title: A monoclonal antibody against pig gastric H+/K(+)-ATPase, which binds to the cytosolic E1.K+ form.
abstract: Monoclonal antibodies were raised against a purified membrane fraction from hog gastric mucosa containing H+/K(+)-ATPase. The properties of one of these monoclonal antibodies (5B6) were further evaluated. On immunoblot it recognized the 95 kDa peptide of the H+/K(+)-ATPase-rich membrane fraction. The K(+)-ATPase activity was inhibited by 65% under standard assay conditions (pH 7.0). At pH 6.0 and 8.0 this enzyme activity was inhibited by 40% and 100%, respectively. The maximal inhibition in inside-out vesicles was also 65% at pH 7.0. The inhibition was uncompetitive with respect to K+ and noncompetitive with respect to ATP. Mg2(+)-ATPase activity and K(+)-dependent p-nitrophenylphosphatase activity were not influenced. The monoclonal antibody lowered the steady-state phosphorylation level at pH 6.0, 7.0 and 8.0 by 30%, 40% and 60% respectively. The rate of the K(+)-stimulated dephosphorylation step was not inhibited. These findings demonstrate that 5-B6 recognizes the E1.K+ dephosphoenzyme at the cytosolic side.
authors.affiliation: Department of Biochemistry, University of Nijmegen, The Netherlands.
authors.forename: T J
authors.initials: TJ
authors.lastname: Van Uem
authors.forename: W H
authors.initials: WH
authors.lastname: Peters
authors.forename: J J
authors.initials: JJ
authors.lastname: De Pont
mesh_terms: D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D005260:Female; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine
chemical_list: D000911:Antibodies, Monoclonal; D009597:4-Nitrophenylphosphatase; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium