A MONOCLONAL-ANTIBODY AGAINST PIG GASTRIC H+/K+-ATPASE, WHICH BINDS TO THE CYTOSOLIC E1.K+ FORM

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 ws inhibited by 40% and 100%, respectively. The maximal inhibition in inside-out vesicles was also 65% at pH 7.). 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. © 1990.