title: Interaction of polypeptides with the gastric (H+ + K+)ATPase: melittin, synthetic analogs, and a potential intracellular regulatory protein.
abstract: The 26 amino acid bee venom toxin, melittin, is an amphipathic helical polypeptide which inhibits the gastric (H+ + K+)ATPase. The site of interaction with the (H+ + K+)ATPase was shown to be the alpha subunit of the (H+ + K+)ATPase in studies using [125I]azidosalicylyl melittin, a radioactive photoaffinity analog of melittin. A synthetic amphipathic polypeptide (Trp3) containing tryptophan, which exhibits a structure similar to that of melittin, also inhibited the gastric (H+ + K+)ATPase, and prevented labeling by [125I]azidosalicylyl melittin. These findings suggested that melittin and the synthetic amphipathic helical polypeptide were bound to the same or overlapping site(s). In the present studies, novel tritiated photoaffinity analogs of Trp3 containing benzoylphenylalanine (in place of tryptophan) were used to photoaffinity label the (H+ + K+)ATPase. These studies help to establish that the (H+ + K+)ATPase contains a binding site for polypeptides which exhibit an amphipathic helical motif. The precise amino acid sequence of the polypeptide appears to be of secondary importance for interaction with the (H+ + K+)ATPase as long as the alpha helical motif is present. The benzoylphenylalanine containing polypeptides are ideal for mapping the binding site on the (H+ + K+)ATPase. Using an antibody which recognizes this amphipathic helical ('melittin-like') motif, we have demonstrated that the gastric parietal cell contains a 67 kDa 'melittin-like' protein. This protein was associated with the gastric parietal cell apical membrane in the stimulated (secreting) state, but not in the resting (non-secreting) state. The binding site for the gastric 'melittin-like' protein appears to overlap with the melittin binding site on the alpha subunit of the (H+ + K+)ATPase. The potential physiological significance of the melittin binding site and the overlapping binding site for this newly identified endogenous 'melittin-like' protein on the (H+ + K+)ATPase to regulated HCl secretion by the parietal cell is presently under investigation.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Physiology and Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0576.
authors.forename: J
authors.initials: J
authors.lastname: Cuppoletti
authors.forename: D H
authors.initials: DH
authors.lastname: Malinowska
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry*; D008555:Melitten / Q000737:chemistry*; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D012995:Solubility; D013270:Stomach / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013552:Swine
chemical_list: D000345:Affinity Labels; D010455:Peptides; D008555:Melitten; D009219:Myosin-Light-Chain Kinase; D017506:H(+)-K(+)-Exchanging ATPase