Absence Of Putrescine Research Articles

Putrescine-dependent re-localization of TvCP39, a cysteine proteinase involved in Trichomonas vaginalis cytotoxicity.

Polyamines are involved in the regulation of some Trichomonas vaginalis virulence factors such as the transcript, proteolytic activity, and cytotoxicity of TvCP65, a cysteine proteinase (CP) involved in the trichomonal cytotoxicity. In this work, we reported the putrescine effect on TvCP39, other CP that also participate in the trichomonal cytotoxicity. Parasites treated with 1,4-diamino-2-butanone (DAB) (an inhibitor of putrescine biosynthesis), diminished the amount and proteolytic activity of TvCP39 as compared with untreated parasites. Inhibition of putrescine biosynthesis also reduced ∼80% the tvcp39 mRNA levels according to RT-PCR and qRT-PCR assays. Additionally, actinomycin D-treatment showed that the tvcp39 mRNA half-life decreased in the absence of putrescine. However, this reduction was restored by exogenous putrescine addition, suggesting that putrescine is necessary for tvcp39 mRNA stability. TvCP39 was localized in the cytoplasm but, in DAB treated parasites transferred into exogenous putrescine culture media, TvCP39 was re-localized to the nucleus and nuclear periphery of trichomonads. Interestingly, the amount and proteolytic activity of TvCP39 was recovered as well as the tvcp39 mRNA levels were restored when putrescine exogenous was added to the DAB-treated parasites. In conclusion, our data show that putrescine regulate the TvCP39 expression, protein amount, proteolytic activity, and cellular localization.

Crescimento e conteúdo de prolina em plântulas de guandu submetidas a estresse osmótico e à putrescina exógena

O objetivo deste trabalho foi verificar o efeito da aplicação da poliamina putrescina no acúmulo de prolina e no crescimento inicial de plântulas de guandu cultivadas sob estresse hídrico ou salino, e avaliar se esta poliamina pode ser usada como atenuadora desses estresses. Sementes de duas cultivares de guandu, BRS Mandarim e Caqui, foram germinadas em caixas gerbox forradas com papel de filtro umedecido com 0,0, -0,2, -0,3, -0,4, -0,5 MPa de PEG 6000 ou 0, 20, 40, 60, 80 e 120 mmol L-1 de NaCl, na ausência (0 mmol L-1) ou na presença (0,5 mmol L-1) de putrescina. Utilizou-se o delineamento inteiramente casualizado, em arranjo fatorial 2x5x2 e 2x6x2 (duas cultivares, cinco níveis de deficiência hídrica ou seis níveis de estresse salino, e presença ou ausência de putrescina), com quatro repetições. O crescimento das plântulas é acentuadamente reduzido pelas condições de restrição hídrica e salinidade. A putrescina exógena incrementa, sob ambos os estresses, os teores de prolina nas plântulas de guandu, especialmente na cultivar BRS Mandarin, e atenua os efeitos da deficiência hídrica moderada nas cultivares estudadas. Os estresses de restrição hídrica e de salinidade causam acúmulo de prolina na parte aérea e nas raízes de ambas as cultivares. A prolina pode ser considerada um bom indicador bioquímico e fisiológico desses estresses em plântulas de guandu.

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance in Escherichia coli

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance were studied using a polyamine biosynthesis and uptake deficient Escherichia coli KK3131 transformed with pACYC184 containing potFGHI or puuP. Putrescine uptake activity of E. coli KK3131 transformed with pACYC184-PotFGHI was higher than that of E. coli 3131 transformed with pACYC-PuuP when cells were cultured in the absence of putrescine. Putrescine uptake by PotFGHI was both ATP and membrane potential dependent, while that by PuuP was membrane potential dependent. Feedback inhibition by polyamines occurred at the PotFGHI uptake system but not at the PuuP uptake system. Expression of PuuP was reduced in the presence of PuuR, a negative regulator for PuuP, and expression of PuuR was positively regulated by glucose, which reduces the level of cAMP. The complex of cAMP and CRP (cAMP receptor protein) inhibited the expression of PuuR in the absence of glucose. Thus, the growth rate of E. coli KK3131 in the presence of both 0.4% (22.2 mM) glucose and 10 mM putrescine was in the order of cells transformed with pACYC-PotFGHI > pACYC-PuuP > pACYC-PuuP + PuuR, which was parallel with the polyamine content in cells. The results indicate that PotFGHI is necessary for rapid cell growth in the presence of glucose as an energy source. When glucose in medium was depleted, however, PuuP was absolutely necessary for cell growth in the presence of putrescine, because accumulation of putrescine to a high level by PuuP was necessary for utilization of putrescine as an energy source.

