REACTION SEQUENCE AND MOLECULAR-MASS OF A CL-TRANSLOCATING P-TYPE ATPASE

The basolateral membranes of Aplysia californica foregut absorptive ce lls contain both Cl--stimulated ATPase and ATP-dependent Cl- transport activities, and each was inhibited by orthovanadate. Both of these or thovanadate-sensitive activities were reconstituted into proteoliposom es. The reaction sequence kinetics were determined by [gamma-P-32]ATP- induced phosphorylation of the reconstituted Cl- pump. Rapid phosphory lation and dephosphorylation kinetics of acyl phosphate bonding were c onfirmed by destabilization of the phosphoprotein by either hydroxylam ine or high pH. Mg2+ caused phosphorylation of the enzyme; Cl- caused dephosphorylation. Orthovanadate almost completely inhibited the Mg2+- driven phosphorylation reaction. The molecular mass of the catalytic u nit (subunit) of the enzyme appeared to be 110 kDa, which is in agreem ent with molecular masses of all other catalytic units (subunits) of P -type ATPases.