Physical Separatino of the Catalytic and Regulatory Proteins of Hepatic

Both the catalytic protein (C) and the guanine nucleotide-binding regulatory protein (G/F) of rabbit hepatic adenylate cyclase were solubilized in active form in sodium cholate at high ionic strength. Upon precipitation by (NH4)*S04, C was selectively aggregated such that it was largely excluded from Ultrogel AcA 34 when chromatographed in cholate solution at high ionic strength. G/F eluted with KO = 0.5. Thus C can be completely resolved from G/F by this procedure. Rabbit hepatic C prepared in this way resembles the C activity previously described by us in the cycmutant of S49 lymphoma cells (Ross, E. M., Howlett, A. C., Ferguson, K. M., and Gilman, A. G. (1978) J Biol. Chern. 253, 6401-6412). Similarities include 7to 10-fold stimulation by Mn‘+ relative to Mg“+ and a complete lack of sensitivity to fluoride or guanine nucleotides. Reconstitution of hepatic C with hepatic G/F causes restoration of stimulation up to 25-fold by either of these activators in the presence of MgZ+. While C is relatively unstable in cholate under these conditions, especially at temperatures over 4°C. it is stabilized by removal of cholate and by addition of phospholipid. These procedures allow the study of the catalytic protein of adenylate cyclase from a hormone-responsive tissue in the absence of endogenous regulatory protein or membrane lipid and also provide a starting point for the purification of the catalyst.