Chemical modification of gastric microsomal potassium-stimulated ATPase

Selective chemical modification was used to examine amino acid residues that might be critical for the operation of the gastric K+-stimulated ATPase. Modification of amino groups with the fluorigenic reagent 2-methoxy-2,4-diphenyl-3-dihydrofuranone resulted in selective inhibition of the K+-stimulated ATPase and H+-transporting activities of the gastric microsomes, while the Mg2+-atpase was not affected. Half-maximal inhibition occurred at about 3 microgram 2-methoxy-2,4-diphenyl-3-dihydrofuranone/ml at pH 8.5. ATP provided complete protection against inhibition; the apparent Km for ATP protection was about 50 microM. Nucleotide selectivity for protection was ATP greater than ADP greater than ITP greater than GTP greater than CTP greater than AMP. Sodium dodecyl sulfate gel electrophoresis of the reacted microsomes showed that virtually all the fluorescent label was on the Mr 100 000 peptide band, a very small peptide, and aminolipids. In the presence of ATP there was about 75% reduction in the fluorescent label on the Mr 100 000 peptide, but no change in the labeling of the other components. The arginine specific reagent, butanedione, inhibited Mg2+-ATPase and K+-ATPase activities, with the former being much less reactive. Similar to 2-methoxy-2,4-diphenyl-3-dihydrofuranone, ATP provided complete protection from butanedione treatment. It is concluded that amino and guanidino groups are critical to the function of the K+-ATPase and may be actually at the ATP binding site.