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.
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 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 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 alpha-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 A 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.
Gatto
C
C
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201-3098, USA.
Lutsenko
S
S
Kaplan
J H
JH
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000737:chemistry* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002414:Cations, Monovalent / Q000737:chemistry; D003488:Cyanogen Bromide; D004285:Dogs; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D008239:Lysine / Q000737:chemistry*; D010449:Peptide Mapping; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000648:ultrastructure*
D002414:Cations, Monovalent; D004791:Enzyme Inhibitors; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008239:Lysine; D003488:Cyanogen Bromide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid