyamine biosynthesizing enzymes in helminth parasites suggests their dependence on hosts for uptake and interconversion of polyamines, providing a potential target for chemotherapy.     

Localization of ornithine decarboxylase and changes in polyamine content in root meristems of Zea mays

Polyamine content and the activity of arginine decarboxylase (EC 4.1.1.19) and ornithine decarboxylase (EC 4.1.1.17) were studied with respect to meristematic activity in primary roots and in developing lateral roots of Zea mays L. (cv. Neve Ya'ar 170) seedlings. Comparative localization of active ornithine decarboxylase and of meristematic activity were determined by labelling roots either with α-[5-¹⁴C]-difluoromethyl ornithine or with [³H]-thymidine, respectively.
Lateral roots were formed during the 72 h post-decapitation period, accompanied by an initial decline in putrescine content and by a significant increase in spennidine con-tent at 48–72 h. High levels of spermidine and lower levels of putrescine were found in the primary root apex as well. A marked increase in ornithine and arginine decarboxylase activity, as measured by ¹⁴CO₂ release, was found during the 72 h post-decapitation period of lateral root development. This increase in ornithine decarboxylase activity was confirmed also by a parallel rise in the incorporation of α-[5-¹⁴C]-difluoromethyl ornithine into trichloroacetic acid-insoluble fractions. Microautoradiographs of longitudinal and cross sections of roots, labelled with α-[5-¹⁴C]-difluoromethyl ornithine, showed that ornithine decarboxylase is localized mainly in the meristematic zones, as evidenced by [³H]-thymidine incorporation. A close correlation between meristematic activity and polyamines was demonstrated in situ , suggesting that polyamine content and biosynthesis may have a role in meristematic activity in corn roots.     

Polyamine biosynthesis during somatic embryogenesis in interior spruce (Picea glauca x Picea engelmannii complex)

Putrescine, spermidine, and spermine levels during somatic embryogenesis of interior spruce (Picea glauca x Picea engelmannii complex) were quantified On abscisic acid supplemented growth medium putrescine and spermidine levels increased two-fold coinciding with maturation of the early somatic embryos to globular embryos. Polyclonal antibodies raised against Escherichia coli arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), following affinity purification specifically recognized spruce ADC and ODC, which corresponded to 85kD and 65kD bands on western blots of total protein extracts from embryogenic masses, Immunoassays using these antibodies showed increased ADC levels corresponding to embryo maturation while ODC levels remained the same. From these results it is concluded that polyamines are involved in the maturation of somatic embryos of interior spruce.Abbreviations ADC arginine decarboxylase - BSA bovine serum albumin - ODC ornithine decarboxylase - PBS phosphate buffered saline - PCA perchloric acid - SDS-PAGE sodium dodecyl sulfateporyacrylamide gel electrophoresis     

Diurnal changes in polyamine content, arginine and ornithine decarboxylase, and diamine oxidase in tobacco leaves

Changes in the contents of polyamines (PAs) in tobacco leaves (Nicotiana tabacum L. cv. Wisconsin 38) grown under 16 h photoperiod were correlated with arginine and ornithine decarboxylase (EC 4.1.1.19 and EC 4.1.1.17) and diamine oxidase (EC 1.4.3.6) activities. The maximum of free and soluble conjugated forms of PAs occurred 1-2 h after the middle of the light period and was followed by two distinct peaks at the end of the light and at the beginning of the dark phase. Putrescine was the most abundant and cadaverine the least abundant PA in both free and PCA-soluble forms. However, cadaverine was predominant in PCA-insoluble conjugates, followed by putrescine, spermidine, and spermine. Both arginine and ornithine decarboxylases are involved in putrescine biosynthesis in tobacco leaves. Light dramatically stimulated the activity of ornithine decarboxylase, while no photoinduction of arginine decarboxylase activity was observed. Ornithine decarboxylase was found mainly in the particulate fraction. Only one peak, just after light induction, occurred in the cytosolic fraction, with 35% of the total ornithine decarboxylase activity. By contrast, the total arginine decarboxylase activity was equally divided between the soluble and pellet fractions. A sharp increase in diamine oxidase activity occurred 1 h after exposure to light, concomitant with the light-induced increase in ornithine decarboxylase activity. After a decline, diamine oxidase activity increased again, together with the rise in the amount of free Put. The roles of both conjugation of PAs with hydroxycinnamic acids and oxidative degradation of putrescine in maintaining free PA levels during the 24 h light/dark cycle are discussed. The presented results have shown that the parameters studied here followed rhythmical changes and were not only affected by light.     

