RESUMO
The inhibitory effect of potassium chloride and ammonium sulphate on purified human skin tryptase and bovine trypsin was studied enzyme-kinetically, using Z-Gly-Pro-Arg-pNA, Z-Gly-Pro-Arg-AMC, benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME) as substrates. With increasing salt concentrations, the curve of reaction velocity vs. substrate concentration changed from hyperbolic to sigmoidal when anilide substrates (Z-Gly-Pro-Arg-pNA or -AMC) were used. Only the Km value increased, while the Vmax value remained unchanged. The trend was similar with BAEE or TAME as the substrates. However, the effect of salt on the hydrolysis of these ester substrates was not as strong as on the hydrolysis of anilide substrates, and sigmoidal kinetics were not observed even at the highest KCl concentration (0.7 M) used. Heparin, used as a stabilizer, had no influence on this phenomenon, but it did slightly decrease the apparent Km and Vmax values in low-salt conditions. By comparison, trypsin was not as strongly affected by salt as tryptase, and the inhibition type was mixed competitive and non-competitive. The present results indicate that the salt acts on tryptase as an allosteric effector, and this should be carefully considered when enzyme kinetic parameters and enzyme activity of skin tryptase are measured.
Assuntos
Mastócitos/enzimologia , Peptídeo Hidrolases/metabolismo , Sulfato de Amônio/farmacologia , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Bovinos , Heparina/farmacologia , Humanos , Cinética , Cloreto de Potássio/farmacologia , Pele/enzimologia , Tosilarginina Metil Éster/metabolismoRESUMO
The urinary excretion of putrescine, spermidine, spermine, and N1- and N8-acetylspermidines was measured in 95 volunteers. The 24-h excretion, split in four consecutive periods, was analyzed for circadian rhythm in eight volunteers. Circadian rhythm was observed in total polyamine and in N1- and N8-acetylspermidine excretions. The excretion rates of these polyamines were highest in the morning. The normal values for 24-h urinary excretion of polyamines were determined in 87 volunteers. Men excreted significantly more spermidine (P less than 0.001), N8-acetylspermidine (P less than 0.05), and spermine (P less than 0.001) than did women; putrescine excretion was higher in women (P less than 0.001). This variation was only partially explained by differences between sexes in body or muscle mass because most differences remained significant even after normalization for creatinine excretion and body weight. No correlation between the polyamine excretions and age or menstrual cycle was found.
Assuntos
Envelhecimento/fisiologia , Peso Corporal , Ritmo Circadiano , Creatinina/urina , Ciclo Menstrual , Poliaminas/urina , Adulto , Cromatografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Putrescina/urina , Espermidina/urina , Espermina/urinaRESUMO
The kinetics of inactivation of adenosylmethionine decarboxylase of rat liver and of baby hamster kidney cells (BHK21/C31) by 1-aminooxy-3-aminopropane was studied. The apparent dissociation constants (Ki) for the hepatic and BHK21/C13 enzymes were 1.5 and 2.0 mM and the times of half-inactivation at infinite concentration of the inhibitor (tau 1/2) were 1.2 and 3.8 min, respectively. Treatment of BHK21/C13 with 0.5 mM 1-aminooxy-3-aminopropane prevented cell growth and depleted the cells of putrescine and spermidine within 1 day. The depletion of spermidine resulted in increased activity of S-adenosylmethionine decarboxylase which was due, at least partly, to the increase in the half-life of the enzyme activity. Because spermine levels were not significantly affected, it appears that spermidine is the principal feedback regulator of S-adenosylmethionine decarboxylase. So, 1-aminooxy-3-aminopropane is a very weak inhibitor of S-adenosylmethionine decarboxylase and the cellular effects can be correlated primarily with its inhibitory effects on ornithine decarboxylase and spermidine synthase. In cell-free systems, however, 1-aminooxy-3-aminopropane is likely to find use in unraveling the reaction mechanism of S-adenosylmethionine decarboxylase.
Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Carboxiliases/metabolismo , Propilaminas/farmacologia , Adenosilmetionina Descarboxilase/antagonistas & inibidores , Animais , Divisão Celular , Sistema Livre de Células , Células Cultivadas , Cricetinae , Meia-Vida , Rim/metabolismo , Cinética , Fígado/citologia , Fígado/metabolismo , Inibidores da Ornitina Descarboxilase , Putrescina/metabolismo , Ratos , S-Adenosilmetionina/metabolismo , Espermidina/metabolismo , Espermidina Sintase/antagonistas & inibidoresRESUMO
Aminooxy analogues of spermidine, 1-aminooxy-3-N-[3-aminopropyl]- aminopropane (AP-APA) and N-[2-aminooxyethyl]-1,4-diaminobutane (AOE-PU), were tested as substrates or inhibitors of the enzymes involved in methionine and polyamine metabolism. Both compounds were good competitive inhibitors and poor substrates of spermine synthase, good substrates of cytosolic polyamine acetyltransferase, inactivators of S-adenosylmethionine decarboxylase and inhibitors of ornithine decarboxylase. AP-APA and AOE-PU showed K1-values of 1.5 and 186 microM as inhibitors of purified spermine synthase, and Km-values of 1.4 and 2.1 mM as substrates of the crude hepatic polyamine acetyltransferase activity. AP-APA was more potent than AOE-PU in crude enzyme preparations. Neither drug had any significant effect at 1 mM concentration on the activities of spermidine synthase, methionine adenosyltransferase, S-adenosylhomocysteine hydrolase, and methylthioadenosine phosphorylase. The results suggest that compounds of this type are valuable tools in unraveling the physiology of polyamines.
Assuntos
Adenosilmetionina Descarboxilase/antagonistas & inibidores , Fígado/enzimologia , Inibidores da Ornitina Descarboxilase , Propilaminas/farmacologia , Espermidina Sintase/antagonistas & inibidores , Espermidina/análogos & derivados , Espermina Sintase/antagonistas & inibidores , Acetilação , Animais , Ligação Competitiva , Metionina/metabolismo , Ratos , Especificidade por SubstratoRESUMO
The uptake, catabolism, and release of H-labeled 1-aminooxy-3-aminopropane, a new putrescine analog shown to be a potent polyamine antimetabolite, into and from baby hamster kidney cells (BHK21/C13) were studied. The results show that [3H]-1-aminooxy-3-aminopropane (APA) is not concentrated in the cell, does not compete with polyamines for transport and reveals no difference in uptake between polyamine-depleted and control cells. After a 12-h culture, 60% of APA was recovered intact in the culture media. At this time point, only 30% of the intracellular radioactivity was intact APA, showing that the drug is catabolized in the cells. This intracellular ratio persisted throughout the 4-day culture period. The metabolites of APA were not characterized further. The results indicate that the drug is not recognized as a polyamine by the cells and does not replace or interfere with the polyamines in cellular functions. Thus, its potent affinity to ornithine decarboxylase and spermidine synthase is likely to be due to close structural similarity with the intermediates formed in these reactions. This has implications for the mechanisms involved.
Assuntos
Rim/metabolismo , Inibidores da Ornitina Descarboxilase , Propilaminas/metabolismo , Animais , Células Cultivadas , Fenômenos Químicos , Química , Cricetinae , Eflornitina/farmacologia , Putrescina/metabolismo , Espermidina/metabolismoRESUMO
1- or 3-methylated derivatives and oximes of 1-aminooxy-3-aminopropane (APA) with pyridoxal (PL) and pyridoxal 5'-phosphate (PLP) were synthesized to examine whether the stability of the parent APA molecule could be increased without loss of its inhibitory capacity towards ornithine decarboxylase. Preformed APA-PLP was more stable than APA and was not a substrate of cellular acetylating activity. The only detectable degradation mechanism of APA-PLP was a slow dephosphorylation to APA-PL, which was a substrate for cellular acetylating activity like the methylated APA derivatives. Methylation at the 1 or 3 position of APA did not increase its stability but markedly changed its inhibitory potency towards S-adenosylmethionine decarboxylase and spermidine synthase. Supplementation of cell growth media with 1 mM aminoguanidine markedly reduced the degradation rate of 1- or 3-Me-APA and APA. All the growth-retarding effects of the drugs were reversed by addition of 10-20 microM putrescine or spermidine to the growth media containing a drug concentration of 1 mM, except with APA-PL, which had signs of emergent toxicity at concentrations above 0.5 mM. APA-PL and APA-PLP were as good as APA and two orders of magnitude more effective than alpha-difluoromethylornithine (DFMO) in inhibiting DNA synthesis by BHK21/C13 cells.
Assuntos
Inibidores da Ornitina Descarboxilase , Propilaminas/farmacologia , Adenosilmetionina Descarboxilase/antagonistas & inibidores , Animais , Butilaminas/síntese química , Butilaminas/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Replicação do DNA/efeitos dos fármacos , Guanidinas/farmacologia , Espermidina Sintase/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Using a synthetic deoxyoligonucleotide mixture constructed for a tryptic peptide of the bovine enzyme as a probe, cDNA coding for the full-length subunit of spermidine synthase was isolated from a human decidual cDNA library constructed on phage lambda gt11. After subcloning into the Eco RI site of pBR322 and propagation, both strands of the insert were sequenced using a shotgun strategy. Starting from the first start codon, which was immediately preceded by a GC-rich region including four overlapping CCGCC consensus sequences, an open reading frame for a 302-amino-acid polypeptide was resolved. This peptide had an Mr of 33,827, started with methionine, and ended with serine. The identity of the isolated cDNA was confirmed by comparison of the deduced amino acid sequence with resolved sequences of the tryptic peptides of bovine spermidine synthase. The coding strand of the cDNA revealed no special regulatory or ribosome-binding signals within 82 nucleotides preceding the start codon and no polyadenylation signal within 247 nucleotides following the stop codon. The coding region, containing a 13-nucleotide repeat close to the 5' end, was longer than, and very different from, that of the bacterial counterpart. This region seems to be of retroviral origin and shows marked homology with sequences found in a variety of human, mammalian, avian, and viral genes and mRNAs. By computer analysis, the first 200 nucleotides of the 5' end of the coding strand appear able to form a very stable secondary structure with a free energy change of -157.6 kcal/mole.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Espermidina Sintase/genética , Transferases/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Clonagem Molecular , Decídua/enzimologia , Feminino , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Gravidez , RNA Mensageiro/genéticaRESUMO
