MECHANISM AND PROPERTIES OF INHIBITION OF PURIFIED RAT-BRAIN ADENYLATE-CYCLASE BY G-PROTEIN BETA-GAMMA-SUBUNITS

The mode of the inhibition of purified rat brain adenylate cyclase by the beta gamma-subunits of G protein (beta gamma) was studied. These s ubunits inhibited the catalytic activity of the cyclase with the maxim al inhibition of 85% and the half-maximal inhibition at about 0.7 nM b eta gamma. The complex of beta gamma and adenylate cyclase isolated by density gradient centrifugation contained 1.8-2.0 mol beta gamma per mol of the cyclase when beta gamma was assayed by immunoblotting and b y its inhibitory activity on adenylate cyclase. However, the beta gamm a concentration-inhibition curves suggest that one of the two Py molec ules bound may be essential for the inhibition. The role for the secon d beta gamma molecule is unknown. As a tentative estimate, 70% of the adenylate cyclase activity remained inhibited by beta gamma when the c omplex was isolated. The inhibition was not dependent on G alpha(s) or calmodulin. Although purified adenylate cyclase contained a protein ( 0.06-0.08 mol/mol of adenylate cyclase) that reacted with anti-G alpha (s) antibody, this protein was not liberated from the cyclase when it formed a complex with beta gamma. In addition, guanine nucleotide anal ogs little affected the cyclase activity or the inhibition by beta gam ma. The inhibition by beta gamma was reversed by the dilution of the c omplex, and the following re-addition of beta gamma suppressed the enz yme activity to about 15% of the initial activity again. These finding s provide strong evidence that beta gamma inhibits adenylate cyclase d irectly and reversibly through the formation of the complex.