Intracellular Concentrations Of Putrescine Research Articles

A combination of proteomics, genetics, and physiology provides insights into the acid-tolerance phenotype of Pseudomonas pergaminensis F77

Acid tolerance is crucial for the effective and persistent mineral weathering by acid-producing bacteria. Here, the molecular basis of the acid tolerance of mineral-weathering Pseudomonas pergaminensis F77 was identified using proteomics analysis of the strain under acid stress. Then, the acid tolerance of strain F77 and its mutants with deletion of the acid tolerance-related genes orf03767, mcp, resR, nueR, yegD, and fxsA, which are involved in the two-component systems, DNA repair, nucleotide binding, and membrane parts, were compared. Finally, the acid tolerance-related physiological mechanisms of strain F77 and its mutants F77ΔnueR and F77ΔresR under acidic conditions were characterized. The significantly upregulated proteins in the acid-adapted and acid-challenged strain F77 included the proteins involved in metabolic pathways associated with ATPase, membrane components, organic acid transmembrane transporters, response to stimulus, nucleotide binding, ABC transporters, and two-component systems. The cell numbers decreased by 24–100% at pH ≤ 4.50, while the membrane fluidity increased by 22–61% at pH ≤ 5.50 for the mutants F77ΔnueR and F77ΔresR, compared with that of strain F77. The intracellular H+-ATPase activities decreased by 29–33% for the mutant F77ΔnueR at pH ≤ 4.50% and 33–79% for the mutant F77ΔresR at all tested pHs (pH ≤ 7.00); meanwhile, the ratios of intracellular NAD+/NADH decreased by 71–91% for the mutant F77ΔresR at all tested pHs (pH ≤ 7.00), compared with that of strain F77. Furthermore, the intracellular putrescine concentrations were reduced by 40–70% for the mutant F77ΔresR at all tested pHs (pH ≤ 7.00) compared with that of strain F77. Our findings suggested that multiple proteins and metabolic pathways were associated with bacterial acid tolerance and revealed that nueR and resR were involved in acid tolerance based on their modulation of multiple acid tolerance-related physiological functions in strain F77.

Novel Inhibitors of Ornithine Decarboxylase of Leishmania Parasite (LdODC): The Parasite Resists LdODC Inhibition by Overexpression of Spermidine Synthase.

Ornithine decarboxylase (LdODC), a key enzyme in polyamine biosynthesis in Leishmania donovani, catalyzes the conversion of ornithine to putrescine that is finally used for synthesis of spermidine and other polyamines. Inhibition of ornithine decarboxylase is likely to deplete the parasite trypanothione and may result in increased reactive oxygen species (ROS). Sequence as well as structure of LdODC and human ODC shows significant difference; therefore, we have identified novel specific inhibitors of LdODC. These inhibitors are able to inhibit recombinant LdODC and decrease intracellular putrescine concentration showing target specificity. The Ki values of LdODC inhibition do not correlate with IC50 values in Leishmania promastigote possibly due to different stability/pharmacokinetics. These inhibitors, except compound M-5, have only minor effect on Leishmania promastigotes, and IC50 values are several folds higher as compared to Ki values. In case of compound M-5, IC50 value is less than Ki value indicating that the compound may have additional targets. Our studies suggest that the parasite resists these LdODC inhibitors by overexpression of spermidine synthase mRNA.

