RESUMO
The effect of several methylputrescines on the activity of insulin-induced ornithine decarboxylase (ODC) was examined in H-35 hepatoma cells. The induction involved both protein and m-RNA synthesis. Actinomycin D inhibited ODC activity when given up to 1 h after insulin treatment. When added to the medium 2 h or 3 h after the insulin, the activity was increased 100% and 80% respectively. Insulin-induced ODC from H-35 cells had a biphasic half-life, a shorter one of 46 min and a longer one of 90 min. 1-Methylputrescine and 2-methylputrescine were found to be competitive inhibitors of the ODC from H-35 cells with Ki values of 2.8 and 0.1 mM respectively. Putrescine itself was found to have a Ki = 2.4 mM. N-Methylputrescine was a very poor inhibitor of the cell free ODC while 1,4-dimethylputrescine did not show any inhibitory effect. When cellular ODC activity was measured, the four methylputrescines assayed as well as putrescine entirely abolished its activity in the H-35 cells when given at a 1 mM concentration together with insulin. 1-Methylputrescine and 1,4-dimethylputrescine abolished 60% of the activity at a 0.1 microM concentration. All the methylputrescines given at 0.1 mM concentrations decreased the putrescine content of the stimulated cells to the levels found in quiescent cells, but only 1-methyl and 2-methylputrescines decreased spermidine and spermine content. 1,4-Dimethyl and 1-methylputrescines showed a strong inhibition of ODC synthesis, while the other diamines were less inhibitory. At concentrations that abolished ODC activity, 1,4-dimethylputrescine decreased 70% of the total immunoreactive ODC bands, while 1-methyl and 2-methylputrescine decreased them by 50%, and N-methylputrescine and putrescine decreased them by 20%. The lack of decrease in immuno-reactive ODC with the latter two compounds was mainly due to the appearance of immunoreactive degradation products of ODC of low molecular weight. Putrescine and N-methylputrescine affected protein synthesis to a small extent in stimulated cells, while 1-methylputrescine decreased it to the level of non-stimulated cells. Insulin (1 microM concentration) stimulated DNA synthesis in the cells, and this stimulation was doubled in the presence of 2-methylputrescine or putrescine. It can be concluded that, among the methylputrescines assayed, 2-methylputrescine was the best inhibitor of cell-free ODC activity, while 1,4-dimethylputrescine and 1-methylputrescine were the best inhibitors of cellular ODC activity.
Assuntos
Insulina/farmacologia , Fígado/enzimologia , Ornitina Descarboxilase/metabolismo , Putrescina/análogos & derivados , Putrescina/farmacologia , Animais , DNA/biossíntese , Dactinomicina/farmacologia , Ativação Enzimática , Cinética , Fígado/efeitos dos fármacos , Neoplasias Hepáticas Experimentais , Ornitina Descarboxilase/biossíntese , Biossíntese de Proteínas , Ratos , Células Tumorais CultivadasRESUMO
Lactogenic and somatogenic receptors present in rat liver have been examined by cross-linking with a derivative of human somatotropin (AP-hGH1) followed by SDS-polyacrylamide gel electrophoresis. AP-hGH1, which has a content of 2.2 azidophenacyl groups per molecule, mainly linked to half Cys-182 and half Cys-189, exerted a specificity similar to that of the native hormone (hGH), with an ability of 46% with respect to hGH to compete with the radiolabelled hormone for the binding sites of microsomal preparations. Photolysis of the 125I-labelled derivative bound to the lactogenic receptors present in either microsomal membranes or Triton X-100 solubilized preparations gave rise to a 63 kDa species. In addition, 30% of the covalent complexes formed in microsomal membranes belonged to a species with a molecular mass of 70 kDa. Incubation of viable rat hepatocytes with the radiolabelled derivative at either 0 degrees C for 3 h or 15 degrees C for 1.5 h and subjection to irradiation, yielded covalent complexes of molecular masses estimated at 130, 73, 63, 45 and 35 kDa. Experiments performed in the presence of 1 mM NaCN, gave rise to the previous species in a similar yield as that obtained in the absence of cyanide. The 130 kDa complex is related to the somatogenic binding sites, since it was not visualized in the presence of unlabelled bovine somatotropin, while the 70-73, 63, 45 and 35 kDa bands disappeared when the incubations were performed in the presence of unlabelled ovine prolactin.
Assuntos
Fígado/análise , Microssomos Hepáticos/análise , Receptores da Prolactina/análise , Receptores da Somatotropina/análise , Animais , Ligação Competitiva , Reagentes de Ligações Cruzadas , Eletroforese em Gel de Poliacrilamida , Feminino , Hormônio do Crescimento/metabolismo , Membranas Intracelulares/análise , Fígado/ultraestrutura , Microssomos Hepáticos/metabolismo , Peso Molecular , Fotólise , Ratos , Ratos Endogâmicos , Receptores da Prolactina/metabolismo , Receptores da Somatotropina/metabolismo , Cianeto de Sódio/farmacologia , SolubilidadeRESUMO
When mice fed on a protein-depleted diet are restored to the normal diet (re-feeding), there is a 2-fold increase in liver RNA polymerase I activity. The results obtained with pactamycin; an inhibitor of protein synthesis, suggest the presence of short-lived proteins which are required for inducing an activated state of transcription. To gain an insight on whether ornithine decarboxylase (ODC)--the first enzyme in polyamine biosynthesis--is the labile protein that regulates rRNA synthesis, we have investigated the correlation between liver ODC and RNA polymerase I activities under different nutritional conditions. We have also studied the effects of alpha-difluormethylornithine (alpha-DFMO)--a specific ODC inactivator--on rRNA transcription. The results indicate that, after re-feeding, there is an abrupt increase in ODC activity that rapidly declines, while RNA polymerase I is still increasing. On the other hand, alpha-DFMO--which inhibits the elevated activity of ODC--has not effect on rRNA transcription.
Assuntos
Dieta , Fígado/enzimologia , Ornitina Descarboxilase/metabolismo , RNA Polimerase I/metabolismo , Animais , Eflornitina , Masculino , Camundongos , Camundongos Endogâmicos , Ornitina/análogos & derivados , Ornitina/metabolismo , Inibidores da Ornitina Descarboxilase , Deficiência de Proteína/metabolismoRESUMO
When mice fed on a protein-depleted diet are restored to the normal diet (re-feeding), there is a 2-fold increase in liver RNA polymerase I activity. The results obtained with pactamycin; an inhibitor of protein synthesis, suggest the presence of short-lived proteins which are required for inducing an activated state of transcription. To gain an insight on whether ornithine decarboxylase (ODC)--the first enzyme in polyamine biosynthesis--is the labile protein that regulates rRNA synthesis, we have investigated the correlation between liver ODC and RNA polymerase I activities under different nutritional conditions. We have also studied the effects of alpha-difluormethylornithine (alpha-DFMO)--a specific ODC inactivator--on rRNA transcription. The results indicate that, after re-feeding, there is an abrupt increase in ODC activity that rapidly declines, while RNA polymerase I is still increasing. On the other hand, alpha-DFMO--which inhibits the elevated activity of ODC--has not effect on rRNA transcription.