Isolation and Characterization of Secretory Vesicles from Bovine Neurohypophyses

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Abstract
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 electronmicrographs 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 .mu.g 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.