Mechanism for Regulation of the Putrescine Utilization Pathway by the Transcription Factor PuuR in Escherichia coli K-12

In Escherichia coli, putrescine is metabolized to succinate for use as a carbon and nitrogen source by the putrescine utilization pathway (Puu pathway). One gene in the puu gene cluster encodes a transcription factor, PuuR, which has a helix-turn-helix DNA-binding motif. DNA microarray analysis of an E. coli puuR mutant, in which three amino acid residues in the helix-turn-helix DNA binding motif of PuuR were mutated to alanine to eliminate DNA binding of PuuR, suggested that PuuR is a negative regulator of puu genes. Results of gel shift and DNase I footprint analyses suggested that PuuR binds to the promoter regions of puuA and puuD. The binding of wild-type PuuR to a DNA probe containing PuuR recognition sites was diminished with increasing putrescine concentrations in vitro. These results suggest that PuuR regulates the intracellular putrescine concentration by the transcriptional regulation of genes in the Puu pathway, including puuR itself. The puu gene cluster is found in E. coli and closely related enterobacteria, but this gene cluster is uncommon in other bacterial groups. E. coli and related enterobacteria may have gained the Puu pathway as an adaptation for survival in the mammalian intestine, an environment in which polyamines exist at relatively high concentrations.

γ-Glutamylputrescine Synthetase in the Putrescine Utilization Pathway of Escherichia coli K-12

Glutamate-putrescine ligase (gamma-glutamylputrescine synthetase, PuuA, EC 6.3.1.11) catalyzes the gamma-glutamylation of putrescine, the first step in a novel putrescine utilization pathway involving gamma-glutamylated intermediates, the Puu pathway, in Escherichia coli. In this report, the character and physiological importance of PuuA are described. Purified non-tagged PuuA catalyzed the ATP-dependent gamma-glutamylation of putrescine. The K(m) values for glutamate, ATP, and putrescine are 2.07, 2.35, and 44.6 mm, respectively. There are two putrescine utilization pathways in E. coli: the Puu pathway and the pathway without gamma-glutamylation. Gene deletion experiments of puuA, however, indicated that the Puu pathway was more critical in utilizing putrescine as a sole carbon or nitrogen source. The transcription of puuA was induced by putrescine and in a puuR-deleted strain. The amino acid sequences of PuuA and glutamine synthetase (GS) show high similarity. The molecular weights of the monomers of the two enzymes are quite similar, and PuuA exists as a dodecamer as does GS. Moreover the two amino acid residues of E. coli GS that are important for the metal-catalyzed oxidation of the enzyme molecule involved in protein turnover are conserved in PuuA, and it was experimentally shown that the corresponding amino acid residues in PuuA were involved in the metal-catalyzed oxidation similarly to GS. It is suggested that the intracellular concentration of putrescine is optimized by PuuA transcriptionally and posttranslationally and that excess putrescine is converted to a nutrient source by the Puu pathway.

Poster 031: Evaluation of the Type of Cell Death Induced by Mitomycin C in Human Oral Squamous Cell Carcinoma Cell Lines

Poster 031: Evaluation of the Type of Cell Death Induced by Mitomycin C in Human Oral Squamous Cell Carcinoma Cell Lines

Spermine and L-Name Pretreatment Effects on Polyamine and Nitric Oxide Metabolism in Rat Brain During Seizures

Spermine and L-Name Pretreatment Effects on Polyamine and Nitric Oxide Metabolism in Rat Brain During SeizuresIn the CNS polyamines can exert opposite effects, depending on the concentration and conditions in the cell. Protective or neurotoxic polyamine effects were documented during seizures and repeated CNS excitation. Intensive research of exogenous polyamines effects during seizures induced by numerous agents did not clear up confusions about the duality of effects and the role of polyamines in seizures. In order to understand polyamine modulatory effects in seizures, the importance of NO and polyamine metabolism interdependence and the possible implication of changes of postulated NO and polyamine equillibrium in seizures, the effects of spermine alone and in combination with L-NAME (NOS inhibitor) on seizures induced by pentazol (PTZ) were investigated. To compare the obtained results, the effects of anticonvulsant midazolam on NO production during seizures were also investigated. Seizures were induced by i.p. application of pentazol (100 mg/kg b.w.). Spermine and L-NAME were administered i.p. before PTZ. In the striatum and hippocampus, spermine induced increased NO production (p<0.001) related to values in the group treated by PTZ. Application of L-NAME before spermine and PTZ caused decrease of NO production in comparison with animals treated only by PTZ or spermine and PTZ. L-NAME given before spermine exerts protective effects related to seizures induced by PTZ and to the group treated by spermine, extending the time of seizure symptoms appearance, thus confirming the NO signaling system involvement in spermine effects during seizures. Highly significant PAO activity increase caused by spermine points out the intensified interconversion of spermine into putrescine, in order to maintain the intracellular putrescine concentration. The obtained results prove a strong relationship between the NO signaling system and polyamine metabolism in the brain during seizures and the importance of their changes in this kind of CNS injury.

Tolerance to Putrescine Toxicity in Chinese Hamster Ovary Cells Is Associated with Altered Uptake and Export

When Chinese hamster ovary (CHO) cells were cultured with low concentrations of putrescine (< 5 mM) their cell cycle time increased significantly and a fraction of the cells died. A cell line tolerant to the cytotoxic and growth inhibitory effects of millimolar concentrations of putrescine was developed by growing CHO cells over many months in increasing concentrations of the polyamine. A putrescine-tolerant cell line was obtained which was capable of growing in concentrations up to 25 mMputrescine and displayed growth and cell division rates similar to the original untreated/parental CHO cells. The tolerant cells grown in putrescine displayed relatively high intracellular putrescine yet the cell-associated putrescine concentration was estimated to be 10-fold less than the culture medium level. This high concentration of cellular putrescine diminished within 60 min when the cells were changed to non-putrescine-containing media. The putrescine-tolerant phenotype was further characterized in regards to the mechanisms involved in putrescine uptake, efflux, and biosynthesis. The parental and tolerant cell lines had similar or identical levels of cellular spermidine and spermine and no differences in the acetylated polyamine pools or diamine oxidase activity. The activity of ornithine decarboxylase was also similar in the two cell lines in both the presence and the absence of ornithine. The tolerant cells, however, had a decreased uptake rate for putrescine. The tolerant cell line also showed a greatly enhanced ability to export putrescine, especially when treated with ornithine, suggesting that an upregulated polyamine export system may be present in the tolerant cells which could be responsible for the increased cell survival in high putrescine concentrations. The data are discussed in regard to the potential for identifying the transport protein(s) responsible for the maintenance of nontoxic intracellular concentrations of putrescine in a tolerant cell line grown in putrescine.

Metabolism of l-arginine through polyamine and nitric oxide synthase pathways in proliferative or differentiated human colon carcinoma cells

HT-29 Glc −/+ cells originate from a human colon adenocarcinoma. These cells have been selected in a glucose-free culture medium and switched back in a glucose-containing medium. In this condition, they can spontaneously differentiate after confluency in enterocyte-like cells according to the activity of the brush-border associated hydrolase dipeptidyl peptidase IV. Since l-arginine can generate polyamines which are necessary for cellular proliferation and also differentiation, and nitric oxide with reported anti-proliferative property, the metabolism of this amino acid was examined in proliferative and differentiated isolated HT-29 cells. Proliferative HT-29 cells were characterized by micromolar intracellular concentration of putrescine and millimolar concentration of spermidine and spermine. In these cells, l-arginine is converted to l-ornithine and putrescine and to a minor part to nitric oxide and l-citrulline. Putrescine was taken up by HT-29 cells, leading to the production of a modest amount of spermidine. The diamine was slightly incorporated into cellular proteins and largely released in the incubation medium. The proliferative HT-29 cells take up spermidine and spermine but do not catabolize these polyamines and slightly released spermidine. Differentiation of HT-29 cells is not associated with change in intracellular polyamine content but is paralleled by an almost complete extinction of de novo synthesis of putrescine (due to a dramatic decrease of ornithine decarboxylase activity) and by a reduced release capacity of putrescine. In contrast, putrescine net uptake and incorporation into cellular proteins remained unchanged after differentiation. Furthermore, spermidine and spermine metabolism as well as the circulation of l-arginine in the nitric oxide synthase pathway were also not modified after differentiation. In conclusion, putrescine is the l-arginine-derived molecule, the metabolism of which is specifically and markedly modified when HT-29 cells move from proliferative to differentiated state.

Selective putrescine export is regulated by insulin and ornithine in Reuber H35 hepatoma cells

Cultured Reuber H35 rat hepatoma cells under highly viable serum-free conditions were found to selectively export putrescine from inside the cell into the culture medium, but not spermidine, spermine, or their acetylated derivatives. Even untreated cells, with very low intracellular putrescine levels, constitutively exported significant amounts of only putrescine for a 12 h period. Administration of the phorbol ester TPA (12- O-tetradecanoylphorbol 13-acetate) which markedly elevates ornithine decarboxylase (ODC), did not potentiate putrescine export over what was measured in the unstimulated cultures. However, addition of 1 mM ornithine to the cultures resulted in increased intracellular putrescine (maximum at 4 h) with a marked concomitant increase in putrescine export between 0 and 8 h, after which putrescine export again stopped. Treatment with 10 −7 M insulin yielded intracellular putrescine levels that remained elevated for 36 h along with a continuous and more rapid export of putrescine over the same 36 h time period. When insulin and ornithine were administered together, even higher levels of intracellular putrescine and putrescine export were observed, with putrescine efflux proceeding over the 36 h time-course at the highest observed rates of 1.5 (0–12 h) and 1.0 (12–36 h) nmol/mg total protein per h. Exposure to DFMO, an inhibitor of ODC, depleted intracellular putrescine stores and effectively suppressed putrescine export. There was not a positive correlation between the time-dependent decreases in the intracellular putrescine concentrations and the respective alterations in the rate of putrescine export under a variety of conditions. Furthermore, the drug verapamil was capable of completely inhibiting putrescine export (IC 50 approx. 1 μM) without any change in the level of intracellular putrescine. This data was not consistent with the involvement of simple diffusion of putrescine through the membrane as the major mechanism for putrescine export. The potential mechanisms involved in putrescine export and the role of this process in regulating intracellular polyamine levels, as well as, possible functions of extracellular putrescine are discussed.

Depletion of polyamines prevents the neurotrophic activity of the GABA-agonist THIP in cultured rat cerebellar granule cells.

Effects of polyamine depletion by alpha-difluoromethylornithine (DFMO) were studied on the GABA-agonist mediated enhancement of the morphological development of cultured rat cerebellar granule cells. An increase in the number of neurite extending cells and in the cytoplasmic density of organelles relevant for protein synthesis was observed upon culturing in the presence of 4,5,6,7-tetrahydro-isoxazole[5,4-c]pyridin-3-ol (THIP) for 4 days. The intracellular concentrations of putrescine, spermidine, and spermine in these cultures were similar to the concentrations of the polyamines observed in cultures grown in a plain culture medium for 1, 2, 3 or 4 days, respectively. Upon culturing in the simultaneous presence of THIP and DFMO, the concentrations of putrescine and spermadine were reduced to less than 20% of the levels in the controls. This depletion was associated with a severely impaired morphological development of the granule cell cultures. Thus, the number of neurite extending cells was reduced to 50% of the number in the control cultures upon culturing in the presence of DFMO alone or in combination with THIP. Moreover, the THIP mediated increase in the cytoplasmic density of rough endoplasmic reticulum, Golgi apparatus and different types of vesicles was prevented by the exposure to DFMO.

Putrescine stimulates DNA synthesis in intestinal epithelial cells

Experiments were designed to examine the effects of exogenously supplied putrescine on the synthesis of DNA, RNA, and protein in cultured epithelial cells (IEC-6). Putrescine increased aphidicolin-sensitive DNA synthesis at concentrations as low as 0.3 microM putrescine with maximal stimulation (267% control) at 10 microM. This response appeared to be an effect of increases in the intracellular concentration of putrescine as the intracellular levels of spermidine and spermine did not change over the time period examined. Furthermore, pulse-chase experiments revealed that putrescine that entered the cell was not metabolized to another polyamine or degraded. In addition, 10 microM putrescine enhanced both cycloheximide-sensitive lysine incorporation and actinomycin D-sensitive uridine incorporation, indexes of protein and RNA synthesis, respectively. Incorporation of both lysine and uridine was maximal 12 h after the addition of putrescine, whereas thymidine incorporation was still increasing at 24 h, the longest time point examined. These data suggest that putrescine synthesis and/or transport during mucosal proliferation is directly involved in the stimulation of epithelial DNA, RNA, and protein synthesis.

Downregulation of T cell growth factor production by ornithine decarboxylase and its product putrescine: d,l-α-Difluoromethylornithine suppresses general protein synthesis but augments simultaneously the production of interleukin-2

Treatment of EL-4 lymphoma cells with tetradecanoylphorbol-acetate (TPA), a well-known activator of protein kinase C, induces the production of the T cell growth factor interleukin-2 (IL-2) and the expression of IL-2-specific mRNA within 4–8 h. This system is an ideal model for studies on the induction of a differentiated function in a homogeneous lymphoid cell population by a defined signal. TPA induces also an increase of ornithine decarboxylase (ODC) activity and elevates the intracellular concentrations of putrescine and polyamines within 4–8 h. A similar increase of intracellular putrescine and polyamine concentrations can be achieved by administration of 2 m M putrescine to the culture medium. However, putrescine cannot induce the production of IL-2 in the absence of TPA and cannot reconstitute the IL-2 production in cultures with PGE 2 or cyclosporine A, i.e., two well-known immunosuppressive substances which inhibit ODC activity. Putrescine has rather a counter-regulatory effect as concluded from the observation that the TPA-induced TCGF production and IL-2-specific mRNA expression are augmented (superinduced) by the ODC inhibitor d,l-α-difluoromethylornithine (DFMO) and again suppressed after the administration of putrescine or polyamines to DFMO-treated cultures. The glycolytic activity, general protein synthesis ([ 3H]leucine incorporation), and the cell cycle progression from G 2/M to G 1, in contrast, are inhibited by DFMO and reconstituted by putrescine. This demonstrates that the cells are able to sacrifice to a large extent several vital functions including their general protein synthesis and to devote themselves at the same time to a fulminant production of their functionally most relevant protein IL-2. This process is downregulated by ODC and its product putrescine. A correlation between increased IL-2 production and accumulation of cells in the G 2/M phase was also observed in cultures treated with hydroxyurea or with a combination of amethopterin and adenosine.

Spermidine level and protein synthesis are coregulated in nonproliferating hepatocytes.

The relationship between polyamines and the rate of protein synthesis was investigated in non-proliferating cells: primary cultures of adult rat hepatocytes maintained in serum-free media, and treated with dexamethasone or dexamethasone + insulin. During the second day of culture, polyamine biosynthesis became induced along with the rate of protein synthesis. While the activity of ornithine decarboxylase and the intracellular concentration of putrescine increased only transiently and that of spermine declined, the rise of the protein synthetic rate was paralleled by that of the intracellular spermidine concentration. The polyamine analogue diamino-propanol specifically decreased spermidine content and the protein synthetic rate. The intracellular concentration of spermidine was found subject to tight homeostatic regulation, e.g. not being altered by the addition of up to 1 mM of this polyamine to the culture medium. In contrast, addition of putrescine or spermine led to an increase in their respective intracellular concentrations. These findings indicate that spermidine specifically of the polyamines is involved in protein synthesis in the intact hepatocyte. Moreover, spermidine may mediate part of the trophic action of dexamethasone and insulin upon cultured hepatocytes.

