title: Soluble and membrane-bound forms of dopamine beta-hydroxylase are encoded by the same mRNA.
abstract: A full length cDNA clone for bovine dopamine beta-hydroxylase was expressed in rat pheochromocytoma PC12 cells by stable transformation of this cell line with a plasmid expression vector. The recombinant protein exhibited dopamine beta-hydroxylase enzyme activity and was found in both the soluble and membrane fractions of the secretory vesicle. Immunoprecipitation of cell extracts from recombinant cell lines with dopamine beta-hydroxylase antisera followed by fractionation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two subunits, which migrated to relative molecular masses of 76 and 78 kDa. The recombinant protein co-fractionated with neurotransmitter when subcellular structures were separated by sucrose gradient density centrifugation, suggesting that the protein was routed to the secretory vesicles. Dopamine beta-hydroxylase immunoreactivity in those sucrose gradient fractions presumed to contain secretory vesicles was resistant to treatment with trypsin unless the nonionic detergent Triton X-100 was also present to disrupt membrane structure. The 76- and 78-kDa isoform were each found in both the membrane and soluble fractions of the secretory vesicle. Treatment of cultured cells with nerve growth factor or 8-(4-chlorophenylthio)-cyclic AMP alters the relative distribution of the subunits such that the 76-kDa form predominates. The subcellular distribution of a dopamine beta-hydroxylase cDNA clone lacking the first 16 nucleotide residues was also determined. The predicted amino acid sequence of the protein encoded by this cDNA would be deleted of the first 13 residues of the signal sequence, which were reported to be present in the membrane-bound form, but not the soluble form, of native dopamine beta-hydroxylase (Taljanidisz, J., Stewart, L., Smith, A. J., and Klinman, J. P. (1989) Biochemistry 28, 10054-10061). Immunoprecipitable dopamine beta-hydroxylase derived from expression of the deleted cDNA was found in both the membrane-bound and soluble fractions of the secretory vesicle. These experiments demonstrate that the membrane-bound and soluble forms of dopamine beta-hydroxylase are derived from one primary translation product, which is also sufficient to produce enzyme activity. In addition, the amino-terminal amino acids encoding residues 1-13, which compose the hydrophilic region of the signal sequence, are not necessary for the biogenesis of membrane-bound dopamine beta-hydroxylase.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97212-3098.
authors.forename: E J
authors.initials: EJ
authors.lastname: Lewis
authors.forename: L P
authors.initials: LP
authors.lastname: Asnani
mesh_terms: D000311:Adrenal Glands / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002458:Cell Fractionation; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004299:Dopamine beta-Hydroxylase / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D021382:Protein Sorting Signals / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D012995:Solubility; D014357:Trypsin
chemical_list: D008565:Membrane Proteins; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA; D004299:Dopamine beta-Hydroxylase; D014357:Trypsin