RESUMO
Total mRNA were extracted from brain of Nereis diversicolor (Annelida, Polychaeta) and were translated in vitro or in ovo. The newly synthesized polypeptides were analyzed through electrophoresis of immunoprecipitated products or the Western blotting technique using polyclonal antibodies raised against mammalian dynorphin 1-17 and mammalian alpha-neo-endorphin. Among the products translated in vitro, only one class of polypeptide of 70 kDa was recognized by anti-dynorphin 1-17 antibodies. Furthermore, some in ovo translated products as well as proteins extracted from brain of worms showed identical immunoreactivity. These polypeptides, 60-70 kDa, reacted with anti-dynorphin 1-17 and anti alpha-neo-endorphin antibodies. These results suggest the existence of epitopes common to in ovo and in vitro translated products, to polypeptides extracted from the brain and to some mammalian opioid peptides of the prodynorphin family. We postulate the presence, in the brain of N. diversicolor, of precursors of peptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Data reported in this investigation do not allow us to propose or even postulate the presence, in the brain of the worm, of one precursor molecule common to polypeptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Furthermore, the Nereis precursor molecules exhibit a clear-cut difference in molecular mass with the mammalian prodynorphin: 70 kDa versus 30 kDa.
Assuntos
Endorfinas/metabolismo , Poliquetos/metabolismo , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Cromatografia de Afinidade , Dinorfinas/imunologia , Eletroforese em Gel de Poliacrilamida , Endorfinas/imunologia , Ensaio de Imunoadsorção Enzimática , Técnicas In Vitro , Dados de Sequência Molecular , Peso Molecular , Testes de Precipitina , Biossíntese de Proteínas , Precursores de Proteínas/imunologia , RNA Mensageiro/isolamento & purificaçãoRESUMO
Total mRNA extracted from the brain of a marine worm, Nereis diversicolor, were in vitro translated using 2 cell-free systems: rabbit reticulocyte lysate and wheat germ extract. Among numerous products newly translated in both systems, only one class of 70 kDa polypeptides immunoprecipitated when we used a mixture of 3 well defined antibodies raised against known sequences of the human prepro-CCK. At the cellular level, using immunocytochemistry techniques, strong and moderate immunoreactivities were seen in perikarya located in various ganglionic nuclei of the worm brain. Immunoreactive nerve fibres were visible in the neuropile but not in the infracerebral region, a neurohemal area. Immunoreactions also appeared on perikarya located in the anterior and medial groups of the ventral nerve cord. Furthermore, immunolabeled cells were observed in the midgut. Interestingly, several co-localizations of materials immunologically related to human prepro-CCK and CCK/gastrin were observed in the brain and the ventral nerve cord both in perikarya and in nerve fibres. We propose that, in Nereis a polypeptide (molecular mass 70 kDa) is the large precursor of molecules related to those of the CCK/gastrin peptide family.
Assuntos
Anelídeos/genética , Colecistocinina/genética , Biossíntese de Proteínas , Precursores de Proteínas/genética , Animais , Colecistocinina/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Imuno-Histoquímica , Biologia Marinha , Precursores de Proteínas/metabolismoRESUMO
Total mRNA extracted from brain of a sea worm Nereis diversicolor (Annelida, Polychaeta) were injected into Xenopus oocytes. The in ovo-translated polypeptides were analyzed through electrophoresis of immunoprecipitated products or Western blotting techniques. Some of these polypeptides cross-reacted antibodies elaborated against three mammalian (human and rat) procholecystokinin (pro-CCK) sequences. Polypeptides of 15, 64, and 70 kDa were determined, but two families of less obvious products, whose molecular masses were between 27-34 kDa and 46-60 kDa, appeared. Furthermore, the Western blotting technique also showed a cross-reaction between some of the polypeptides (64 and 70 kDa) extracted from brains and the antibodies used. From this work, evidence is given of the presence of epitopes shared by cellular polypeptides extracted from the brain, in ovo-translated products and mammalian pro-CCK. Furthermore, these data suggest the existence of a CCK precursor in the brain of N. diversicolor.
