The K+ affinity of gastric H+,K+-ATPase is affected by both lipid composition and the beta-subunit

It is generally assumed that negatively charged residues present in the alp ha-subunit of gastric H+,K+-ATPase are involved in K+ binding and transport . Despite the fact that there is no difference between various species rega rding these negatively charged residues, it was observed that the apparent K+ affinity of the pig enzyme was much lower than that of the rat H+,K+-ATP ase. By determining the K+-stimulated dephosphorylation reaction of the pho sphorylated intermediate K-0.5 values for K+ of 0.12 +/- 0.01 and 1.73 +/- 0.03 mM were obtained (ratio 14.4) for the rat and the pig enzyme, respecti vely. To investigate the reason for the observed difference in K+ sensitivi ty, both enzymes originating from the gastric mucosa were either reconstitu ted in a similar lipid environment or expressed in Sf9 cells. After reconst itution in K+-permeable phosphatidylcholine/cholesterol liposomes K-0.5 val ues for K+ of 0.16 +/- 0.01 and 0.35 +/- .0.05 mM for the rat and pig enzym e respectively were measured (ratio 2.2). After expression in Sf9 cells the pig gastric H+,K+-ATPase still showed a 4.1 times lower K+ sensitivity tha n that of the rat enzyme. This means that the difference in K+ sensitivity of the rat and pig gastric H+,K+-ATPase is not only due to a different lipi d composition but also to the structure of either the alpha- or beta-subuni t. Expression of hybrid enzymes in Sf9 cells showed that the difference in K+ sensitivity between the rat and pig gastric H+,K+-ATPase is primarily du e to differences in the beta-subunit. (C) 2000 Elsevier Science B.V. All ri ghts reserved.