Chemical modification of gastric microsomal potassium-stimulated ATPase

Abstract

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 Mg 2+-ATPase was not affected. Half-maximal inhibition occurred at about 3 μg 2-methoxy-2,4-diphenyl-3-dihydrofuranone/ml at pH 8.5. ATP provided complete protection against inhibition; the apparent K m for ATP protection was about 50 μM. Nucleotide selectivity for protection was ATP > ADP > ITP > GTP > CTP > AMP. Sodium dodecyl sulfate gel electrophoresis of the reacted microsomes showed that virtually all the fluorescent label was on the M r 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 M r 100 000 peptide, but no change in the labeling of the other components. The arginine specific reagent, butanedione, inhibited Mg 2+-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. © 1980.