Properties of Detergent‐Dispersed Adenylate Cyclase from Cerebral Cortex. Presence of an Inhibitor Protein

Abstract

Hormone-sensitive adenylate cyclase [EC 4.6.1.1] was solubilized from washed particulate fraction of rabbit cerebral cortex with the nonionic detergent Lubrol 12A9, and subjected to either gel filtration on Ultrogel AcA 34 or chromatography on DEAE Bio-Gel A. By both procedures the enzyme was resolved into 2 components: one was insensitive to guanyl-5''-yl imidodiphosphate [Gpp(NH)p] and NaF but stimulated by Ca2+ and calmodulin, and another that was sensitive to Gpp(NH)p and NaF but relatively insensitive to Ca2+ and calmodulin. Two independent forms of adenylate cyclase may exist in cerebral cortex, one regulated by guanine nucleotide regulatory protein and another by Ca2+-calmodulin. Fractions containing the guanylnucleotide-sensitive activity contain a factor that inhibited basal and Ca2+-stimulated adenylate cyclase in the Ca2+-sensitive fraction. The inhibitor was inactivated by heating at 60.degree. C and by incubation with trypsin. Inhibition was not time-dependent and it was not due to destruction of cAMP by phosphodiesterase or of ATP by ATPase. Inhibitory action was not reversed by calmodulin and it does not appear to be a calmodulin binding protein. Sucrose density gradient sedimentation indicated a sedimentation coefficient of 4S for the inhibitor; by this technique it co-sedimented with the adenylate cyclase sensitive to Gpp(NH)p and NaF.