Polyamine metabolism in an obligately alkalophilic Bacillus alcalophilus that grows at pH 11.0

Bacillus alcalophilus, an obligately alkalophilic bacterium that grows at pH 11.0, has an intracellular pH of 9.5 or less. Unlike all other living organisms, polyamines (putrescine, spermidine and spermine) in B. alcalophilus, if present, will be largely unprotonated. HPLC analysis indicated that spermidine is the major polyamine in B. alcalophilus, accounting for more than 90% of total polyamines, and the level of spermidine varies during growth. Ornithine decarboxylase activity was not detectable in B. alcalophilus under all conditions examined. When [3H]arginine was added to the culture medium, the radioactivity can be recovered from polyamine pool; the distribution is 3% for putrescine, 94% for spermidine, and 3% for spermine, suggesting the presence of arginine pathway for polyamine biosynthesis. The polyamine transport system in B. alcalphilus appears to be Na+-dependent and is highly sensitive to the inhibition of gramicidin S and valinomycin.     

Polyamines as physiological substrates for transglutaminases

When normal human blood lymphocytes are treated with mitogen in the presence of [3H]putrescine, label is incorporated into a few cellular proteins. Labeled N-(gamma-glutamyl) putrescine, N1-(gamma-glutamyl)spermidine, and N8-(gamma-glutamyl)spermidine were identified in exhaustive proteolytic digests of the cellular protein fraction. The enzyme-mediated clotting of rat seminal plasma to which 14C-labeled spermidine and spermine are added is accompanied by incorporation of the polyamines into a number of seminal plasma proteins. Proteolytic digests of the protein fraction from this clotted seminal plasma contain labeled N1-(gamma-glutamyl)spermidine, N8-(gamma-glutamyl)spermidine, N1,N8-bis(gamma-glutamyl)spermidine, N1-(gamma-glutamyl)spermine, and N1,N12-bis(gamma-glutamyl)spermine. These findings support a proposal that polyamines serve as substrates for transglutaminases both in cells and in an extracellular fluid. They show differences in cellular and extracellular substrate properties of the polyamines and indicate cross-linking through these amines in the extracellular system, but provide no evidence for such cross-linking in the cells.     

New Metabolites of Streptomyces alboniger

A mutant unable to grow under acidic conditions was isolated from the acidophilic heterotrophic bacterium Acidiphilium facilis 24R. The growth of the mutant could be fully restored by the addition of spermidine or lysine at the concentration of 100 μm. The HPLC analysis of polyamine composition showed that spermidine and putrescine were major polyamine components in the parental strain. In the mutant strain, putrescine was replaced by cadaverine. It was found that some polyamines in the cells were conjugated with the other cell components. The growth of the bacterium in the medium below pH 4.5 was inhibited in the presence of α-methylornithine or methylglyoxal-bis(guanylhydrazone), which are inhibitors of rate-limiting enzymes involved in the biosynthesis of polyamines. The growth of the bacterium that had been inhibited in the presence of inhibitors could be fully restored by the addition of putrescine or spermidine. On the basis of these results, it was concluded that polyamines have a significant role in the growth of Acidiphilium facilis 24R under acidic conditions.     

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.     

Androgenic control of polyamine concentrations in rat epididymis.

Unilateral orchidectomy resulted in a significant decrease in tissue content of putrescine and polyamines. However, no differences were detected when the results were expressed in terms of ng g-1 tissue. At 48 h after bilateral orchidectomy, a significant decrease in putrescine content was observed, but spermidine and spermine content were unaffected. The observed decrease in putrescine was prevented by treatment with testosterone propionate, but neither spermidine nor spermine were affected. Bilateral orchidectomy resulted in a significant decrease in the tissue content of putrescine, spermidine and spermine after 7 days. Treatment with testosterone propionate increased the content of putrescine, spermidine and spermine in the epididymis by about 200%, 92% and 34%, respectively. When results were expressed as nmol g-1, a significant decrease after castration in putrescine and spermidine, but not in spermine, was observed. Treatment with testosterone propionate restored putrescine concentration, but had no effect on spermidine and spermine concentrations. In castrated rats treated with testosterone propionate, the anti-androgen flutamide abolished the effect of the androgen on putrescine and spermidine content, but there was no effect on spermine. Acetylputrescine was not detected in the epididymis, while acetylpolyamines were detected at much lower concentrations than polyamines. After bilateral orchidectomy there was a decrease in the tissue content of all acetylpolyamines and an increase in their tissue concentration. The effect of castration on acetylpolyamine content was reversed by testosterone propionate treatment. We conclude that an active synthesis of polyamines occurs in the rat epididymis, and that this process depends upon the androgen environment. Regulation of ornithine decarboxylase activity appears to be the main step that is controlled by androgens.     

