Membrane Association of Soluble Protein Activators of Rat Liver Adenylate Cyclase Evidence for Distinctness from the Guanine Nucleotide-binding Stimulating Protein (NS)

Sonication of a crude rat liver membrane preparation and centrifugation at 100,000 x g yielded a supernatant which activated basal and hormone-sensitive adenylate cyclases [ATP pyrophospha telyase (cyclizing), EC 4.6.1.1]. The membrane origin of the stimulatory activity was confirmed by the use of lactate dehydrogenase as a marker for contamination by cytosol. The solubility of the activating factors was verified by their passage through 0.05 μm diameter pores of Millipore filters. The membrane-derived activators were nondialyzable and destroyed by heat and trypsin in the same manner as adenylate cyclase activators detectable in cytosol. Stimulation by factors from membranes and cytosol was not additive. The amount of the activators which could be freed from membranes by sonication was 12-15% of that contained in cytosol previously separated from the membranes. Soluble activators from the two sources had limited ability to restore adenylate cyclase activity to membranes from the cycclone of S49 mouse lymphoma cells which are deficinet in the enzyme's guanine nucleotide-binding stimulatory protein, NS. Cytosol did not contain a substrate for ADP-ribosylation by cholera toxin that corresponded electrophoretically to NS. Furthermore, purified NS did not affect adenylate cyclase activity in preparations stimulated by the soluble activators. These findings suggest that the activating factors found in cytosol may be released±