MEASUREMENT OF GTP GAMMA-S BINDING TO SPECIFIC G-PROTEINS IN MEMBRANES USING G-PROTEIN ANTIBODIES

We developed a novel method to quantitatively measure GTP-gamma-S binding to specific G proteins in crude membranes using G-protein antibodies. The basic strategy was that the materials were initially incubated with [S-35]GTP-gamma-S at 37-degrees-C. After 4-degrees-C incubation in the wells of an ELISA plate precoated with G-protein antibodies, the radioactivity of each well was counted. This method, using an anti-G(i) antiserum and an anti-G(i) antiserum, quantitatively and specifically detected the binding of GTP-gamma-S to purified G(i2) and G(s). In S49 cell membranes. GTP-gamma-S binding to immunoreactive G(s) was observed in a time-dependent manner that obeyed first-order kinetics, and the rate constant was stimulated approximately twofold in response to isoproterenol. The effect of isoproterenol was not observed in unc mutant membranes. The present method thus makes it possible to quantitatively measure GTP-gamma-S binding to specific G proteins in cell membranes.