Use of photoaffinity nucleotide analogs to determine the mechanism of ATP regulation of a membrane-bound, cAMP-activated protein kinase.
Using a radioactively tagged, photoaffinity analog of cAMP, 8-azidoadenosine-3',5'-cyclic monophosphate (8-N3 cAMP) and [gamma32P]ATP, the membrane-binding properties of both the regulatory and catalytic subunits of the cAMP-activated protein kinase of human erythrocyte membranes were investigated. [32P]8-N3 cAMP was used to locate and quantify regulatory subunits. Increased phosphorylation of specific membrane proteins by [gamma32P]ATP was used to determine the presence of the catalytic subunit. The data support a mechanism which operates through a tight membrane-bound regulatory subunit and a catalytic subunit that is released from the membrane when cAMP is present and the Mg.ATP concentration is below approximately 10 micrometer. The catalytic subunit is not required for the Mg.ATP inhibition of 8-N3 cAMP binding. Experiments with a photoaffinity analog of ATP, 8-azidoadenosine triphosphate (8-N3ATP), support the hypothesis that ATP hydrolysis and phosphorylation are not involved in the regulation. The data indicate that the regulatory subunit contains an ATP regulatory site which inhibits 8-N3 cAMP binding and the release of the catalytic subunit. These results indicate that the membrane-bound type I enzyme (type IM) differs significantly from the soluble (type IS) enzyme studied on other tissues. These enzymes are compartmentalized by being in different cellular locations and are regulated differently by Mg.ATP.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D000494:Allosteric Regulation; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D008027:Light; D046911:Macromolecular Substances; D008954:Models, Biological; D011494:Protein Kinases / Q000378:metabolism*
D000345:Affinity Labels; D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases