Identification of calmodulin-sensitive and calmodulin-insensitive adenylate cyclase in rat kidney.
Adenylate cyclase from rat kidney membranes solubilized with Lubrol-PX, was resolved into calmodulin-insensitive and calmodulin-sensitive forms using DEAE-Sephacel and calmodulin-Sepharose affinity chromatography. The major fraction, 90% of the activity recovered, did not bind to the calmodulin-Sepharose in the presence of Ca2+, and was insensitive to activation by calmodulin. The calmodulin-sensitive enzyme, approximately 10% of the recovered activity, bound to the affinity column and was eluted with buffer containing 2 mM EGTA. In the presence of free Ca2+, calmodulin increased the specific activity of the calmodulin-sensitive adenylate cyclase from 15.2 to 60.4 pmol/mg protein-1 min-1. Maximum stimulation occurred at 0.035-0.076 mM Ca2+. The apparent Ka for calmodulin was 8 nM. The calmodulin-mediated increase in activity was inhibited by trifluoperazine, but not by its analog trifluoperazine-5-oxide. In contrast, trifluoperazine did not inhibit the calmodulin-insensitive activity. The GTP analog, guanyl-5'-yl imidodiphosphate, did not activate either fraction. Furthermore, activation by calmodulin did not require the presence of a guanyl nucleotide. The present finding of a calmodulin-sensitive form of adenylate cyclase in kidney raises the possibility that a calmodulin-mediated mechanism is involved in the formation of cAMP in this organ.
D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation / Q000187:drug effects; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology*; D008297:Male; D051381:Rats; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D014268:Trifluoperazine; D006165:Guanylyl Imidodiphosphate; D000262:Adenylyl Cyclases; D002122:Calcium Chloride