Binding and inhibition of human spermidine synthase by decarboxylated S-adenosylhomocysteine.

Aminopropyltransferases are essential enzymes that form polyamines in eukaryotic and most prokaryotic cells. Spermidine synthase (SpdS) is one of the most well-studied enzymes in this biosynthetic pathway. The enzyme uses decarboxylated S-adenosylmethionine and a short-chain polyamine (putrescine) to make a medium-chain polyamine (spermidine) and 5'-deoxy-5'-methylthioadenosine as a byproduct. Here, we report a new spermidine synthase inhibitor, decarboxylated S-adenosylhomocysteine (dcSAH). The inhibitor was synthesized, and dose-dependent inhibition of human, Thermatoga maritima, and Plasmodium falciparum spermidine synthases, as well as functionally homologous human spermine synthase, was determined. The human SpdS/dcSAH complex structure was determined by X-ray crystallography at 2.0 Å resolution and showed consistent active site positioning and coordination with previously known structures. Isothermal calorimetry binding assays confirmed inhibitor binding to human SpdS with K(d) of 1.1 ± 0.3 μM in the absence of putrescine and 3.2 ± 0.1 μM in the presence of putrescine. These results indicate a potential for further inhibitor development based on the dcSAH scaffold.

Putrescine differently influences the effect of salt stress on polyamine metabolism and ethylene synthesis in rice cultivars differing in salt resistance

Effects of salt stress on polyamine metabolism and ethylene production were examined in two rice (Oryza sativa L.) cultivars [I Kong Pao (IKP), salt sensitive; and Pokkali, salt resistant] grown for 5 d and 12 d in nutrient solution in the presence or absence of putrescine (1 mM) and 0, 50, and 100 mM NaCl. The salt-sensitive (IKP) and salt-resistant (Pokkali) cultivars differ not only in their mean levels of putrescine, but also in the physiological functions assumed by this molecule in stressed tissues. Salt stress increased the proportion of conjugated putrescine in salt-resistant Pokkali and decreased it in the salt-sensitive IKP, suggesting a possible protective function in response to NaCl. Activities of the enzymes ornithine decarboxylase (ODC; EC 4.1.1.17) and arginine decarboxylase (ADC; EC 4.1.1.19) involved in putrescine synthesis were higher in salt-resistant Pokkali than in salt-sensitive IKP. Both enzymes were involved in the response to salt stress. Salt stress also increased diamine oxidase (DAO; 1.4.3.6) and polyamine oxidase (PAO EC 1.5.3.11) activities in the roots of salt-resistant Pokkali and in the shoots of salt-sensitive IKP. Gene expression followed by reverse transcription-PCR suggested that putrescine could have a post-translational impact on genes coding for ADC (ADCa) and ODC (ODCa and ODCb) but could induce a transcriptional activation of genes coding for PAO (PAOb) mainly in the shoot of salt-stressed plants. The salt-resistant cultivar Pokkali produced higher amounts of ethylene than the salt-sensitive cultivar IKP, and exogenous putrescine increased ethylene synthesis in both cultivars, suggesting no direct antagonism between polyamine and ethylene pathways in rice.

Conformational Stabilization of Rat S-Adenosylmethionine Decarboxylase by Putrescine

The activity and processing of mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is stimulated by putrescine. To obtain new insights into the mechanism through which putrescine stimulates AdoMetDC, we investigated conformational changes in rat prostate AdoMetDC in the presence or absence of putrescine. We examined the reactivity of purified rat prostate AdoMetDC to the SH-reagent iodoacetic acid (IAA) and its susceptibility to proteolysis in the presence or absence of putrescine using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The activity of AdoMetDC treated with IAA in the absence of putrescine was reduced, but about 80% of its activity remained after treatment with IAA in the presence of putrescine. In the presence of putrescine, IAA incorporation was 1.9 mol IAA/mol of AdoMetDC α-subunit, while there was no incorporation of IAA in the β-subunit of AdoMetDC. In the absence of putrescine, 5.0 mol of IAA/mol of α-subunit and 0.9 mol of IAA/mol of β-subunit were incorporated. Only Cys292 and Cys310 were carboxymethylated by IAA in the presence of putrescine. In contrast, in the absence of putrescine all cysteines were carboxymethylated by IAA. In addition, putrescine slowed the rate of AdoMetDC degradation by trypsin. These results demonstrate that the conformation of AdoMetDC purified from rat prostate is stabilized by putrescine.

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.

Cloning and kinetic characterization of the Trypanosoma cruziS-adenosylmethionine decarboxylase

The gene for S-adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in the biosynthesis of polyamines, has been cloned from a Trypanosoma cruz cDNA library. The cDNA clone contains a 1.1 kb open reading frame predicted to encode a 42 kDa protein that shares 31% sequence identity to the human proenzyme. T. cruzi AdoMetDC expressed and purified from E. coli is auto-catalytically processed into two subunits of 32 kDa ( α) and 10 kDa ( β). The catalytic activity of the purified recombinant enzyme is activated by the addition of putrescine to the reaction. To determine the effect of putrescine on the kinetics of the reaction, the velocity data collected at various substrate and putrescine concentrations were fit to the rate equation describing a non-essential activator. In the presence of fully saturating putrescine, k cat increases by 9-fold over the unactivated rate to 0.06 s −1. The model derived K m for AdoMet is 0.05 mM in the absence of putrescine and the model-derived K d for putrescine binding to free enzyme is 2.5 mM. The K m for AdoMet increases by ≈2-fold when the enzyme is fully saturated with putrescine. Unlike human AdoMetDC, cadaverine activates the T. cruzi enzyme to a similar extent as putrescine.

Homospermidine synthase of Rhodopseudomonas viridis: Substrate specificity and effects of the heterologously expressed enzyme on polyamine metabolism of Escherichia coli.

Homospermidine synthase (HSS) catalyses the formation of the polyamine homospermidine from 2 mol of putrescine. The general and kinetic properties of purified HSS from Rhodopseudomonas viridis are given and compared with those of the respective enzymes from other sources. The R. viridis enzyme is shown to catalyse a number of side reactions: (I) In the presence of putrescine or spermidine as donors of the 4-aminobutyl moiety, various homologous diamines are transformed into the respective N-(4-aminobutyl)derivatives. (II) In the absence of putrescine, spermidine as a substrate yields homospermidine, putrescine and diaminopropane as reaction products. The mechanism of the reactions catalysed by HSS and its role in the formation of uncommon bacterial polyamines are discussed. Overexpression of the homospermidine synthase (hss) gene in Escherichia coli revealed the formation of two HSS-products, homospermidine and N-(4-aminobutyl)-cadaverine, which are absent from wild-type E. coli. Expression of the hss gene in E. coli does not dramatically affect the pool concentrations of the cellular polyamines.

Expression of mammalian S-adenosylmethionine decarboxylase in Escherichia coli. Determination of sites for putrescine activation of activity and processing.

Mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is known to be regulated by putrescine in two ways: (a) acceleration of the rate of conversion of the proenzyme into the mature enzyme in a reaction that forms the pyruvate prosthetic group and (b) activation of the mature enzyme activity. To determine sites of putrescine interaction with AdoMetDC, putrescine stimulation of both proenzyme processing and catalytic activity was tested with mutant AdoMetDCs in which specific amino acid residues, conserved between mammalian and yeast AdoMetDCs, had been altered by site-directed mutagenesis. Mutations E178Q or E256Q (and the previously reported mutation E11Q (Stanley, B. A., and Pegg, A. E. (1991) J. Biol. Chem. 266, 18502-18506)) abolished stimulation by putrescine without an effect on the processing rate in the absence of putrescine. Mutations E11K, as well as Y112A and L259Stop, completely abolished processing regardless of putrescine concentration, whereas mutation E133Q conferred an absolute putrescine requirement for processing to occur. Mutation E132Q, E135Q, E183Q, or D185N had no effect on proenzyme processing. The effects of mutations on enzyme activity were determined using AdoMetDC protein produced in Escherichia coli and purified by affinity chromatography. Mutation E11Q completely inactivated the enzyme, mutation E133Q reduced the catalytic constant by > 10(4), and mutation E256Q produced a 20-fold decrease. Putrescine did not stimulate the activity of mutants E178Q and E256Q but did activate mutants E133Q and E183Q. It is concluded that residues Glu-11, Glu-178, and Glu-256 are critical residues in the putrescine stimulation of AdoMetDC proenzyme processing and that Glu-178 and Glu-256 are critical for putrescine stimulation of AdoMetDC catalytic activity.

Biosynthesis of pyrrolizidine alkaloids: putrescine and spermidine are essential substrates of enzymatic homospermidine formation

sym-Homospermidine, the first pathway-specific intermediate of pyrrolizidine alkaloid (PA) biosynthesis, is formed from two moles of putrescine in an NAD+-dependent reaction catalyzed by homospermidine synthase (HSS). In the overall process catalyzed by HSS, NAD+ seems to function as hydride acceptor in the first part of the reaction and subsequently as hydride donor in the second part. This was proved with chirally Cl-deuterated putrescines. Both a bacterial HSS (isolated from Rhodopseudomonas viridis) and the enzyme from PA-producing Eupatorium cannabinum transformed (R)-[1-2H]putrescine and (S)-[1-2H]-putrescine into homospermidine with a 100% retention of deuterium. Studies with deuterated and 14C-labeled substrates revealed that in the presence of putrescine spermidine is a substrate of HSS. The putrescine semialdehyde moiety of spermidine is combined with putrescine and 1,3-diaminopropane is released. Kinetic studies with plant HSS documented that the putrescine semialdehyde moiety of spermidine is incorporated into homospermidine with the same affinity (Km) and activity (Vmax) as putrescine. Plant HSS is highly specific for putrescine and spermidine; spermidine is not a substrate in the absence of putrescine. It is suggested that in the biosynthesis of homospermidine and thus PAs, one of the two C4 units is derived from spermidine and the other one from putrescine. A scheme of the HSS-catalyzed reaction is proposed that fully accounts for the enzymatic data and the results of previous tracer studies. Bacterial HSS is less substrate specific and not as well adapted as plant HSS to accept spermidine as a substrate.

Selectivity of polyamines in triplex DNA stabilization.

Triplex DNA has been recently studied as an anti-gene strategy to suppress the transcription of specific genes. A major challenge in this area is to stabilize triplex DNA at physiological conditions. We studied the effects of putrescine, spermidine, spermine, their synthetic homologs, and their acetyl derivatives on triple- and double-stranded structures formed from poly(dA) and poly(dT) by measuring their respective melting temperatures. In the presence of polyamines, the absorbance (A) versus temperature (T) profile showed two transitions: Tm1, corresponding to triplex-->duplex+single-stranded DNA, and Tm2, corresponding to duplex melting. In the presence of 0.5 mM putrescine, Tm1 and Tm2 were 44.8 and 71 degrees C, respectively, in 10 mM sodium cacodylate buffer (pH 7.2). In contrast, triplex DNA was not detectable when the A versus T profile of the polynucleotides was monitored in the absence of putrescine. Tm2 was also lower (55 degrees C) in the absence of putrescine. With 2.5 microM spermidine or 0.1 microM spermine, Tm1 values were 42.8 and 54.4 degrees C and Tm2 values were 65 and 82 degrees C, respectively. As the concentration of polyamine was increased, the difference between Tm1 and Tm2 decreased, and both melting transitions merged into one Tm, corresponding to the melting of triplex DNA to single strands. In a series of putrescine homologs, H2N (CH2)nNH2 where n = 2-6 (n = 4 for putrescine), H2N(CH2)3NH2 was the most effective diamine to stabilize the poly(dA).2poly(dT) triplex. At 10 mM concentration, diaminopropane stabilized the triplex DNA such that the Tm1 was 10 degrees C higher than that in the presence of an equimolar concentration of putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Ornithine Decarboxylase Gene Expression, Polyamine Levels, and DNA Synthetic Rates by All-Trans-Retinoic Acid in Cultured Human Keratinocytes