Excretion of putrescine and spermidine by the protein encoded by YKL174c (TPO5) in Saccharomyces cerevisiae

The properties of the protein encoded by YKL174c (TPO5) were studied. It was found that TPO5 excretes putrescine effectively and spermidine less effectively. Gamma-aminobutyric acid slightly inhibited the excretion of putrescine, but basic amino acids did not affect excretion, suggesting that TPO5 preferentially recognizes polyamines. Accordingly, yeast cells transformed with the plasmid encoding YKL174c (TPO5) were resistant to toxicity caused by 120 mm putrescine or by 3 mm spermidine, and a mutant with a disrupted YKL174c (TPO5) gene was sensitive to toxicity by 90 mm putrescine. The growth of this mutant was faster than that of the wild-type strain. In parallel, there was an increase in putrescine and spermidine content of the YKL174c (TPO5) mutant compared with wild-type. It is noted that TPO5 functions as a suppressor of cell growth by excreting polyamines. The level of YKL174c (TPO5) mRNA was increased by the addition of polyamines to the medium. The degree of induction of the mRNA was spermine > spermidine > putrescine. The subcellular localization of TPO5 was determined by immunostaining of hemagglutinin-tagged TPO5, and it was found on Golgi or post-Golgi secretory vesicles. Excretion of putrescine and spermidine by TPO5 was reduced in cells that have mutations in the secretory or endocytic pathways, indicating that both processes are involved in the excretion of polyamines.     

Hydroxyl radical scavenging and singlet oxygen quenching properties of polyamines

Polyamines (cadaverine, putrescine, spermidine, spermine) have been shown to be present in all prokaryotic and eukaryotic cells, and proposed to be important anti-inflammatory agents. Some polyamines at high concentrations are known to scavenge superoxide radicals in vitro. We have investigated the possible antioxidant properties of polyamines and found that polyamines, e.g., cadaverine, putrescine, spermidine and spermine do not scavenge superoxide radicals at 0.5, 1.0 and 2 mM concentrations. However, polyamines were found to be potent scavengers of hydroxyl radicals. Hydroxyl radicals were produced in a Fenton type reaction and detected as DMPO-OH adducts by electron paramagnetic resonance spectroscopic technique. Spermine, spermidine, putrescine and cadaverine inhibited DMPO-OH adduct formation in a dose dependent manner, and at 1.5 mM concentration virtually eliminated the adduct formation. The *OH-dependent TBA reactive product of deoxyribose was also inhibited by polyamines in a dose-dependent manner. Polyamines were also found to inhibit the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxy 1 (TEMPO) formation. 1O2 was produced in a photosensitizing system using Rose Bengal or Methylene Blue as photosensitizers, and was detected as TEMP-1O2 adduct by EPR spectroscopy. Spermine or spermidine inhibited the 1O2-dependent TEMPO formation maximally to 50%, whereas putrescine or cadaverine inhibited this reaction only up to 15%, when used at 0.5 and 1 mM concentrations. These results suggest that polyamines are powerful. OH scavengers, and spermine or spermidine also can quench singlet oxygen at higher concentrations.     

NaCl处理下大麦根系质膜微囊结合多胺与Na+/H+逆向运输的关系

NaCl10 0mmol/L处理结合外施Spd和Put以及多胺代谢抑制剂邻二氮杂菲和MGBG ,以改变大麦根系质膜结合多胺种类和数量 ,研究了大麦根系质膜上两种形态多胺与质子泵和Na /H 逆向运输活性的关系。结果发现 ,NaCl处理后大麦根系质膜微囊上存在Na /H 逆向运输活性。质膜H ATPase活性与膜上非共价键结合多胺数量间呈显著正相关 ,其中 ,Spd对H ATPase的激活程度大于Put。膜蛋白上共价键结合多胺数量与Na /H 逆向运输活性间呈极显著正相关关系 ,说明大麦根系质膜Na /H 逆向运输的盐诱导似乎与Na /H 逆向运输蛋白的从头合成有关。此外 ,质膜Na /H 逆向运输活性仅与膜蛋白上共价键结合多胺数量有关 ,而与多胺种类关系不大。     

Cloning and characterization of spermidine synthase and its implication in polyamine biosynthesis in Helicobacter pylori strain 26695

The HP0832 (speE) gene of Helicobacter pylori strain 26695 codes for a putative spermidine synthase, which belongs to the polyamine biosynthetic pathway. Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), which serves as an aminopropyl donor. The deduced amino acid sequence of the HP0832 gene shares less than 20% sequence identity with most spermidine synthases from mammalian cells, plants and other bacteria. In this study, the HP0832 open reading frame (786 bp) was cloned into the pQE30 vector and overexpressed in Escherichia coli strain SG13009. The resulting N-terminally 6xHis-tagged HP0832 protein (31.9 kDa) was purified by Ni-NTA affinity chromatography at a yield of 15 mg/L of bacteria culture. Spermidine synthase activity of the recombinant protein was confirmed by the appearance of spermidine after incubating the enzyme with putrescine and dcSAM. Substrate specificity studies have shown that spermidine could not replace putrescine as the aminopropyl acceptor. Endogenous spermidine synthase of H. pylori was detected with an antiserum raised against the recombinant HP0832 protein. H. pylori strain 26695 contains putrescine and spermidine at a molar ratio of 1:3, but no detectable spermine or norspermidine was observed, suggesting that the spermidine biosynthetic pathway may provide the main polyamines in H. pylori strain 26695.     

Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.     

Active transport and metabolic characteristics of polyamines in the rat lens

Putrescine, spermidine and spermine were transported into the rat lens against a concentration gradient. This process appeared to be energy-dependent and involved a carrier system different from those for amino acids. Competition experiments suggested that the three polyamines were transported by the same system or very similar systems. Incorporated spermine was converted to spermidine and putrescine, and spermidine was converted to putrescine. In contrast, the conversion of putrescine to spermidine and spermine, or the conversion of spermidine to spermine was not observed. Furthermore, ornithine was not utilized for the synthesis of putrescine. These metabolic characteristics of the polyamines in the rat lens were correlated with the extremely low activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase. Other enzymes of polyamine metabolisms, however, were relatively active. In conclusion, the lens has a very low ability for the de novo synthesis of polyamines. The polyamines in the lens are considered to be supplied form the surrounding intraocular fluid by an active transport system specific for polyamines.     

Polyamines and the Accumulation of Ribonucleic Acid in Some Polyauxotrophic Strains of Escherichia coli

