ATP-INDUCED DYNAMIC FLUORESCENCE CHANGES OF A N-[P-(2-BENZIMIDAZOLYL)PHENYL]MALEIMIDE PROBE AT CYS(241) IN THE ALPHA-CHAIN OF PIG STOMACH H-ATPASE(,K+)

H+,K+-ATPase preparations from pig stomach were modified with a sulfhy dryl fluorescence reagent, N-[p-(2-benzimidazolyl)phenyl] maleimide (B IPM). The addition of ATP to the modified enzyme preparations in the p resence of Mg2+ decreased the BIPM fluorescence but increased the Trp fluorescence, After exhaustion of ATP, the fluorescence intensities in creased and decreased to the original levels, respectively. The result s of stopped flow and rapid quenching experiments suggested that the d ecrease in BIPM fluorescence (36/s) was accompanied by binding of Mg2 and ATP or phosphorylation (35-36/s) which was followed by slower inc reases in Trp fluorescence (24/s) and light scattering (20/s). Tosylph enylalanyl chloromethyl ketone-trypsin treatment of the modified prepa rations, which showed an about 1% decrease in BIPM fluorescence accomp anying phosphorylation, gave one major fluorescent peptide peak on rev erse-phase chromatography. Amino acid sequence analysis of the peptide revealed the following sequence, Ser-Pro-Glu-X-Thr-His-Glu-Ser-Pro-Le u-Glu-Thr-Arg. On comparison with the amino acid sequence deduced from cDNA from pig stomach [Maeda, M., Ishizaki, J., and Futai, M. (1988) Biochem. Biophys. Res. Commun. 157, 203-209], X was shown to correspon d to Cys(241) of the alpha-chain in H+,K+-ATPase. These data and other s suggest that the decrease in BIPM fluorescence at Cys(241) reflects some molecular event triggered by the binding of ATP with Mg2+ and/or phosphorylation, whereas the increases in the intrinsic Trp fluorescen ce and light scattering reflect one after phosphorylation.