[125I]azidosalicylyl melittin binding domains: evidence for a polypeptide receptor on the gastric (H+ + K+)ATPase.
We have previously shown that melittin, a bee venom peptide, potently inhibited the catalytic and transport functions of rabbit gastric (H+ + K+)ATPase. A radioactive photoaffinity analog of melittin, ([125I]azidosalicylyl melittin), labeled the (H+ + K+)ATPase. These results suggested that melittin exerted inhibitory effects through direct interaction with the (H+ + K+)ATPase. In this study we attempt to define the melittin-binding domain of the (H+ + K+)ATPase using conformation-dependent proteolytic fragmentation of [125I]azidosalicylyl melittin-labeled hog gastric (H+ + K+)ATPase. In the presence of KCl (E2 form) the 95,000-Da [125I]-azidosalicylyl melittin-labeled (H+ + K+)ATPase was cleaved by trypsin to a 40,000-Da NH2-terminal tryptic fragment and a 56,000-Da COOH-terminal fragment through cleavage at Arg 454 of the (H+ + K+)ATPase. The 40,000-Da fragment was labeled by [125I]-azidosalicylyl melittin. The 56,000-Da fragment was not labeled. When unmodified (H+ + K+)ATPase was trypsinized in the presence of KCl, and the fragments were then reacted with [125I]azidosalicylyl melittin, similar tryptic fragmentation results were obtained. In the absence of KCl (E1 form), the 56,000- and 40,000-Da fragments did not accumulate. Chymotryptic hydrolysis of [125I]azidosalicylyl melittin-labeled (H+ + K+)-ATPase was very slow in the presence of KCl (E2 form). In the absence of KCl (E1 form), chymotryptic hydrolysis was more rapid, with accumulation of a major 42,000-Da fragment which was radiolabeled. The melittin-binding region on the (H+ + K+)ATPase is N-terminal to Arg 454 of the (H+ + K+)ATPase. This region is known to contain the aspartyl phosphate residue (Asp 385), the site of phosphoenzyme formation on the (H+ + K+)ATPase. Melittin is also known to bind to calmodulin and other proteins. Another known calmodulin-binding peptide with a different sequence but similar structure, Trp-3, (Leu-Lys-Trp-Lys-Lys-Leu-Leu-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Leu-Gly) also inhibited the (H+ + K+)ATPase and label incorporation by [125I]azidosalicylyl melittin. These Trp-3 results suggested that the (H+ + K+)ATPase contains a peptide-binding domain which is similar to the peptide-binding domains found on other melittin-binding proteins.
Cuppoletti
J
J
Department of Physiology and Biophysics, University of Cincinnati College of Medicine, OH 45267-0576.
D009597:4-Nitrophenylphosphatase / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001514:Bee Venoms / Q000378:metabolism*; D002918:Chymotrypsin; D004789:Enzyme Activation / Q000187:drug effects; D005753:Gastric Mucosa / Q000187:drug effects / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase; D006868:Hydrolysis; D008555:Melitten / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D011188:Potassium / Q000494:pharmacology; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013552:Swine; D014357:Trypsin / Q000494:pharmacology
D001514:Bee Venoms; D011956:Receptors, Cell Surface; C061558:azidosalicylylmelittin; D008555:Melitten; D009597:4-Nitrophenylphosphatase; D002918:Chymotrypsin; D014357:Trypsin; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium