Purification of bovine adrenal-cortex plasma-membrane vesicles containing a highly corticotropin-sensitive adenylate-cyclase system and angiotensin-II-binding sites.
The purpose of this experimental investigation was to provide a purified plasma membrane fraction containing a highly hormone-responsive adenylate cyclase system. Bovine adrenal cortex was homogenised and a washed pellet (450 000 X g - min) was fractionated by zonal centrifugation in a sucrose and dextran gradient. Adenylate cyclase activity was purified up to 60-fold to a specific activity of 55, 340 and 210 pmol of adenosine 3':5'-monophosphate (cyclic AMP) produced/minute per mg of protein at 38 degrees C for the basal, adrenocorticotrophin and fluoride-activated states, respectively. The time course of the adenylate cyclase activity is linear. The concentration necessary for half-maximal stimulation by adrenocorticotrophin-(1-24)-tetracosipeptide is 0.5 muM. The high hormone-responsiveness of the membrane preparation allows one to demonstrate activation of adenylate cyclase by very weakly agonistic adrenocorticotrophin fragments. The F- activated state can be detergent-dispersed by Lubrol and shows a Km (ATP) different from that of either the basal or adrenocorticotrophin-stimulated state. Other marked enzymes such as 5'-nucleotidase, glucose-6-phosphatase and cytochrome oxidase were followed during purification. The plasma membrane fraction shows rather homogeneous, relatively large vesicles (mean diameter 0.5 mum). It contains high-affinity binding sites for angiotensin II (about 2 pmol per mg protein) with an apparent association constant of 2 X 10(7) (1/mol) at 12 degrees C. The yield, 20 mg of membrane protein per preparation, may make it a tool in either affinity-labelling studies with the peptide hormones mentioned or the starting point for solubilisation and purification of adenylate cyclase.
D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism*; D000302:Adrenal Cortex / Q000187:drug effects / Q000378:metabolism*; D000311:Adrenal Glands / Q000378:metabolism*; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology*; D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D002501:Centrifugation, Zonal; D007700:Kinetics; D008854:Microscopy, Electron; D011955:Receptors, Drug / Q000378:metabolism*
D011955:Receptors, Drug; D000804:Angiotensin II; D000324:Adrenocorticotropic Hormone; D000262:Adenylyl Cyclases