Diethylglyoxal bis(guanylhydrazone): A novel highly potent inhibitor of S-adenosylmethionine decarboxylase with promising properties for potential chemotherapeutic use

Diethylglyoxal bis(guanylhydrazone) (DEGBG), a novel analog of the antileukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) was synthesized. It was found to be the most powerful inhibitor of yeast S-adenosylmethionine decarboxylase (AdoMetDC) so far studied ( K i approx. 9 nM). This property, together with the finding that the compound is a weaker inhibitor of intestinal diamine oxidase than are MGBG and its glyoxal, ethylglyoxal and ethylmethylglyoxal analogs, makes the compound a promising candidate as a polyamine antimetabolite for chemotherapy studies. DEGBG was also found to potentiate the antiproliferative effect of the ornithine decarboxylase inhibitor α-difluoromethyl ornithine against mouse L1210 leukemia cells in vitro. DEGBG increased several-fold the intracellular putrescine concentration of cultured L1210 cells, just as MGBG and its ethylglyoxal analog are known to do. The results strongly suggest that DEGBG is worth further studies. Combined with previous studies, they also made possible the construction of some empirical rules concerning the structure-activity relationships of bis(guanylhydrazone) type inhibitors of AdoMetDC. The identity of DEGBG was confirmed by a single-crystal X-ray analysis and by 1H- and 13C-NMR spectroscopy. It consisted of the same isomer as MGBG and several of its analogs are known to consist of.

Diamine oxidase is important in assessment of polyamine effects on hemopoietic cell proliferation in vitro.

A difference was observed in the effect of difluoromethlyornithine (DFMO), a specific inhibitor of ornithine decarboxylase, on human and murine granulocyte-macrophage precursor cell (CFU-C) proliferation in vitro, in the presence of fetal bovine serum (FBS) and horse serum (HS). A dose of DFMO which almost totally abolished CFU-C colonies in cultures containing FBS had no effect or very little effect on CFU-C in cultures supplemented with HS. This effect could be reversed by aminoguanidine reacting with diamine oxidase (DAO), which is present in FBS but not in HS. The importance of DAO in the assessment of polyamine effects is also suggested by decreased colony formation in cultures containing HS and DFMO only after the addition of this enzyme. Additionally, Mo T cell line cultures containing DFMO demonstrated a substantially lower intracellular concentration of putrescine in the presence of FBS rather than HS.

Role of spermidine in the expression of late markers of adipose conversion. Effects of growth hormone.

Confluent Ob1771 cells treated with an inhibitor of spermidine and spermine synthesis, methylglyoxyal bis(guanylhydrazone), were dependent on putrescine addition for the expression of glycerol-3-phosphate dehydrogenase and acyl-CoA synthetase, which behaved as late markers of adipose conversion. A similar dependence was observed with drug-treated Ob17MT18 and 3T3-F442A preadipocyte cells, but not with non-differentiating 3T3-C2 cells. Studies in drug-treated Ob1771 cells at the mRNA level showed that the parallel expression of mRNAs encoding for glycerol-3-phosphate dehydrogenase and an homologue of serine proteinases of Mr 28,000 [Cook, Groves, Min & Spiegelman (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6480-6484] was also dependent on putrescine addition. Double-isotope experiments with [14C]putrescine and [3H]spermidine, as well as analysis of the polyamine content in drug-treated Ob1771 cells under various conditions, demonstrate after putrescine addition that the expression of late markers of adipose conversion was highly correlated with a 2-fold increase in the intracellular concentration of spermidine. No correlation was observed with changes in the intracellular concentrations of putrescine and spermine. Long-term exposure of untreated Ob1771 cells to growth hormone, which led to the expression of late markers of adipose conversion [Doglio, Dani, Grimaldi & Ailhaud (1986) Biochem. J. 238, 123-129] was also accompanied by the same increase in spermidine concentration, which attained values identical with those determined in drug-treated cells supplemented with putrescine. This observation suggests that the permissive effect of growth hormone on the terminal differentiation of adipose cells might e related to changes in the intracellular concentration of spermidine.

