ATP-induced dynamic fluorescence changes of a N-[p-(2-benzimidazolyl)phenyl]maleimide probe at Cys241 in the alpha-chain of pig stomach H+,K+-ATPase.
H+,K+-ATPase preparations from pig stomach were modified with a sulfhydryl fluorescence reagent, N-[p-(2-benzimidazolyl)phenyl] maleimide (BIPM). The addition of ATP to the modified enzyme preparations in the presence of Mg2+ decreased the BIPM fluorescence but increased the Trp fluorescence. After exhaustion of ATP, the fluorescence intensities increased and decreased to the original levels, respectively. The results of stopped flow and rapid quenching experiments suggested that the decrease in BIPM fluorescence (36/s) was accompanied by binding of Mg2+ and ATP or phosphorylation (35 36/s) which was followed by slower increases in Trp fluorescence (24/s) and light scattering (20/s). Tosylphenylalanyl chloromethyl ketone-trypsin treatment of the modified preparations, which showed an about 1% decrease in BIPM fluorescence accompanying phosphorylation, gave one major fluorescent peptide peak on reverse-phase chromatography. Amino acid sequence analysis of the peptide revealed the following sequence, Ser-Pro-Glu-X-Thr-His-Glu-Ser-Pro-Leu-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 correspond to Cys241 of the alpha-chain in H+,K+-ATPase. These data and others suggest that the decrease in BIPM fluorescence at Cys241 reflects some molecular event triggered by the binding of ATP with Mg2+ and/or phosphorylation, whereas the increases in the intrinsic Trp fluorescence and light scattering reflect one after phosphorylation.
Eguchi
H
H
Biological Chemistry, Graduate School of Science, Hokkaido University, Sapporo.
Kaya
S
S
Shimada
A
A
Ootomo
Y
Y
Nomoto
K
K
Kikuchi
M
M
Usida
Y
Y
Taniguchi
K
K
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism* / Q000494:pharmacology; D017506:H(+)-K(+)-Exchanging ATPase / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008301:Maleimides / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011487:Protein Conformation; D012542:Scattering, Radiation; D013270:Stomach / Q000187:drug effects / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000378:metabolism* / Q000494:pharmacology; D013552:Swine
D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008301:Maleimides; D013439:Sulfhydryl Reagents; C000245:N-(4-(2-benzimidazolyl)phenyl)maleimide; D000255:Adenosine Triphosphate; D017506:H(+)-K(+)-Exchanging ATPase; D008274:Magnesium; D003545:Cysteine