Mechanism of biosynthesis of soluble and membrane-bound forms of dopamine beta-hydroxylase in PC12 pheochromocytoma cells.
Dopamine beta-hydroxylase was present as 2 subunit forms (apparent Mr = 77,000 and 73,000) in the PC12 pheochromocytoma cell line as detected by immunoprecipitation from [35S]methionine-labeled cultures, and analyzed by sodium dodecyl sulfate gel electrophoresis and fluorography. The Mr = 77,000 form was present in a crude membrane fraction, while the Mr = 73,000 form was soluble. Both forms appeared to be present in approximately equal amounts, and both were glycosylated. Treatment of PC12 cells with tunicamycin, a potent inhibitor of core glycosylation in the endoplasmic reticulum, completely inhibited the appearance of the Mr = 77,000 and Mr = 73,000 forms, and 2 new immunoreactive polypeptides were obtained (apparent Mr = 67,000 and 63,000). Pulse-chase experiments suggested that the Mr = 77,000 form is initially synthesized (by 5 min) and a portion is converted in 15-90 min to the Mr = 73,000 form. Thereafter, the ratio between forms remains relatively constant, at least for several hours. Translation of mRNA from bovine and rat adrenals, and immunoprecipitation, indicated that dopamine beta-hydroxylase is initially synthesized as a single polypeptide (apparent Mr = 67,000). The subcellular site of biosynthesis of dopamine beta-hydroxylase was determined by isolation of mRNA from free and membrane-bound polysomes from bovine adrenal medulla. Translation in a cell free system and immunoprecipitation localized the synthesis of dopamine beta-hydroxylase on membrane-bound polysomes. These experiments suggest that both soluble and membrane-bound forms of dopamine beta-hydroxylase are synthesized and core glycosylated in the endoplasmic reticulum, and that there probably is a precursor-product relationship between the Mr = 77,000 and the Mr = 73,000 subunit forms of dopamine beta-hydroxylase.
D000310:Adrenal Gland Neoplasms / Q000201:enzymology*; D000311:Adrenal Glands / Q000378:metabolism; D000313:Adrenal Medulla / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004299:Dopamine beta-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D010673:Pheochromocytoma / Q000201:enzymology*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D012333:RNA, Messenger; D004299:Dopamine beta-Hydroxylase