Effect of urethan on polyamine and DNA synthesis in the regenerating rat liver

When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of ornithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14–15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37–42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis.     

Regulation of growth and polyamine biosynthesis of the ciliated protozoan Tetrahymena thermophila. Effects of L-arginine metabolites and polyamines

Growth of Tetrahymena thermophila in a synthetic nutrient medium with or without the essential amino acid L-arginine was studied in the presence or absence of the arginine metabolites L-citrulline and L-ornithine and the polyamines putrescine, spermidine, and spermine. The effects of the growth conditions on the stimulations of the enzymes of the arginine metabolic and polyamine biosynthetic pathway, arginine deiminase (ADI), citrulline hydrolase (CH), ornithine decarboxylase (ODC), and ornithine-oxo-acid aminotransferase were determined. Tetrahymena cells were unable to grow in the absence of L-arginine and the amino-acid utilization was greatly impaired. None of the metabolites or polyamines was able to substitute for arginine. In the presence of arginine, Tetrahymena cultures grew well and citrulline and ornithine did not alter the growth behaviour in any way. In the presence of putrescine, the lag period was decreased from 3 h to 2 h. Spermidine and spermine acted similar to putrescine but less pronounced. The stimulation of the activity of ADI, the key enzyme of arginine degradation, was absolutely dependent upon the presence of arginine in the medium: in the absence of arginine, the low ADI activity which was present in the cells before inoculation was decreased to zero levels within 30 min. In the presence of arginine, the stimulation of ADI was not altered by citrulline and ornithine but putrescine, spermidine, and spermine decreased ADI-stimulation to half of the control values. The stimulation of CH activity in the presence of arginine was not altered by any added metabolite or polyamine. In the media without arginine, stimulation of CH was greatly reduced, in the presence of ornithine more than in its absence, and even more in the presence of putrescine and spermidine. Stimulation of ODC activity in the presence of arginine was not affected by citrulline and ornithine but in the presence of polyamines it was rapidly decreased to unstimulated levels after an initial ca. 10-fold increase. The "hyperstimulation" of ODC in the absence of free arginine was reduced to normal in the presence of citrulline, the stimulation was decreased even below normal levels in the presence of ornithine and polyamines decreased ODC activity to zero levels. O delta T activity was stimulated more in the presence of arginine than in its absence. In both cases the stimulation was enhanced in the presence of polyamines and only in the absence of arginine--by ornithine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of inhibitors of protein synthesis on rat liver spermidine N-acetyltransferase

The increase in spermidine N-acetyltransferase activity in rat liver produced by carbon tetrachloride was completely prevented by simultaneous treatment with inhibitors of protein and nucleic acid synthesis suggesting that the increase results from the synthesis of new protein rather than the release of the enzyme from a cryptic inactive form. Treatment with cycloheximide 2 h after carbon tetrachloride also completely blocked the rise in spermidine N-acetyltransferase seen 4 h later. Such treatment completely prevented the fall in spermidine and rise in putrescine in the liver 6 h after carbon tetrachloride confirming the importance of the induction of spermidine N-acetyltransferase in the conversion of spermidine into putrescine. When cycloheximide was administered to rats in which spermidine N-acetyltransferase activity had been stimulated by prior treatment with carbon tetrachloride or thioacetamide, the activity was lost rapidly showing that the enzyme protein has a rapid rate of turnover. The half-life for the enzyme in thioacetamide-treated rats was 40 min, whereas the half-life for ornithine decarboxylase (which is well known to turn over very rapidly) was 27 min. In carbon tetrachloride-treated rats the rate or protein degradation was reduced and the half-life of spermidine N-acetyltransferase was 155 min and that for ornithine decarboxylase was 65 min. It appears that three of the enzymes involved in the synthesis and interconversion of putrescine and spermidine namely, ornithine decarboxylase, S-adenosylmethionine decarboxylase and spermidine N-acetyltransferase have rapid rates of turnover and that polyamine levels are regulated by changes in the amount of these enzymes.