Comparison of chemical properties of purified plasma membranes and secretory vesicle membranes from the bovine adrenal medulla

A highly enriched fraction of plasma membranes from the bovine adrenal medulla has been isolated by differential and sucrose gradient centrifugation. The membranes were found to occur as 0.1-0.5μ diameter vesicles and to equilibrate at a density of 1.13-1.14 g/ml. This fraction was characterized by 4-fold elevated levels of adenylate cyclase and 20-fold elevated levels of 5′-nucleotidase. Secretory vesicle membranes, isolated by repeated hypotonie and hypertonic shocks of whole vesicles, were found to equilibrate between d = 1.08 and d = 1.12 on a sucrose density step gradient. These membranes were highly enriched in cytochrome b₅₆₂ and dopamine-β-hydroxylase. Proteins in the two membranes were compared by SDS gel electrophoresis. All protein size classes found in the vesicle membrane fraction were also represented in the plasma membrane fraction, though in different proportions on the basis of staining intensity. The plasma membrane fraction contained prominent bands co-migrating with the α- and β-bands of tubulin, as well as a component co-migrating with actin. These bands were absent from the vesicle membranes. Fingerprint analysis of stained bands from the membrane fraction demonstrated that the components were indeed tubulin and actin. The plasma membranes contained twice as much sialic acid residues as did the chromaffin granule membranes, but had only half the cholesterol content on a weight basis. The cholesterol:phospholipid ratio in the plasma membranes was 0.63, while in the secretory vesicle membranes it was 1.04. These results show that plasma membranes and secretory vesicle membranes are functionally and structurally different.