Melittin inhibition of the gastric (H+ + K+) ATPase and photoaffinity labeling with [125I]azidosalicylyl melittin.
Melittin is a 26-amino acid amphipathic polypeptide toxin from bee venom which forms anion-selective ion channels in bilayers and biological membranes under the influence of membrane potential. Melittin has been shown to interact with a number of membrane proteins. We found that melittin inhibited K+-stimulated ATP hydrolysis by the (H+ + K+) ATPase in parietal cell apical membrane vesicles derived from histamine-stimulated rabbit gastric mucosa with a KIapp of 0.5 micron. Melittin also inhibited K+-stimulated p-nitrophenyl hydrolysis activity which is associated with the gastric (H+ + K+) ATPase in a dose-dependent manner with a KIapp of 0.95 micron. ATP-driven, K+-dependent H+ transport was inhibited over this same concentration range, even in the absence of a membrane potential. Melittin did not appear to increase the H+ leak from vesicle with preformed H+ gradients when the H+ pump was arrested by Mg2+ chelation, but all possible membrane perturbation effects were difficult to rule out. However, the data suggest that melittin exerts its inhibitory effect through interaction with the (H+ + K+) ATPase. In order to determine whether direct interactions between the (H+ + K+) ATPase and melittin occurred, a radioactive derivative of melittin, [125I]azidosalicylyl melittin, was prepared and photoreacted with sealed rabbit gastric membranes and highly purified hog gastric membrane containing the (H+ + K+) ATPase. In the purified hog preparation only a 95,000-Da band, the (H+ + K+) ATPase was labeled, while in the rabbit preparation a 95,000-Da band and one other membrane protein of 70,000 Da were labeled with this reagent. Label incorporation into the (H+ + K+) ATPase and the 70,000-Da band was greatly reduced by addition of excess unlabeled melittin, suggesting specificity of the interaction. Label incorporation occurred in the absence of ATP or added salts and was not reduced by SCH28080 (a K+ site inhibitor) suggesting that the melittin binding site was distinct from the luminal K+ site of action of SCH28080.
Cuppoletti
J
J
Department of Physiology, University of Cincinnati College of Medicine, Ohio 45267-0576.
Blumenthal
K M
KM
Malinowska
D H
DH
D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001514:Bee Venoms / Q000378:metabolism* / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology; D017506:H(+)-K(+)-Exchanging ATPase; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008555:Melitten / Q000378:metabolism* / Q000494:pharmacology* / Q000031:analogs & derivatives; D010295:Parietal Cells, Gastric / Q000201:enzymology*; D011817:Rabbits
D001514:Bee Venoms; C061558:azidosalicylylmelittin; D008555:Melitten; D009597:4-Nitrophenylphosphatase; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase