Identification of N-[p-(2-benzimidazolyl)phenyl]maleimide-modified residue participating in dynamic fluorescence changes accompanying Na+,K+-dependent ATP hydrolysis.
Na+,K+-ATPase from pig kidney was specifically modified with a sulfhydryl fluorescent reagent, N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM), by pretreatment of N-ethylmaleimide. The preparation thus obtained retained 100% of initial Na+,K+-ATPase activity and contained 1 BIPM residue/alpha-chain, and it showed almost 2-fold larger fluorescence changes accompanying ATP hydrolysis than the previous preparations which retained 60% of initial activity and contained 3-4 BIPM residues/alpha-chain (Taniguchi, K., Suzuki, K., and Iida, S. (1982) J. Biol. Chem. 257, 10659-10667). Extensive trypsin (Sigma type I) treatment of the new preparation produced mainly two different fluorescent peptide peaks in both ion-exchange and reverse-phase chromatography. Amino acid sequence analysis of both peptides showed that they had the same common sequence, Ser-Tyr-X-Pro-Gly-Met-Gly-Val, except that the larger one contained Ala-Leu next to the Val residue. From the comparison of the amino acid sequence deduced from cDNA from sheep kidney (Shull, G. E., Schwartz, A., and Lingrel, J. B. (1985) Nature 316, 691-695), X was shown to correspond to Cys-964 of the alpha-chain in Na+,K+-ATPase. The data suggest that the microenvironment of the BIPM residue covalently bound to the sulfhydryl group of Cys-964 changes accompanying sequential appearance of reaction intermediates of Na+,K+-ATPase.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005033:Ethylmaleimide / Q000494:pharmacology; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D008301:Maleimides / Q000378:metabolism*; D008970:Molecular Weight; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D008301:Maleimides; C000245:N-(4-(2-benzimidazolyl)phenyl)maleimide; D000255:Adenosine Triphosphate; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D005033:Ethylmaleimide