Inactivation of the Na,K-ATPase by modification of Lys-501 with 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS)

AbstractThe sodium pump or Na,K-ATPase, maintains the Na+ and K+ gradients across eukaryotic cell membranes at the expense of ATP. Incubation of purified canine renal Na,K-ATPase with 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS) inhibited the ATPase activity. Both the labeling of the protein and the loss or ATPase activity were prevented by co-incubation with ADP (acting as an ATP analog) or KCI. Only the α-subunit was labeled by SITS. The α-subunit from the inhibited enzyme was extensively digested with trypsin, and SITS-labeled peptides were purified by reverse-phase HPLC and sequenced. The amino acid sequence determined, His-Leu-Leu-Val-Met-X-Gly-Ala-Pro-Glu, indicated that SITS modifies Lys-501 (X) on the α-subunit of Na,K-ATPase