RESUMO
Total poly (A+) RNA has been extracted from rat pheochromocytoma and translated in vitro by means of a reticulocyte lysate system. We show that two antisera, prepared against pig kidney DOPA decarboxylase (DDC) or rat pheochromocytoma DDC, immunoprecipitate an in vitro synthetized 50 kDa polypeptide identified as DDC by competition experiments with pure DDC. The proportion of specific mRNA has been calculated and represents 0.05% of total poly A+ mRNA. Its size has been established by electrophoresis in methylmercuric hydroxide containing agarose gel, corresponding to a 2.2 kb length mRNA.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/genética , Dopa Descarboxilase/genética , Feocromocitoma/enzimologia , RNA Mensageiro/análise , Animais , Dopa Descarboxilase/biossíntese , Técnicas In Vitro , Rim/enzimologia , Poli A/análise , Testes de Precipitina , Ratos , SuínosRESUMO
Antisera were raised in rabbits against dopamine or noradrenaline conjugated to thyroglobulin with glutaraldehyde. These antisera, tested in enzyme linked immunosorbent assay and immunohistochemistry specifically recognized their homologous antigens. With the aid of anti-tyrosine hydroxylase, anti-aromatic aminoacid decarboxylase, anti-dopamine-beta-hydroxylase, anti-dopamine, and anti-noradrenaline antisera, immunohistochemical reactions were performed on glutaraldehyde fixed sections of sheep diencephalon in order to determine the presence of dopamine in the catecholaminergic group A15. Perikarya of this nucleus were stained with anti-tyrosine hydroxylase, anti-aromatic aminoacid decarboxylase and anti-dopamine, but not with anti-dopamine-beta-hydroxylase or anti-noradrenaline. Both of these latter antisera stained fibers within this area. So as recently found in the rat, we could conclude that dopamine is present in group A15 of the sheep.
Assuntos
Tronco Encefálico/análise , Diencéfalo/análise , Dopamina/análise , Animais , Feminino , Imuno-Histoquímica/métodos , Norepinefrina/análise , OvinosRESUMO
The L-DOPA decarboxylase mRNA levels were determined by a sensitive S1 nuclease method in four organs and one tumor of adult rat. S1 mapping analysis, with probes corresponding to the mRNA coding region, showed that this region is conserved in all L-DOPA decarboxylase mRNA of neuronal and non-neuronal tissues. The mRNA was not very abundant; its representation varies approximately from 0.00035% of total RNA in the mid brain to 0.013% of total mRNA in the pheochromocytoma. A strong correlation between mRNA level and enzyme amount was observed (correlation coefficient = 0.99). The results indicate that the level of mRNA is a primary factor determining the L-DOPA decarboxylase level.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/metabolismo , Dopa Descarboxilase/metabolismo , RNA Mensageiro/análise , Neoplasias das Glândulas Suprarrenais/enzimologia , Glândulas Suprarrenais/enzimologia , Animais , Encéfalo/enzimologia , Dopa Descarboxilase/genética , Rim/enzimologia , Fígado/enzimologia , Feocromocitoma/enzimologia , RNA Neoplásico/análise , Ratos , Distribuição Tecidual , Células Tumorais Cultivadas/enzimologiaRESUMO
L-DOPA decarboxylase was purified from rat pheochromocytoma. Tryptic digestion of this enzyme permitted obtaining fourteen peptides. The comparison of the sequence of L-DOPA decarboxylase from other species with one of these peptides demonstrates a great preservation of this protein.