Chemical modification with dihydro-4,4'-diisothiocyanostilbene-2,2'- disulfonate reveals the distance between K480 and K501 in the ATP- binding domain of the Na,K-ATPase

Craig Gatto, Svetlana Lutsenko, Jack H. Kaplan

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

Abstract

Dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonate (H2DIDS) inactivates the renal Na,K-ATPase in an ATP- and K-preventable fashion; inactivation results in the covalent incorporation of a single [3H2]DIDS molecule into the Na pump α-subunit. K+ protection is observed at low concentrations (<2 mM) and reversed at higher concentrations. The biphasic effect is also seen with Rb+, to a lesser extent by Cs+, and not at all by Na+ or choline. After extensive tryptic digestion of 3H2DIDS-inactivated enzyme, a single radiolabeled peptide is seen in 16.5% Tricine gels. N- terminal amino acid sequencing revealed two sequences 470IVEIPFNSTNxYQLS and 495HLLVMxGAPER, the unidentified residues were K480 and K501, respectively. These data provide suggestive evidence of cross-linking by H2DIDS between the two lysines. CNBr digestion of 3H2DIDS-labeled α- subunit produced a single radioactive band of the predicted 15-kDa mass for cross-linking between K480 an K501 produced by cleavage at known methione residues. The 15-kDa band combined two N-terminal sequences 464RDRYAKIVEI and 501xGAPERILDR which include K480 and K501. Thus K480 and K501 are within approximately 14 Å of each other in the Na- bound form of the enzyme and information about the occupancy of the cation binding domain is transmitted to the ATP binding loop of the Na,K-ATPase.

Original languageEnglish (US)
Pages (from-to)90-100
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume340
Issue number1
DOIs
StatePublished - Apr 1 1997
Externally publishedYes

Keywords

  • Na pump
  • active transport
  • chemical modification
  • cross- linking
  • isothiocyanates

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Fingerprint

Dive into the research topics of 'Chemical modification with dihydro-4,4'-diisothiocyanostilbene-2,2'- disulfonate reveals the distance between K480 and K501 in the ATP- binding domain of the Na,K-ATPase'. Together they form a unique fingerprint.

Cite this