The cellular accumulations of polyamines and ribonucleic acid (RNA) were compared in the polyauxotrophic mutants of Escherichia coli strain 15 TAU and E. coli K-12 RC(re1) met(-) leu(-). Putrescine, spermidine, and their monoacetyl derivatives were the main polyamines in both strains, when grown in glucose-mineral medium. No significant degradation of either (14)C-putrescine or (14)C-spermidine was found in growing cultures of strain 15 TAU, which requires thymine, arginine, and uracil for growth. Experiments with this organism showed that in a variety of different incubation conditions, which included normal growth, amino acid starvation, inhibition by chloramphenicol or streptomycin, or thymine deprivation, a close correlation was seen between the intracellular accumulation of unconjugated spermidine and RNA. In the presence of arginine, the antibiotics stimulated the production of putrescine and spermidine per unit of bacterial mass. Deprivation of arginine also resulted in an increase in the production of putrescine per unit of bacterial mass, most of which was excreted into the growth medium. However, in this system the antibiotics reduced the synthesis of putrescine. Furthermore, streptomycin caused a rapid loss of cellular putrescine into the medium. The latter effect was not seen in anaerobic conditions or in a streptomycin-resistant mutant of 15 TAU. Methionine added to the growth medium of growing TAU not only markedly increased the total production of spermidine, but also increased both the intracellular concentration of spermidine and the accumulation of RNA. Exogenous spermidine extensively relaxed RNA synthesis in amino acid-starved cultures of 15 TAU. Analysis in sucrose density gradients showed that the RNA accumulated in the presence of spermidine was ribosomal RNA.Cells of E. coli K-12 RC(rel) met(-) leu(-), grown in a complete medium, had approximately the same ratio of free spermidine to RNA as did strain 15 TAU. However, the relaxed strain showed a much lower ratio of putrescine to spermidine than the stringent 15 TAU. Omission of methionine stopped spermidine synthesis and markedly increased both the intracellular accumulation and the total production of putrescine. It seems that a high intracellular level of spermidine acts as a feedback inhibitor in the biosynthesis of putrescine in this strain. The hypothesis that the intracellular concentration of polyamines may participate in the control of the synthesis of ribosomal RNA in bacteria is discussed.     

Evidence for the existence of distinct transporters for the polyamines putrescine and spermidine in B16 melanoma cells

The uptake of intracellular putrescine and spermidine was examined in B16 melanoma cells. It was found that difluoromethylornithine preferentially induced putrescine transport (28-fold) compared to that for spermidine (3.5-fold). Putrescine uptake was partially Na+ dependent, whereas spermidine uptake was not. Inhibition studies with the two polyamines showed that putrescine was a poor competitive inhibitor of spermidine uptake, exhibiting a Ki of 69-75 microM, whereas the estimated Km for putrescine uptake was only 5.36 microM. By contrast, spermidine inhibition of putrescine transport produced a non-linear Eadie-Scatchard plot suggesting that putrescine was taken up by a spermidine-sensitive and a spermidine-insensitive process. The estimated spermidine Ki for inhibition of the spermidine-sensitive process was 0.125 microM. Using a series of polypyridinium quaternary salts to inhibit transport, no correlation between inhibition of putrescine uptake and inhibition of spermidine uptake was seen. Finally, the photoaffinity label, 1,12-di(N5-azido-2-nitrobenzoyl)spermine selectively inactivated the putrescine transporter(s) without affecting spermidine uptake. From these observations, it was concluded that multiple polyamine transporters are present on B16 melanoma cells and that separate, distinct transporter(s) account for the uptake of putrescine and spermidine in this cell-line following induction with difluoromethylornithine. The present of different transporters for the two polyamines indicates that expression of uptake activity for putrescine and spermidine may be under separate cellular control.     

Synthesis and content of polyamines in bloodstream Trypanosma brucei

The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [3H]ornithine, [14C]arginine and [14C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [4C]methionine. Putrescine and sperimidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous 14C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Localization and biosynthesis of polyamines in insulin-producing cells.

Two recently developed fluorescence cytochemical methods, specific for spermidine and spermine, were used to localize polyamines in the endocrine pancreas. The polyamines were restricted to the insulin-producing beta-cells and were mainly associated with the secretory granules. Chemical polyamine determinations carried out on isolated rat and mouse pancreatic islets revealed large amounts of polyamines. Compared with extracts of whole pancreas, the islets contained very high concentrations of spermine relative to spermidine. Biosynthesis of polyamines from [3H]ornithine or from [3H]putrescine in isolated islets was significantly stimulated at high glucose concentrations. Moreover, significant incorporation of label from [3H]putrescine was also detected in gamma-aminobutyric acid. This incorporation, however, was not stimulated by high glucose. Possible roles for polyamines associated with the secretory granules in insulin-producing cells are discussed.     

Changes in endogenous polyamine levels are associated with differentiation in Dictyostelium discoideum.

This is the first report correlating levels of polyamines and its fractions with differentiation in Dictyostelium discoideum. Temporal changes in endogenous levels of free, conjugated and bound putrescine, spermidine and spermine were analysed at critical stages of morphogenesis in this organism. No spermine was found at any given stage and putrescine was the most abundant polyamine. There was a sharp increase in the levels of both free (and total) and conjugated forms of putrescine and spermidine at the slug stage as compared to the growth phase. The levels of putrescine and spermidine were found to be higher in isolated prespore cells as compared to the prestalk cells. Remarkably, the levels of polyamine decreased at the early culminant stage. Data suggest that a moderate level of polyamines is needed for growth but it is important to have high levels of polyamines at the time of differentiation.     

Studies on the accumulation of putrescine and spermidine in Escherichia coli

The rate of accumulation of the polyamines spermidine and putrescine by E. coli depended on growth rate. Spermidine accumulation was faster in chemostat cultures with high dilution rates than in those with low dilution rates and was slower in bacteria that had been grown for several generations with either putrescine or spermidine, suggesting that the spermidine-uptake system was repressed by exogenous polyamines. The uptake of spermidine required metabolic energy. Thus accumulation occurred in an energy-starved unc strain only upon addition of glucose (or D-lactate to a smaller extent). With glucose present accumulation occurred in an unc, frd strain under anaerobic conditions, suggesting that ATP drives uptake. However, accumulation was generally sensitive to carbonylcyanide m-chlorophenylhydrazone (CCCP), indicating that the proton motive force was involved in uptake. Unlike spermidine, putrescine accumulation was faster in slow-growing than in fast-growing cultures. This may have been due to greater efflux of putrescine at faster growth rates. Accumulation of putrescine was faster following prolonged growth with either putrescine or spermidine, suggesting induction of the putrescine-uptake system by exogenous polyamines. Like spermidine accumulation, putrescine accumulation required metabolic energy. Accumulation was insensitive to CCCP and occurred only when glucose was added to energy-starved unc bacteria, suggesting that high-energy bonds may drive the uptake of putrescine.     

Formation of Polyamines in the Rumen of Goats During Growth

Ornithine decarboxylase (E.G. 4.1.1.17) and S-adenosylmethionine decarboxylase (E.G. 4.1.1.50) and their products putrescine, spermidine and spermine were estimated in the rumen liquid from 3 groups of growing kids and 23 adult goats. Polyamines were also estimated in the feedstuff used. Marked differences in polyamine synthesis in rumen liquid were observed between the different groups of kids. Two groups of kids growing up together with adult goats had at an age of 2–4 months a peak of a few days duration in enzyme activity as well as in polyamine concentration. In these groups ornithine decarboxylase activity reached maximal values of 158±79 s (n = 4) and 100 (66–117) (n = 3) nmol[14CO2]/ml rumen liquid/h at an age of 120 and 77 days, respectively. The corresponding activity in rumen liquid from kids who were isolated from other animals was only about 1/10 of this value. By comparison ornithine decarboxylase activity in adult goats was 30.7±20 (n = 43) nmol[14CO]/ml/h. In rumen liquid from kids grown up together with adults, concentrations of the polyamines reached maximum at about the same time as ornithine decarboxylase activity. The mean maximal concentration of putrescine in the 2 groups was about 350 and 500 nmol/ml, while the corresponding value for spermidine was about 200 nmol/ml in both groups. Relatively constant and high concentration of polyamines were present in the feedstuff used. However, in growing kids the ruminai putrescine and spermidine concentration at times far exceeded those that could be accounted for by the estimated intake of polyamines by the food. The results therefore strongly indicate that polyamines are formed in considerable amounts in rumen content of kids during the phase of rapid growth. Results from a few experiments with calves also indicate that this may be true for cattle. polyamines; putrescine; spermidine; spermine; ornithine-decarboxylase; rumen liquid.