Selective regulation of carboxypeptidase peptide hormone-processing enzyme during enkephalin biosynthesis in cultured bovine adrenomedullary chromaffin cells.
Bovine adrenomedullary chromaffin cells in culture were incubated with reserpine or forskolin, two agents acting through different mechanisms, which increase cellular [Met]enkephalin levels by 2-fold after 72 h. Cells were harvested and chromaffin granules were purified on a linear sucrose gradient. After reserpine treatment, carboxypeptidase-processing enzyme specific activity in chromaffin granule fractions was stimulated 1.9-fold, and Co2+-stimulated carboxypeptidase specific activity was stimulated 3-fold. The increase in enzyme activity was dependent on the time of reserpine treatment. Forskolin, on the other hand, had no significant effect on carboxypeptidase activity. The differential effects of reserpine and forskolin suggest that the carboxypeptidase-processing enzyme may be selectively regulated during periods of elevated enkephalin formation. Kinetic studies revealed that in cells exposed to reserpine, the Km value for [Met]enkephalin-Arg6 for the Co2+-stimulated carboxypeptidase activity was lowered to 0.136 from 0.447 mM, but there was no change in the Km values of the non-Co2+-stimulated carboxypeptidase activity from reserpine and control groups. Cellular levels of immunoreactive carboxypeptidase-processing enzyme, measured by a radioimmunoassay method, were not altered after reserpine treatment. These data suggest that while the total number of carboxypeptidase enzyme molecules remained constant, there may be a conversion of existing enzyme molecules to a more active form which displays a higher affinity for [Met]enkephalin-Arg6 in the presence of Co2+.
D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002837:Chromaffin Granules / Q000378:metabolism; D005576:Colforsin; D004224:Diterpenes / Q000494:pharmacology; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives / Q000096:biosynthesis; D004745:Enkephalins / Q000096:biosynthesis*; D007700:Kinetics; D010455:Peptides / Q000378:metabolism*; D012110:Reserpine / Q000494:pharmacology
D004224:Diterpenes; D004745:Enkephalins; D010455:Peptides; D005576:Colforsin; D004744:Enkephalin, Methionine; C028719:enkephalin-Met, Arg(6)-; D012110:Reserpine; D002268:Carboxypeptidases