Excretion of polyamines first increases and then decreases in patients with multiple trauma receiving total parenteral nutrition (TPN). To separate the effects of trauma and TPN on polyamine excretion, we studied 12 patients with multiple trauma and 14 patients after surgery for colorectal malignancy. Patients were randomized to receive either TPN or hypocaloric glucose infusion. Urinary excretion of total and free polyamines, putrescine (PU), spermidine (SPD), and spermine (SP), and their metabolites, N1-acetylspermidine (N1-AcSPD) and N8-acetylspermidine (N8-AcSPD), and energy and nitrogen balance were measured. Polyamine excretion, excluding SP, markedly increased after trauma and surgery, exceeding the normal values by twofold to 10-fold. In patients receiving TPN, the excretion of total polyamines was 48% higher (P < .01), PU was 34% higher (P < .05), SPD was 35% higher (P < .05), and SP was 350% higher (P < .05) than in patients receiving hypocaloric glucose. Urinary excretion of SP was only 17% of the reference value during hypocaloric glucose (P < .05), but was normal during TPN. The difference in polyamine excretion between nutrition groups was more pronounced when normalized for nitrogen or energy balance. Patients receiving TPN were more hypermetabolic than patients receiving hypocaloric glucose (resting energy expenditure, 1.36 +/- 0.06 [SE] and 1.16 +/- 0.04 times predicted values, respectively; P < .025). Statistically, energy expenditure could explain the difference in polyamine excretion between nutrition groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Neoplasias Colorretais/urina , Traumatismo Múltiplo/urina , Poliaminas/urina , Adulto , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/cirurgia , Metabolismo Energético , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Traumatismo Múltiplo/metabolismo , Nitrogênio/metabolismo , Fenômenos Fisiológicos da Nutrição , Período Pós-OperatórioRESUMO
Two recently devised spermidine analogues, N-[2-aminooxyethyl]-1,4-diaminobutane (AOEPU) and 1-aminooxy-3-N-[3-aminopropyl]-aminopropane (APAPA), were used to elucidate the role of charge distribution in the functions of spermidine in cultured baby hamster kidney cells. The drugs did not affect cell proliferation nor did they relieve the growth-arrest but potentiated the metabolic disturbances caused by DL-alpha-difluoromethyl-ornithine (DFMO). Neither drug affected spermidine uptake but both competed with putrescine uptake. Neither drug could replace spermidine in the control of S-adenosylmethionine decarboxylase and accumulation of the reaction product. APAPA prevented spermine synthesis and showed that modest putrescine synthesis take place in the presence of DFMO. AOEPU, but not APAPA, interfered with cellular constituents resulting in enzymatic formation, accumulation and excretion to culture medium of UV-absorbing catabolites.
Assuntos
Poliaminas/farmacologia , Espermidina/análogos & derivados , Espermidina/fisiologia , Animais , Poliaminas Biogênicas/metabolismo , Poliaminas Biogênicas/farmacocinética , Transporte Biológico , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Cricetinae , Eflornitina/farmacologia , Embrião de Mamíferos , Rim/efeitos dos fármacos , Rim/metabolismo , Poliaminas/metabolismo , Putrescina/biossíntese , Putrescina/farmacocinética , S-Adenosilmetionina/biossíntese , S-Adenosilmetionina/metabolismo , Espermidina/metabolismo , Espermidina/farmacocinética , Espermidina/farmacologia , Relação Estrutura-AtividadeRESUMO
Urinary excretion of polyamines increases in patients with trauma and infection. To separate the effect of infection from the general metabolic response to sepsis, we studied 7 patients with sepsis and 13 patients with multiple trauma in the intensive-care unit. Urinary excretion of total and free polyamines, putrescine, spermidine, spermine, and their metabolites N1-acetylspermidine (N1-AcSPD) and N8-acetylspermidine (N8-AcSPD), and energy and nitrogen balance were measured. The patients were randomized to receive either hypocaloric glucose alone or with amino acids for 2 days. The excretion of individual polyamines, except spermine, significantly exceeded normal values in both patient groups; the excretion of total polyamines was 530 and 323% higher than normal in patients with sepsis and trauma, respectively. The excretion of N1-AcSPD and total spermidine was 141 and 74% higher in patients with sepsis than in patients with trauma, respectively (p < 0.05), whereas the excretion of N8-AcSPD was equal in both patient groups. This was also reflected as a significantly increased urinary ratio of N1-AcSPD to N8-AcSPD in septic patients (6.37 +/- 1.61; mean +/- SE) compared with patients after injury (2.69 +/- 0.27, p < 0.01) or a healthy population (1.08 +/- 0.04, p < 0.001). Amino acid infusion had no effect on polyamine excretion. The mean energy balance was -17.0 +/- 1.1 and -19.1 +/- 1.1 kcal.kg-1.day-1, and the mean nitrogen balance was -0.17 +/- 0.03 and -0.15 +/- 0.02 g.kg-1.day-1 in patients with sepsis and trauma, respectively (NS).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Infecções Bacterianas/metabolismo , Traumatismo Múltiplo/metabolismo , Poliaminas/urina , Adulto , Análise de Variância , Bacteriemia/metabolismo , Bacteriemia/terapia , Infecções Bacterianas/terapia , Candidíase/metabolismo , Candidíase/terapia , Metabolismo Energético/fisiologia , Feminino , Fungemia/metabolismo , Fungemia/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Traumatismo Múltiplo/terapia , Nitrogênio/metabolismo , Nutrição Parenteral , Putrescina/urina , Espermidina/urina , Espermina/urinaRESUMO