Regulation of ornithine decarboxylase (ODC) gene expression and cell growth by all-trans-retinoic acid in the presence and absence of exogenous putrescine were examined in normal keratinocyte cultures maintained in serum-free medium containing 0.15 mM Ca++. Putrescine and the higher polyamines are negative feedback regulators of ODC synthesis and are essential for cell growth. Human keratinocytes were incubated with and without 1 microM putrescine and the effects of 5 x 10(-7) M retinoic acid on ODC mRNA levels, ODC activity, polyamine levels, and DNA synthetic rates were determined. Northern blot analysis of total RNA isolated from breast reduction keratinocytes treated with retinoic acid up to 24 h showed a time-dependent suppression of ODC mRNA levels that was unaffected by putrescine. ODC activity was suppressed more rapidly in keratinocytes grown in the absence of putrescine; however, at 24 h, ODC activity was suppressed to an equal extent under both culture conditions. The effect of retinoic acid on polyamine levels was determined in the absence of exogenous putrescine. Retinoic acid treatment markedly suppressed putrescine and N1-acetylspermidine levels, whereas spermidine and spermine levels were relatively unaffected. The effect of retinoic acid on DNA synthetic rates, as measured by 3H-thymidine incorporation, was variable. Retinoic acid either stimulated or had little effect on keratinocyte DNA synthetic rates in cells derived from breast reductions and cultured in the absence of putrescine; these effects were not opposed by the presence of exogenous putrescine. In contrast, DNA synthesis in keratinocytes derived from neonatal foreskins was consistently suppressed by retinoic acid, independent of the polyamine status. Our data, therefore, suggest that the effect of retinoic acid on cell growth, as indicated by DNA synthetic rates, does not necessarily parallel its effect on ODC activity and mRNA levels.

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.

Identification of the polyamine-induced protein as a periplasmic oligopeptide binding protein.

The physiological function of the polyamine-induced protein (PI protein), whose synthesis is stimulated at an early stage after the addition of putrescine to growing cells of a polyamine-requiring mutant of Escherichia coli (Mitsui, K., Igarashi, K., Kakegawa, T., and Hirose, S. (1984) Biochemistry 23, 2679-2683), has been studied. The following findings clearly show that the PI protein is a binding protein of an oligopeptide transport system. (a) PI protein was found in a periplasmic fraction. (b) When the restriction map of a clone for the PI protein gene was compared with Kohara's physical map (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508), the gene was found at 27 min of the E. coli chromosome, where genes for an oligopeptide transport system were located. (c) The clone contained a 1,629-nucleotide open reading frame encoding a 543-amino acid protein whose calculated Mr was 60,901, and the predicted amino acid sequence from this open reading frame was quite similar to that of an oligopeptide binding protein of Salmonella typhimurium. (d) When the transport activity of a tripeptide, Gly-Leu-125I-Tyr, was measured in a polyamine-requiring mutant of E. coli growing both in the presence and absence of putrescine, the activity was higher in the cells growing in its presence. (e) Polyamine stimulation of cell growth was greater when an oligopeptide rather than corresponding amino acids was added to the medium. These results suggest that the polyamine stimulation of PI protein synthesis at the early stage after the addition of putrescine contributes to the polyamine stimulation of cell growth through the supply of nutrients.

Polyamine stimulation of ribosomal synthesis and activity in a polyamine-dependent mutant of Escherichia coli

A polyamine-dependent mutant of Escherichia coli KK101 was isolated by treatment of E. coli MA261 with N-methyl- N′-nitro- N-nitrosoguanidine. In the absence of putrescine, doubling time of the mutant was 496 min. The mutation was accompanied by a change in the nature of the 30 S ribosomal subunits. Addition of putrescine to the mutant stimulated the synthesis of proteins and subsequently, this led to stimulation of RNA and DNA synthesis. Under these conditions, we determined which proteins were preferentially synthesized. Putrescine stimulated the synthesis of ribosomal protein S1 markedly, but stimulated ribosomal proteins S4, L20, and X1, and RNA polymerase slightly. The amounts of initiation factors 2 and 3 synthesized were not influenced significantly by putrescine. The preferential stimulation of the synthesis of ribosomal protein S1 occurred as early as 20 min after the addition of putrescine, while stimulation of the synthesis of the other ribosomal proteins and RNA polymerase appeared at 40 min. The stimulation of the synthesis of ribosomal RNA also occurred at 40 min after addition of putrescine. Our results indicate that putrescine can stimulate both the synthesis and the activity of ribosomes. The increase in the activity of ribosomes was achieved by the association of S1 protein to S1-depleted ribosomes. The early stimulation of ribosomal protein S1 synthesis after addition of putrescine may be important for stimulation of cell growth by polyamines.

