Assuntos
Catecolaminas/biossíntese , Genes , Tirosina 3-Mono-Oxigenase/genética , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/enzimologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , RNA Mensageiro/genética , Ratos , Reserpina/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
A procedure is described for the detection of opiate binding sites synthesized during in vitro translation of various mRNA preparations. RNA were isolated from membrane bound polysomes which were prepared from NG 108-15 hybridoma, C6BU1 glioma cells, as well as from N18TG2, NB2aAg and NB41A3 neuroblastoma cells. Polyadenylated [poly(A)(+)] RNA were purified, translated in vitro in a rabbit reticulocyte lysate and the translation products assayed for their ability to bind [(3)H] bremazocine. Bound and free ligands were separated by column chromatography. After translation of poly(A)(+) RNA obtained from NG 108-15 cells we demonstrated a stereospecific, saturable binding of [(3)H]bremazocine (displaced by levorphanol and not by dextrorphan) with a K(d) of 2.4 +/- 1.0 nM. The total amount of opiate binding sites synthesized was 6.2 +/- 0.5 fmol per ?g of poly(A)(+) RNA. Opiate binding sites were undetectable at zero time and a plateau was reached after translation had proceeded for 20 min. Five time less opiate binding sites were synthesized when the poly(A)(+) RNA purified from N18TG2 neuroblastoma cells were used under the same experimental conditions. There was no detectable binding of opiate ligands with poly(A)(+) RNA obtained from C6BU1 glioma cells, NB2aAg or NB41A3 neuroblastoma cells.
RESUMO
Choline acetyltransferase (ChAT) mRNA and ChAT enzymatic activity have been compared in different regions of the rat central nervous system. mRNA was assayed by exploiting the Xenopus oocyte system which was first tested by measuring the electrophysiological response to glycine after injection of mRNA derived from the ventral part of the spinal cord (VSC). This tissue was found to contain the highest ChAT mRNA level. The striatum, which yielded the maximal enzymatic activity, contained 10 times less ChAT mRNA than the VSC. These results are discussed in terms of the neuroanatomical differences between the two structures.
Assuntos
Sistema Nervoso Central/enzimologia , Colina O-Acetiltransferase/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Animais , Células Cultivadas , Sistema Nervoso Central/análise , Oócitos/metabolismo , RNA Mensageiro/isolamento & purificação , Ratos , Xenopus laevisRESUMO
Several clones specific for tyrosine hydroxylase [tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] have been identified from a rat PC12 library by using the previously characterized clone pTH-1. The most complete of these, pTH-51, is 1758 base pairs long and covers most of the length of the mRNA, including the entire coding and 3' untranslated region. The polypeptide has an estimated molecular weight of 55,903 and some of its characteristic features are discussed.