Properties of the separated catalytic and regulatory units of brain adenylate cyclase.

Abstract

Adenylate cyclase from bovine brain cortex was solubilized with 14 mM cholate and 1 M (NH4)2SO4. Gel filtration over a column of Sepharose 6B separated the catalytic unit (CU) from a factor (G/F) that confers responsiveness to 5''-guanylyl imidophosphate (p[NH]ppG) or fluoride. The separated CU, which elutes with a Kav of 0.48 .+-. 0.01 (n = 5), is not responsive to p[NH]ppG or fluoride and is relatively inactive when Mg .cntdot. ATP is the substrate but activated 8- to 15-fold by Mn2+. The separated G/F elutes with a Kav of 0.70 .+-. 0.02 (n = 4). It restores the responsiveness of the CU to p[NH]ppG and fluoride. Activation of the enzyme by p[NH]ppG before solubilization does not decrease the amount of G/F eluting with a Kav of 0.7. The G/F is probably present in brain cortex in excess over the CU. p[NH]ppG stabilizes the G/F but not the CU against thermal inactivation, evidently it interacts with G/F and not with CU. Incubation of the G/F with p[NH]ppG before addition of CU markedly increases the rate of activation of the reconstituted enzyme by p[NH]ppG. The rate-limiting step in adenylate cyclase activation may be a process in G/F alone and not a slow conformational change in CU or a slow association of G/F with CU. Binding of p[NH]ppG to the isolated G/F appears to be readily reversible; the ability of fully activated G/F to stimulate CU can be blocked if GDP is added before CU. After the CU was activated by interaction with G/F, GDP cannot reverse the activation. Association with the CU apparently increases the affinity of G/F for p[NH]ppG.