Polyamines, synthesized by all mammalian cells, are involved in protein and energy metabolism. We measured urinary excretion of polyamines, putrescine, spermidine, spermine, and their metabolites N1-acetylspermidine and N8-acetylspermidine, resting energy expenditure, and nitrogen excretion in 12 depleted patients with gastrointestinal malignancy during preoperative and postoperative parenteral nutrition and in 7 patients with multiple trauma receiving similar parenteral nutrition. During preoperative nutrition support, the excretion of putrescine (p less than .05) and total polyamines (p less than .01) increased by 420% and 60%, respectively. Increases in energy balance and resting energy expenditure during nutrition could entirely explain the observed changes in polyamine excretion. Preoperatively, the excretion of N1-acetylspermidine (p less than .05), N8-acetylspermidine (p less than .001) and total polyamines (p less than .05) was higher in patients with a surgically noncurable tumor than in those with a surgically curable tumor. The energy balance and resting energy expenditure could also explain the differences in polyamine excretion between patients with surgically curable and noncurable disease, excluding the increased N8-acetylspermidine. Postoperatively, the excretion of N8-acetylspermidine in patients with multiple trauma without malignancy and in patients with palliative operation was similar, and was higher than in patients with a totally resected malignancy (p less than .01). Our results suggest that the excretion of polyamines reflects the activity of energy metabolism in general and that polyamine excretion is not specific for any particular disease.
Assuntos
Metabolismo Energético , Neoplasias Gastrointestinais/urina , Nutrição Parenteral Total , Poliaminas/urina , Adulto , Idoso , Feminino , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Traumatismo Múltiplo/urina , Nitrogênio/metabolismoRESUMO
Selenomethionine metabolism and the biochemical basis for its cytotoxicity were analyzed in cultured human and murine lymphoid cells. The metabolic pathways were also addressed, using purified mammalian enzymes and crude tissue extracts. Selenomethionine was found to be effectively metabolized to S-adenosylmethionine analog, and that analog was further metabolized in transmethylation reactions and in polyamine synthesis, similarly to the corresponding sulphur metabolites of methionine. Selenomethionine did not block these pathways, nor was there a specific block on the synthesis of DNA, RNA, or proteins when added to the culture medium. Selenomethionine showed cytotoxicity at above 40 microM levels. Yet, low selenomethionine levels (10 microM) could replace methionine and support cell growth in the absence of methionine. Selenomethionine toxicity took place concomitantly with changes in S-adenosylmethionine pools. D-form was less cytotoxic than L-form. Methionine concentration modified the cytotoxicity. Together, this indicates that selenomethionine uptake and enzymic metabolism are involved in the cytotoxicity in a yet unknown way.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Metionina/metabolismo , Selenometionina/metabolismo , Animais , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , Humanos , Cinética , Leucemia Mielogênica Crônica BCR-ABL Positiva , Linfoma , Metionina Adenosiltransferase/metabolismo , Metilação , Modelos Biológicos , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/biossíntese , S-Adenosilmetionina/metabolismo , Selenometionina/análogos & derivados , Selenometionina/farmacologiaRESUMO
A series of structural analogs of putrescine, spermidine, and spermine with the aminomethylene fragment substituted by the aminooxy group was suggested. The synthesis of the new aminooxy analogs of spermine was described. Biochemical aspects of the activity of the aminooxy analogs of polyamines were discussed in respect of their selective inhibition of normal and leukemic cells.
Assuntos
Putrescina/análogos & derivados , Espermidina/análogos & derivados , Espermina/análogos & derivados , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Leucemia L1210/patologia , Putrescina/farmacologia , Espermidina/farmacologia , Espermina/farmacologia , Células Tumorais CultivadasRESUMO
The tissue distribution of S-adenosylmethionine, S-adenosylhomocysteine, methionine adenosyltransferase and S-adenosylhomocysteine hydrolase was explored in the rat. Also the effects of methionine administration on the accumulation of S-adenosylmethionine, S-adenosylhomocysteine and polyamines were studied in rat liver, brain and kidney. The tissue distribution of S-adenosylmethionine, S-adenosylhomocysteine, methionine adenosyltransferase and S-adenosylhomocysteine hydrolase was similar in both sexes, and was only slightly changed with age. The specific activity of S-adenosylhomocysteine hydrolase greatly exceeded that of methionine adenosyltransferase, and the concentration of S-adenosylmethionine was higher than that of S-adenosylhomocysteine in all tissues examined. However, the hepatic S-adenosylmethionine/S-adenosylhomocysteine ratio was dependent on food supply and on the age of the animal. No correlation was noticed between the activity of methionine adenosyltransferase and the concentrations of the adenosyl compounds in different tissues. Intraperitoneal administration of methionine resulted in a profound but transient increase in the hepatic concentrations of S-adenosylmethionine and S-adenosylhomocysteine. The concentration of S-adenosylmethionine was elevated also in the brain during the first 2h after methionine injection. The rise of S-adenosylmethionine concentration after methionine treatment could be diminished by simultaneous glycine administration. The results support the view that the rate-limiting factor of S-adenosylmethionine synthesis is the tissue concentration of methionine. They further suggest that glycine N-methyltransferase may have a regulatory role in the utilization of S-adenosylmethionine in the liver.
Assuntos
Homocisteína/análogos & derivados , Metionina/farmacologia , Poliaminas/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Fatores Etários , Animais , Encéfalo/metabolismo , Glicina/farmacologia , Hidrolases/metabolismo , Rim/metabolismo , Fígado/metabolismo , Metionina Adenosiltransferase/metabolismo , Ratos , Fatores SexuaisRESUMO
S-Adenosylmethionine metabolism and its relation to the synthesis and accumulation of polyamines was studied in rat liver under various nutritional conditions, in adrenalectomized or partially hepatectomized animals and after treatment with cortisol, thioacetamide or methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidine)dinitrilo]diguanidine}. Starvation for 2 days only slightly affected S-adenosylmethionine metabolism. The ratio of spermidine/spermine decreased markedly, but the concentration of total polyamines did not change significantly. The activity of S-adenosylmethionine decarboxylase initially decreased and then increased during prolonged starvation. This increase was dependent on intact adrenals. Re-feeding of starved animals caused a rapid but transient stimulation of polyamine synthesis and also increased the concentrations of S-adenosylmethionine and S-adenosylhomocysteine. Similarly, cortisol treatment enhanced the synthesis of polyamines, S-adenosylmethionine and S-adenosylhomocysteine. Feeding with a methionine-deficient diet for 7-14 days profoundly increased the concentration of spermidine, whereas the concentrations of total polyamines and of S-adenosylmethionine showed no significant changes. The results show that nutritional state and adrenal function play a significant role in the regulation of hepatic metabolism of S-adenosylmethionine and polyamines. They further indicate that under a variety of physiological and experimental conditions the concentrations of S-adenosylmethionine and of total polyamines remain fairly constant and that changes in polyamine metabolism are not primarily connected with changes in the accumulation of S-adenosylmethionine or S-adenosylhomocysteine.
