Direct interaction between the catalytic subunit of the calmodulin-sensitive adenylate cyclase from bovine brain with 125I-labeled wheat germ agglutinin and 125I-labeled calmodulin.
A calmodulin-sensitive adenylate cyclase has been purified to apparent homogeneity from bovine cerebral cortex using calmodulin-Sepharose followed by forskolin-Sepharose and wheat germ agglutinin-Sepharose. The final product appeared as one major polypeptide of approximately 135,000 daltons on sodium dodecyl sulfate-polyacrylamide gels. This polypeptide was a major component of the protein purified through calmodulin-Sepharose. The catalytic subunit was stimulated 3-4-fold by calmodulin (CaM) with a turnover number greater than 1000 min-1 and was directly inhibited by adenosine. The catalytic subunit of the enzyme interacted directly with 125I-CaM on a sodium dodecyl sulfate-polyacrylamide gel overlay system, and this interaction was Ca2+ concentration dependent. In addition, the catalytic subunit was shown to directly bind 125I-labeled wheat germ agglutinin using a sodium dodecyl sulfate-polyacrylamide gel overlay technique, and N-acetylglucosamine inhibited binding of the lectin to the catalytic subunit. Calmodulin did not inhibit binding of wheat germ agglutinin to the catalytic subunit, and the binding of calmodulin was unaffected by wheat germ agglutinin. These data illustrate that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is a glycoprotein which interacts directly with calmodulin and that adenosine can inhibit the enzyme without intervening receptors or G coupling proteins. It is concluded that the catalytic subunit of adenylate cyclase is a transmembrane protein with a domain accessible from the outer surface of the cell.
Minocherhomjee
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Department of Pharmacology, University of Washington, Seattle 98195.
Selfe
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D000818:Animals; D001921:Brain / Q000201:enzymology*; D002147:Calmodulin / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D005576:Colforsin; D007457:Iodine Radioisotopes; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D012685:Sepharose / Q000031:analogs & derivatives
D002147:Calmodulin; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; C042758:wheat germ agglutinin-sepharose 6MB; D005576:Colforsin; D012685:Sepharose