Adenylate cyclase from rabbit small intestine: activation by cholera toxin and interaction with calcium.
The stimulation of adenylate cyclase in various fractions of plasma membranes from rabbit small intestinal epithelium has been studied. In crude plasma membranes cholera toxin activated 5-fold at 10 micrograms/ml; vasoactive intestinal peptide (VIP) activated at concentration from 10(-8) to 10(-7) M, the maximal stimulation being 6-fold. Fluoride activated 10-fold at 10 mM. VIP-stimulated enzyme was inhibited by Ca2+ concentrations in the micromolar range. In the presence of calmodulin a biphasic response was obtained. At low Ca2+ concentration (4 x 10(-9)-6 x 10(-8) M) the enzyme was activated. As the Ca2+ concentration was increased the enzyme was concomitantly inhibited. We have investigated the mechanism by which cholera toxin activates intestinal adenylate cyclase. We have found that cholera toxin catalyzed incorporation of 32P into proteins located in the brush-border membrane whose molecular weights are in the range of 40-45kDa. These membranes bind [3H]GTP with a Kd of 1.8 x 10(-7) M. In contrast, basal lateral membranes do not contain any protein which becomes labeled in a toxin-dependent manner when incubated with cholera toxin and [32P]NAD. The modification of brush-border membrane protein occurred in spite of the absence of adenylate cyclase in these membranes. Adenylate cyclase in basal lateral membranes was poorly activated by cholera toxin as compared to crude plasma membranes. On the other hand, the ability of VIP and fluoride to activate the enzyme was enhanced in basal lateral membranes with respect to crude membranes. The results are discussed in relation to the mechanism by which cholera toxin activates adenylate cyclase in intact intestinal cells.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001485:Basement Membrane / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology; D002458:Cell Fractionation; D002772:Cholera Toxin / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000378:metabolism; D007421:Intestine, Small / Q000201:enzymology* / Q000648:ultrastructure; D008871:Microvilli / Q000201:enzymology; D008954:Models, Biological; D011817:Rabbits
D002147:Calmodulin; D000246:Adenosine Diphosphate Ribose; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D000262:Adenylyl Cyclases; D002118:Calcium