Vitronectin is a substrate for transglutaminases

Vitronectin (VN) was found to be a substrate for both plasma transglutaminase (Factor XIIIa) and guinea pig liver transglutaminase (TG). Incorporation of [3H]-putrescine indicated the presence of reactive glutaminyl residues in VN. When VN was incubated with TG or Factor XIIIa, in the absence of putrescine, multimeric covalent complexes were identified, indicating that VN can also contribute lysyl residues to the bond catalyzed by transglutaminases. Cross-linking of VN by TG and Factor XIIIa may modulate the effects of VN on the complement and coagulation systems in hemostatic plugs and extracellular matrix.

Properties of human and rodent S-adenosylmethionine decarboxylase.

S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine. Its product, decarboxylated S-adenosylmethionine (dcAdoMet) is used as an aminopropyl donor by spermidine synthase and spermine synthase (Williams-Ashman and Pegg, 1981; Pegg and McCann, 1982; Tabor and Tabor, 1984a). Once decarboxylated by the action of AdoMetDC, S-adenosylmethionine (AdoMet) becomes committed to polyamine production since methyltransferases use dcAdoMet very poorly, if at all (Pegg, 1984, 1986). In fact, the only known metabolic route for further metabolism of dcAdoMet apart for its use as an aminopropyltransferase substrate is its acetylation (Wagner et al., 1985; Pegg et al., 1986) The supply of dcAdoMet is normally regulated very tightly by the cellular polyamine content and this regulation is brought about by changes in the activity of AdoMetDC (Pegg, 1984). In this way, the cellular content of dcAdoMet is usually kept very low (about 1–3% of AdoMet content) as its synthesis is linked to the ability of the aminopropyltransferases to use it to form polyamines. Only when cellular polyamine metabolism is deranged by inhibition of the other enzymes in the polyamine biosynthetic pathway does the dcAdoMet content rise. Increases of several hundred fold occur when ornithine decarboxylase (ODC) activity is inhibited by drugs such as α-difluoromethylornithine (DFMO) and, only under these conditions, is the acetyl derivative of dcAdoMet formed in significant amounts (Pegg, 1986). The increased content of dcAdoMet is due to both an increase in the activity of AdoMetDC (Alhonen-Hongisto, 1980; Mamont et al., 1981; Pegg, 1984) and to the inability of the aminopropyltransferases to utilize the dcAdoMet formed by it because of the absence of putrescine and spermidine to serve as aminopropyl acceptors.

Altered heat-shock response in polyamine-depleted bacteria

A polyamine-auxotrophic mutant of E. coli was cultivated in the presence or absence of putrescine and submitted to heat shock over 3 different ranges of temperature. In all cases, protein synthetic capacity measured in comparison to that of cultures at the preshift temperature was much higher in polyamine-depleted bacteria under thermic stress. Addition of putrescine only before the shift-up was able to restore gradually normal control of the relative protein synthetic capacity.

Characterization of a carrot callus line resistant to high concentrations of putrescine

A mutant carrot callus line resistant to a high concentration (1 mM) of putrescine has been isolated. A high level of endogenous putrescine, about 13-fold higher than in the controls grown in the absence of putrescine, characterized this resistant mutant. Ornithine-, arginine- and S-adenosylmethionine decarboxylase activities were similar in the resistant line and in the controls by the fifth subculture. The uptake of putrescine when supplied to the medium at high concentration (1 mM), was similar in both the putrescine-treated calli and in the untreated controls. At low concentrations (0.64 μM) however the putrescine absorbed by the resistant calli was less than that absorbed by the controls. Putrescine uptake took place almost always against a concentration gradient and might be due to an active mechanism.