Isolation and characterization of secretory vesicles from bovine neurohypophyses

A procedure is described for the isolation of secretory vesicles from bovine neurohypophyses by differential centrifugation followed by density gradient centrifugation on iso-osmolal gradients of percoll/sucrose. Only negligible contamination of the secretory vesicle fraction with markers for mitochondria, microsomes and plasma membranes could be detected. The amount of Ca2-ATPase in the isolated neurohypophysial secretory vesicles was of the same low order of magnitude as that of (Na, K)-ATPase. Thin-section electromicrographs confirmed the high purity of the isolated secretory vesicle fractions, In freeze-fracture electronmicrographs, vesicle fusion was demonstrated after incubation with Ca2. As shown in dodecyl sulfate-gel electrophoresis and subsequent autoradiography secretory vesicles exhibited an endogenous phosphorylation activity. The secretory vesicles contained an average of 23.1 microgram vasopressin/mg of protein. On incubation in media differing in ionic strength, pH and Ca2 concentration the vesicles were stable for at least 1 h.