Assuntos
Fígado/metabolismo , Poliaminas/biossíntese , S-Adenosilmetionina/metabolismo , Adenosilmetionina Descarboxilase/metabolismo , Adrenalectomia , Animais , Feminino , Hepatectomia , Hidrocortisona/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Metionina/deficiência , Mitoguazona/farmacologia , Ratos , S-Adenosil-Homocisteína/biossíntese , S-Adenosilmetionina/biossíntese , Inanição/metabolismo , Tioacetamida/farmacologiaRESUMO
The fate of S-adenosyl-L-methionine was studied in rat liver extracts by analysing the distribution of radioactivity from labelled adenosylmethionine in decomposition products, which were separated from each other by chromatographic and electrophoretic means. Marked non-enzymic degradation to adenine, pentosylmethionine, methylthioadenosine and homoserine was evident at pH 6.9-7.8. Enzymic cleavage to methylthioadenosine was stoichiometric with the accumulation of spermidine and could be totally prevented by inhibiting S-adenosyl-L-methionine decarboxylase. The results rule out the existence of adenosylmethionine cyclotransferase in rat liver and indicate that only two quantitatively significant enzymic processes are involved in hepatic adenosylmethionine degradation. Excluding nonenzymic decomposition, more than 99% of adenosylmethionine is demethylated and exclusively catabolized further by S-adenosyl-L-homocysteine hydrolase. Less than 1% of adenosylmethionine is decarboxylated and immediately utilized totally for polyamine biosynthesis.
Assuntos
Desoxiadenosinas , Fígado/metabolismo , S-Adenosilmetionina/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Radioisótopos de Carbono , Feminino , Técnicas In Vitro , Proteínas/metabolismo , Ratos , Ratos Endogâmicos , Tionucleosídeos/metabolismoRESUMO
1. The hepatic synthesis and accumulation of polyamines, RNA and S-adenosylmethionine were studied in normal and oestrogen-treated immature male chicks. 2. Ornithine decarboxylase activity in chick liver and in whole chick embryo homogenate was preferentially located in the soluble supernatant fraction. 3. In general the activities of the enzymes involved in the synthesis of polyamines and S-adenosylmethionine decreased with increasing age.
Assuntos
Estradiol/farmacologia , Fígado/metabolismo , Poliaminas/metabolismo , RNA/biossíntese , S-Adenosilmetionina/biossíntese , Adenosilmetionina Descarboxilase/metabolismo , Fatores Etários , Animais , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Galinhas , Cicloeximida/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , Fígado/efeitos dos fármacos , Masculino , Metionina Adenosiltransferase/metabolismo , Ácidos Nucleicos/biossíntese , Ornitina Descarboxilase/metabolismo , Espermidina/biossíntese , Espermidina Sintase/metabolismo , Espermina/biossíntese , Espermina Sintase/metabolismo , InaniçãoRESUMO
Weanling rats were fed a casein-based diet supplemented to give dietary methionine (Met) concentrations of 0.41, 0.61, and 1.50%. After 2 weeks of feeding, the rats received intraperitoneally 800 nCi of 2-14C-labeled and/or methyl-3H-labeled L-Met. The animals were killed 20 min, 1 hr, or 2 hr after the isotope injection and the specific radioactivity of adenosylmethionine (AdoMet) as well as the total acid-soluble radioactivity was analyzed in the liver and skeletal muscle. Met concentrations of the liver and skeletal muscle were increased 20-fold by the diet containing 1.50% of Met. In the liver, but not in skeletal muscle, accumulation of AdoMet closely followed changes in Met concentration. Within 2 hr after intraperitoneal injection, the rate of disappearance of 3H label from the acid-soluble fraction was slow in both tissues; increasing in the liver and decreasing in skeletal muscle with increasing dietary Met concentration. At the same time, disappearance of 14C label was slow in both tissues in the rats fed the toxic Met diet, and also in the liver of the rats fed the Met-deficient diet. Decline of the specific radioactivity of the AdoMet pool with respect to 3H label was similar to that of 14C label in the skeletal muscle at all dietary Met concentrations. In the liver, the rate of disappearance of 14C label from the AdoMet pool was markedly increased and that of the 3H label slightly decreased with increasing dietary Met supply. Met deprivation resulted in rapid disappearance of 3H label from the hepatic AdoMet pool, whereas the disappearance of the 14C label was very slow. The results indicate that hepatic Met recycling is very effective with deficient or adequate dietary Met concentrations. In skeletal muscle, the capacity to catabolize extra Met is very limited and continuous flow of Met to liver takes place. Unlike in the liver, in skeletal muscle the transsulfuration route is not adaptable to changes in Met supply and plays a minor role in Met catabolism. The approach used to determine the efficacy and adaptation of methionine salvage pathways by following simultaneously the decline of the specific radioactivities of the methyl group and the methionyl carbon chain of AdoMet following intraperitoneal injection of double-labeled Met has several advantages over that used in literature reports. It offers a reliable means of observing these metabolic pathways in whole animals without disruption of metabolite fluxes.
