Membrane proteins of chromaffin granules, dopamine  -hydroxylase, a major constituent.
1. Soluble lysates and membranes were prepared from chromaffin granules isolated from bovine adrenal medulla. The detergent N-cetylpyridinium chloride was used for solubilizing the membrane proteins, including the membrane-bound dopamine (2,4-dihydroxyphenethylamine) beta-hydroxylase. The solubilized proteins were fractionated by Sephadex chromatography in the presence of N-cetylpyridinium chloride. The major component of the membrane proteins, i.e. chromomembrin A, was identified as the enzyme dopamine beta-hydroxylase. 2. The addition of N-cetylpyridinium chloride to the soluble lysate caused precipitation of up to 96% of the proteins, but only a small proportion of the dopamine beta-hydroxylase activity was precipitated. The only protein demonstrable in the supernatant by polyacrylamide-gel electrophoresis was the protein that has a lower mobility than chromogranin A in disc gel electrophoresis. This component has been identified previously as dopamine beta-hydroxylase. Thus, this method provides an extremely simple isolation procedure for dopamine beta-hydroxylase. 3. A comparison of the membrane-bound and soluble dopamine beta-hydroxylases revealed the identity of these two preparations. Both were activated by N-cetylpyridinium chloride, they migrated identically in polyacrylamide-gel electrophoresis, their amino acid composition was very similar and an immunological cross-reaction could be demonstrated.
D000313:Adrenal Medulla / Q000166:cytology* / Q000201:enzymology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002594:Cetylpyridinium; D002864:Chromogranins; D003429:Cross Reactions; D003594:Cytoplasmic Granules / Q000201:enzymology*; D004299:Dopamine beta-Hydroxylase / Q000032:analysis* / Q000302:isolation & purification; D004589:Electrophoresis, Disc; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D008566:Membranes / Q000201:enzymology; D011506:Proteins / Q000032:analysis*; D014316:Tritium; D014439:Tyramine / Q000378:metabolism
D000596:Amino Acids; D002864:Chromogranins; D011506:Proteins; D014316:Tritium; D002594:Cetylpyridinium; D004299:Dopamine beta-Hydroxylase; D014439:Tyramine