Chemical Modification with Dihydro-4,4′-diisothiocyanostilbene-2,2′-disulfonate Reveals the Distance between K480and K501in 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 [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 of3H2DIDS-inactivated enzyme, a single radiolabeled peptide is seen in 16.5% Tricine gels. N-terminal amino acid sequencing revealed two sequences470IVEIPFNSTNxYQLS and495HLLVMxGAPER, the unidentified residues were K480and K501, respectively. These data provide suggestive evidence of cross-linking by H2DIDS between the two lysines. CNBr digestion of3H2DIDS-labeled α-subunit produced a single radioactive band of the predicted 15-kDa mass for cross-linking between K480an K501produced by cleavage at known methione residues. The 15-kDa band combined two N-terminal sequences464RDRYAKIVEI and501xGAPERILDR which include K480and K501. Thus K480and K501are 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.