Reconstitution of cholera toxin-activated adenylate cyclase.

Abstract

Reconstitution of adenylate cyclase activity responsive to stimulation by guanylyl-5''-imidodiphosphate or NaF may be achieved by mixing dilute Lubrol 12A9-solubilized extracts of wild-type [murine lymphoma] S49 membranes with membranes of an adenylate cyclase-deficient variant. Experiments using N-ethylmaleimide to inactivate components of the adenylate cyclase system indicate that distinct components from both wild-type detergent extracts and adenylate cyclase-deficient membranes are essential for reconstitution. These results and conclusions confirm those of others. Detergent extracts of cholera toxin-treated wild-type membranes yield a reconstituted adenylate cyclase as responsive to GTP as to guanylyl-5''-imidodiphosphate whereas, in the absence of cholera toxin treatment, GTP has little or no effect. Cholera toxin-treated adenylate cyclase-deficient membranes and Lubrol 12A9 extracts from them fail to yield a reconstituted adenylate cyclase that responds to GTP with an increase in cyclase activity. Because treatment of the adenylate cyclase-deficient variants with cholera toxin is without effect on the reconstituted cyclase, the cholera toxin substrate is apparently absent or altered in the adenylate cyclase-deficient phenotype.