Effect of portal branch ligation on polyamine metabolism in rat liver

The activity of ornithine decarboxylase and the intracellular concentrations of putrescine and spermidine in the non-ligated lobes of the liver increased after portal branch ligation. These changes were followed by increased [ 3H]thymidine uptake into the acid-insoluble fraction of the liver. The induction of ornithine decarboxylase and changes in intracellular polyamines are important biochemical events in liver regeneration, so our results suggest that portal branch ligation causes formation of some stimuli that trigger liver regeneration. Changes were less with ligation than with partial hepatectomy.

Ornithine Decarboxylase Activity and Polyamine Content during Zoospore Germination and Hormone-induced Sexual Differentiation of Achlya ambisexualis

The activity of ornithine decarboxylase (EC 4.1.1.17) and the intracellular pools of putrescine and spermidine were determined during zoospore germination and hormoneinduced sexual morphogenesis in Achlya ambisexualis. The specific activity of ornithine decarboxylase increased approximately 6-fold during zoospore germination and outgrowth in an enriched medium. Ornithine decarboxylase activity exhibited a rapid and transient 5·2-fold increase during hormone-induced differentiation of mycelium cultured in enriched media. In contrast, the enzyme activity did not increase following hormone treatment of mycelium cultured in a minimal medium, yet mycelial differentiation occurred normally. Fluctuations in intracellular concentrations of putrescine and spermidine, in general, correlated with changes in ornithine decarboxylase activity during hormone-induced differentiation and zoospore germination.

An ornithine decarboxylase-deficient mutant of Chinese hamster ovary cells.

We have selected a mutant of Chinese hamster ovary cells that is severely deficient in ornithine decarboxylase activity and is auxotrophic for putrescine. The mutant we obtained (C54) has only 3% of the maximum inducible ornithine decarboxylase activity of the parental Chinese hamster ovary cells and the rate of incorporation of [3H]ornithine into acid-soluble material is correspondingly reduced. The defect in the mutant was recessive in somatic cell hybrids. The mutant requires at least 10(-5) M putrescine in the medium to maintain a normal growth rate. Spermidine and spermine can also serve as a polyamine source, and very high (millimolar) concentrations of ornithine can support a normal growth rate. In the absence of polyamine supplementation the cells stop growing after about 3 population doublings and begin to die after 5 or 6 days. The intracellular concentrations of putrescine and spermidine are depleted after 24 and 48 h, respectively. Spermine levels remain essentially constant for 4 days. Unexpectedly, the Km for ornithine of the enzyme from mutant cells was consistently somewhat lower than the Km for the enzyme from wild type cells. It is possible but not yet certain that the mutant is the result of a mutation in the structural gene coding for ornithine decarboxylase.

Putrescine and the regulation of [formula omitted] decarboxylase in cultured mouse mammary gland

The activity of S- adenosyl- l-methionine decarboxylase S- adenosyl- l-methionine carboxy-lyase, EC 4.1.1.50) purified from mouse mammary gland can be stimulated by physiological concentrations of putrescine ( K a = 5 · 10 -7 M). In contrast, the elevation of the intracellular concentrations of putrescine in cultured mouse mammary tissue by exogenous addition of putrescine or by cultivation of the tissue in hypotonic medium results in the decrease in both the activity and the amount of the enzyme in tissue. Conversely, the addition of 1 mM α-methyl ornithine, an inhibitor of ornithine decarboxylase, causes augmentation of the activity and the amount of S- adenosyl- l-methionine decarboxylase, despite its inhibitory action on insulin-stimulated increase in the concentration of putrescine. These data suggest that putrescine can act as a ‘negative’ regulator of the enzyme in this system.