Chemical modification with dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonate reveals the distance between K-480 and K-501 in the ATP-binding domain of the Na,K-ATPase

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 [H-3(2)]DIDS molecule into the Na pump alpha-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 (H2DIDS)-H-3-inactivated enzyme, a single radiolabeled peptide is seen in 16.5% Tricine gels. N-terminal amino acid sequencing revealed two sequences (470)IVEIPFNSTNxYQLS and (495)HLLVMxGAPER, the unidentified residues were K-480 and K-501, respectively. These data provide suggestive evidence of cross-linking by H2DIDS between the two lysines. CNBr digestion of (H2DIDS)-H-3-labeled alpha-subunit produced a single radioactive band of the predicted 15-kDa mass for cross-linking between K-480 an K-501 produced by cleavage at known methione residues. The 15-kDa band combined two N-terminal sequences (464)RDRYAKIVEI and (501)xGAPERLLDR which include R(480) and K-501. Thus K-480 and K-501 are within approximately 14 Angstrom 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. (C) 1997 Academic Press.