Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin

A factor (ARF) that is required for the cholera toxin-dependent ADP-ribosylation of the stimulatory, GTP-binding regulatory component (G(s)) of adenylate cyclase has been purified about 2000-fold from cholate extracts of rabbit liver membranes. ARS is an intrinsic membrane protein with M(r) = 21,000. The final product can be resolved into two polypeptides with very similar molecular weights; each of these has ARF activity. The ADP-ribosylation of G(s) can now be studied with defined components. GTP and ARF are both necessary cofactors. The data imply that the substrates for the activated toxin are NAD and a GTP·G(s)·ARF complex, and the reaction proceeds in a lipid environment. The apparent ability of ARF to bind to the α subunit of G(s) suggests that it may play another, unknown role in the regulation of adenylate cyclase activity.