Purification of Adenohypophyseal Plasma Membranes and Properties of Associated Adenylate Cyclase

Abstract

Abstract Plasma membranes were purified from bovine anterior pituitary tissue by differential and isopycnic centrifugation according to a modification of Neville's technique (Biophys. Biochem. Cytol. 8, 413, 1960). Plasma membranes, rough endoplasmic reticulum, and secretory granules sedimented at the interfaces of sucrose layers of different densities and were thus simultaneously obtained in high yield by this technique. The yields of these different fractions were, respectively, 1, 2.5, and 10 mg per g of original pituitary tissue. As shown by electron microscopy, the plasma membrane fraction was not contaminated by other identifiable subcellular components. Growth hormone and prolactin accounted for, respectively, 10 to 30% and 60 to 80% of the total proteins of secretory granules. There was a 7- to 14-fold enrichment of Mg2+-dependent-(Na+-K+)-stimulated ATPase and 5'-nucleotidase activities in the plasma membrane fraction relative to the total homogenate. Adenylate cyclase activity was approximately 10-fold higher in the plasma membrane fraction than in the original homogenate. NaF, 10 mm, gave a 4- to 10-fold stimulation of enzymatic activity in all fractions. Maximal activation of plasma membrane adenylate cyclase activity by Mg2+ and Mn2+ was observed, respectively, at concentrations of 3.5 and 2 mm. Ca2+ inhibited cyclic adenosine 3' : 5'-monophosphate formation at all concentrations studied.