ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 340, No. 1, April 1, pp. 90–100, 1997 Article No. BB979879 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 1 Craig Gatto, 2 Svetlana Lutsenko, and Jack H. Kaplan 3 Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201-3098 Received November 18, 1996, and in revised form December 23, 1996 The Na,K-ATPase (EC 3.6.1.37) (also known as the Na pump) is an integral plasma membrane protein that Dihydro-4,4-diisothiocyanostilbene-2,2 -disulfonate (H 2 DIDS) inactivates the renal Na,K-ATPase in an employs energy derived from ATP hydrolysis to ac- ATP- and K-preventable fashion; inactivation results tively transport Na and K ions against their electro- in the covalent incorporation of a single [ 3 H 2 ]DIDS chemical potential gradients. The Na pump is a func- molecule into the Na pump a-subunit. K / protection tional heterodimeric protein, consisting of a catalytic is observed at low concentrations (õ2mM) and re- a-subunit (Ç112 kDa) and a smaller glycosylated b- versed at higher concentrations. The biphasic effect subunit (Ç55 kDa). Both subunits have been cloned is also seen with Rb / , to a lesser extent by Cs / , and and the primary structure determined for several iso- not at all by Na / or choline. After extensive tryptic forms from a variety of species (see 1). The Na pump digestion of 3 H 2 DIDS-inactivated enzyme, a single ra- is a member of a large family of cation transporting diolabeled peptide is seen in 16.5% Tricine gels. N-ter- proteins, the P-type ATPases, which includes such pro- minal amino acid sequencing revealed two sequences teins as the Ca-ATPases of the plasma membrane and 470 IVEIPFNSTNxYQLS and 495 HLLVMxGAPER, the un- sarco/endoplasmic reticulum, the gastric H,K-ATPase, identified residues were K 480 and K 501 , respectively. and the Neurospora H-ATPase (for review, see 2). The These data provide suggestive evidence of cross-link- term ‘‘P-type’’ refers to the formation of a covalent phos- ing by H 2 DIDS between the two lysines. CNBr diges- phorylated intermediate during the enzymatic cycle; tion of 3 H 2 DIDS-labeled a-subunit produced a single the overall reaction mechanism shows great similari- radioactive band of the predicted 15-kDa mass for ties among the different members of the family (for cross-linking between K 480 an K 501 produced by cleav- review, see 3). age at known methione residues. The 15-kDa band Although the Na pump has been the subject of exten- combined two N-terminal sequences 464 RDRYAKIVEI sive study for several decades, little precise information and 501 xGAPERILDR which include K 480 and K 501 . Thus is available about the specific amino acid residues in- K 480 and K 501 are within approximately 14 A ˚ of each volved in forming the binding sites for the physiological other in the Na-bound form of the enzyme and infor- ligands. One approach to determine the identity of the mation about the occupancy of the cation binding do- amino acids involved in ligand binding is to employ main is transmitted to the ATP binding loop of the modification with chemical reagents. These may be ei- Na,K-ATPase.  1997 Academic Press ther residue-specific or affinity-based reagents. In this Key Words: isothiocyanates; chemical modification; strategy, a labeled reagent is covalently attached to the Na pump; active transport; cross-linking. protein and by an analysis of the kinetics of inactiva- tion, ligand-based protection, properties of modified en- zyme, stoichiometry, etc., some sense of the likely mode 1 This work was supported by NIH Grant HL 30315 to J.H.K. of action of the reagent and role of the target amino 2 C.G. is an American Heart Association, Oregon Affiliate Research acid emerges. The final step is isolation of specifically Fellow. labeled fragments of the protein and identification of 3 To whom correspondence should be addressed. Fax: (503) 494- 1002. E-mail: kaplanj@ohsu.edu. the modified amino acid within the primary structure. 90 0003-9861/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.