Assuntos
Adaptação Fisiológica , Metionina/metabolismo , S-Adenosilmetionina/metabolismo , Animais , Dieta , Meia-Vida , Fígado/metabolismo , Metionina/administração & dosagem , Músculos/metabolismo , RatosRESUMO
Several studies suggest that polyamines may stabilize chromatin and play a role in its structural alterations. In line with this idea, we found here by chromatin precipitation and micrococcal nuclease (MNase) digestion analyses, that spermidine and spermine stabilize or condense the nucleosomal organization of chromatin in vitro. We then investigated the possible physiological role of polyamines in the nucleosomal organization of chromatin during the cell cycle in Chinese hamster ovary (CHO) cells deficient in ornithine decarboxylase (ODC) activity. An extended polyamine deprivation (for 4 days) was found to arrest 70% of the odc- cells in S phase. MNase digestion analyses revealed that these cells have a highly loosened and destabilized nucleosomal organization. However, no marked difference in the chromatin structure was detected between the control and polyamine-depleted cells following the synchronization of the cells at the S-phase. We also show in synchronized cells that polyamine deprivation retards the traverse of the cells through the S phase already in the first cell cycle. Depletion of polyamines had no significant effect on the nucleosomal organization of chromatin in G1-early S. The polyamine-deprived cells were also capable of condensing the nucleosomal organization of chromatin in the S/G2 phase of the cell cycle. These data indicate that polyamines do not regulate the chromatin condensation state during the cell cycle, although they might have some stabilizing effect on the chromatin structure. Polyamines may, however, play an important role in the control of S-phase progression.
Assuntos
Ciclo Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/fisiologia , Poliaminas/farmacologia , Fase S/efeitos dos fármacos , Fase S/fisiologia , Animais , Células CHO , Ciclo Celular/fisiologia , Cricetinae , Fase G2/efeitos dos fármacos , Fase G2/fisiologia , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Ornitina Descarboxilase/deficiência , Poliaminas/metabolismo , Espermina/farmacologia , Fatores de TempoRESUMO
The hepatic synthesis and accumulation of S-adenosylhomocysteine, S-adenosylmethionine and polyamines were studied in normal and vitamin B-6-deficient male albino rats. A method involving a single chromatography on a phosphocellulose column was developed for the determination of S-adenosylhomocysteine and S-adenosylmethionine from tissue samples. Feeding the rat with pyridoxine-deficient diet for 3 or 6 weeks resulted in a four- to five-fold increase in the concentration of S-adenosylhomocysteine, whereas that of S-adenosylmethionine was only slighly elevated. The concentration of putrescine was decreased to half, that of spermidine was somewhat decreased and that of spermine remained fairly constant. The activities of L-ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, L-methionine adenosyltransferase and S-adenosyl-L-homocysteine hydrolase were moderately increased. S-Adenosylmethionine decarboxylase showed no requirement for pyridoxal 5'-phosphate. The major effect of pyridoxine deficiency of S-adenosylmethionine metabolism seems to be a block in the utilization of S-adenosylhomocysteine, resulting in the accumulation of this metabolite to a concentration that may inhibit biological methylation reactions.