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 pyrophosphate-lyase (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 micron 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 cyclone of S49 mouse lymphoma cells which are deficient 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 from membranes during tissue homogenization. Because these protein activators can be obtained from membranes without use of detergents and can neither substitute for nor be substituted for by Ns in functional assays, they are distinct from Ns.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D004837:Epinephrine / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000494:pharmacology; D005934:Glucagon / Q000494:pharmacology; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051381:Rats; D013010:Sonication
D008565:Membrane Proteins; D005934:Glucagon; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D004837:Epinephrine