The asymmetric orientation of cytochrome b561 in bovine chromaffin granule membranes.

The topological arrangement of cytochrome b561 in the bovine adrenal medullary chromaffin granule membrane was investigated by radiolabeling and immunoprecipitation techniques using antibody raised against the purified cytochrome. The first labeling procedure involved a membrane-permeable amino group labeling reagent, ethyl acetimidate, and two membrane-nonpermeable amino group labeling reagents, isethionyl acetimidate and trinitrobenzenesulfonic acid. The second radiolabeling procedure involved lactoperoxidase-catalyzed iodination of the exposed tyrosines on the membrane-bound proteins. The labeled cytochrome b561 was isolated by immunoprecipitating detergent extracts of treated membranes, followed by electrophoresis of the precipitated cytochrome in polyacrylamide-dodecyl sulfate. From the analysis of both labeling techniques, cytochrome b561 appeared to be a transmembrane protein and a major portion of this protein was cytoplasmically exposed.