Solubilization of calcitonin-responsive renal cortical adenylate cyclase.
Purification of pork renal cortex membranes yielded a particulate adenylate cyclase retaining good sensitivity to stimulation by parathyroid hormone and glucagon and a modest but significant response to porcine calcitonin. Treatment of this partially purified membrane fraction with 0.5% Lubrol PX and 5 mM NaF released adenylate cyclase activity into a fraction which was not sedimented by centrifugation for 20 min at 37,000 X g or for 2 hours at 100,000 X g and passed through a Millipore filter (0.22 mum pore). This solubilized adenylate cyclase was stimulated by porcine calcitonin and NaF but not by parathyroid hormone or glucagon. On gel filtration (Sephadex G-200) in the presence of 1mM dithiothreitol and 5mM NaF, the major portion of the adenylate cyclase activity eluted with the void volume of the column and showed 2.0-fold stimulation with 10 muM calcitonin. Binding of 125I-labeled porcine calcitonin was demonstrated in the 37,000 X g and the 100,000 X g supernatants. From 74 to 86% of the observed binding could be blocked by the addition of unlabeled porcine calcitonin to the reaction mixture. Addition of salmon calcitonin, parathyroid hormone, or glucagon blocked only 12 to 18% of the binding. The dose-response curves for inhibition of binding of iodinated calcitonin by unlabeled calcitonin and the activation of adenylate cyclase by the hormone each showed 50% maximal effect at a concentration between 4.5 and 8 muM porcine calcitonin and maximal effect at a concentration between 33 and 66 muM porcine calcitonin.
D000262:Adenylyl Cyclases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002116:Calcitonin / Q000378:metabolism / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005459:Fluorides / Q000494:pharmacology; D007672:Kidney Cortex / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D011485:Protein Binding; D011956:Receptors, Cell Surface; D012995:Solubility; D013501:Surface-Active Agents; D013552:Swine
D011956:Receptors, Cell Surface; D013501:Surface-Active Agents; D002116:Calcitonin; D000262:Adenylyl Cyclases; D005459:Fluorides