Sulfation and constitutive secretion of dopamine β-hydroxylase from rat pheochromocytoma (PC12) cells

E. M. McHugh, R. McGee, P. J. Fleming

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31 Scopus citations

Abstract

The biosynthesis and secretion of dopamine β-hydroxylase were investigated by radiolabeling rat pheochromocytoma (PC12) cells in culture. Intracellular dopamine β-hydroxylase from a crude chromaffin vesicle fraction and secreted dopamine β-hydroxylase from culture medium were immunoprecipitated using antiserum made against purified bovine soluble dopamine β-hydroxylase. Analysis of the immunoprecipitated enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that: 1) the membrane-bound form of the hydroxylase from crude secretory vesicle membrane extracts contained two nonidentical subunits in approximately stoichiometric amounts [M(r) = 77,000 and 73,000]; 2) the soluble hydroxylase from the lysate of these secretory vesicles was composed predominantly of a single subunti [M(r) = 73,000]; and 3) the hydroxylase secreted into the medium under resting conditions was also composed of a single subunit [approximate M(r) = 73,000]. All subunits of the multiple forms of hydroxylase were glycoproteins. Under resting conditions, the rate of secretion of hydroxylase was approximately 6% of total cellular enzyme/15 min. The secreted form of the hydroxylase incorporated [35S]sulfate, whereas no significant [35S]sulfate was incorporated into the cellular forms of enzyme. We propose that in addition to the dopamine β-hydroxylase which is found in catecholamine storage vesicles and released during stimulus-coupled exocytosis, PC12 cells also have a constitutive secretory pathway for dopamine β-hydroxylase and that the enzyme released by this second pathway is sulfated.

Original languageEnglish (US)
Pages (from-to)4409-4417
Number of pages9
JournalJournal of Biological Chemistry
Volume260
Issue number7
StatePublished - 1985

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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