Exploitation of hormone-induced conformational changes to label selectively a component of rat liver plasma membranes.
The kinetics for inactivation of rat liver plasma membrane adenylate cyclase by iodoacetic acid and iodoacetamide has been measured in the presence and absence of glucagon. Glucagon stimulated the rate of iodoacetic acid inhibition by a factor 9f 2.3-fold and iodoacetamide inhibition by 10-fold. These results suggest that interaction of glucagon with its receptor in the membrane resulted in conformational changes which increased either the exposure or nucleophilicity of one or more sulfhydryl groups crucial for adenylate cyclase activity. Membranes were treated with radioactively labeled iodoacetamide or iodoacetic acid in the presence or absence of glucagon and run on 5 and 7.5% sodium dodecylsulfate polyacrylamide gels. These labeling experiments revealed that two membrane components were more extensively labeled in the presence of glucagon. The first component had an apparent molecular weight of 240,000 on sodium dodecyl sulfate gels and stained positive with Coomassie blue and periodate Schiff reagent. This polypeptide accounted for approximately 1.3% of the total membrane protein. The second component had an apparent molecular weight less than 10,000 and could not be correlated directly with a well defined protein band on sodium dodecyl sulfate polyacrylamide gels. The enhancement in labeling of the 240,000 molecular weight component seen in the presence of glucagon agreed very well with that predicted from the kinetics for inhibition of adenylate cyclase activity in the presence and absence of glucagon. This correlation suggests that the component selectively labeled by this technique may be an integral component of the adenylate cyclase system and that hormone-induced conformational changes may be used to selectively label components of the adenylate cyclase system in mammalian membranes.
D000262:Adenylyl Cyclases / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004837:Epinephrine / Q000494:pharmacology; D005934:Glucagon / Q000494:pharmacology; D007461:Iodoacetates; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D009928:Organ Specificity; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011956:Receptors, Cell Surface; D013045:Species Specificity
D007461:Iodoacetates; D011956:Receptors, Cell Surface; D005934:Glucagon; D000262:Adenylyl Cyclases; D004837:Epinephrine