Na,K-ATPase (Na-pump) that catalyzes the active transport of Na + and K + across the plasma membrane of animal cells consists of two subunits: α-subunit with molecular mass of about 110 kD and β-subunit that is a glycoprotein. The molecular mass of the protein part of the β-subunit is about 35 kD, and after glycosylation it increases to 55-60 kD. Both subunits are integral proteins of the plasma membrane. The α-subunit is the catalytic one: it hydrolyses ATP, forms channel for Na + and K + , and couples ATP hydrolysis to the transport of cations against the electrochemical gradient through the channel [1]. The β-subunit does not participate directly in the catalysis and in the translocation of the cations. It plays an important role in the transportation of newly synthe- sized subunits from endoplasmic reticulum to plasma membrane [2]. The β-subunit appears to have also anoth- er function: it was identified as a factor responsible for cell adhesion in nervous tissues [3]. In various tissues Na,K-ATPase is involved in differ- ent functions: maintenance of intracellular electrolyte homeostasis, creation of transmembrane potential, con- duction of action potential in neurons and contraction of muscles, transepithelial transport of Na + , and Na + - dependent transport of different substances into the cells (Na + /Ca 2+ and Na + /H + -exchange, Na + -dependent transport of sugars and amino acids) (for review see [4]). All these suggest complex mechanisms of Na-pump reg- ulation, including the insertion of Na,K-ATPase mole- cules into the membrane and their removal from the membrane, concentration of pump molecules in certain parts of the plasma membrane (such as basolateral mem- brane of kidney epithelial cells), and interaction of Na- pump molecules with different protein regulators. Na,K-ATPase is not only a pump but also a receptor for steroid compounds like ouabain and its derivatives. First ouabain and similar steroids (cardiac glycosides) were found in plants that do not have Na,K-ATPase, but later cardiac glycosides were revealed in animal tissues. Ouabain binding to Na,K-ATPase in animal cells results in its interaction with certain intracellular proteins that triggers different signal transduction pathways and results finally in the increase of intracellular concentration of some secondary messengers and in the expression of some genes [5]. Thus, the interaction of Na,K-ATPase with tissue specific sets of proteins is necessary for the normal func- tion of the enzyme in different tissues. Four Na,K-ATPase α-subunit isoforms (referred to as α 1 , α 2 , α 3 , and α 4 ) and three types of β-subunit isoforms (β 1 , β 2 , and β 3 ) have Biochemistry (Moscow), Vol. 68, No. 9, 2003, pp. 1040-1047. Translated from Biokhimiya, Vol. 68, No. 9, 2003, pp. 1271-1279. Original Russian Text Copyright © 2003 by Akimova, Dolgova, Mast, Rubtsov, Lopina. 0006-2979/03/6809-1040$25.00 ©2003 MAIK “Nauka / Interperiodica” Abbreviations: DTSP) 3,3′-dithio-bis(propionic acid N-hydroxy- succinimide ester); DTT) dithiothreitol; TBST) Tris-buffered saline containing Tween-20; PMSF) phenylmethylsulfonyl flu- oride; EGS) ethylene glycol-bis(succinic acid N-hydroxysuc- cinimide ester). * To whom correspondence should be addressed. Revealing of Proteins Interacting with Na,K-ATPase O. A. Akimova, N. V. Dolgova, N. V. Mast, A. M. Rubtsov, and O. D. Lopina* Department of Biochemistry, Faculty of Biology, Lomonosov Moscow State University, Moscow 119992, Russia; fax: (095) 939-3955; E-mail: od_lopina@mail.ru Received December 11, 2002 Revision received March 12, 2003 Abstract—Proteins interacting with α1β1-type of Na,K-ATPase were revealed in pig kidney outer medulla and duck salt glands using three different methods (immunoprecipitation, protein overlay, and chemical cross-linking). Immunoprecipitation was performed after solubilization of protein homogenate with Triton X-100 so that both membrane and cytosol proteins bound to Na,K-ATPase could be revealed. Two other methods were used to study the interaction of cytosol proteins with purified Na,K-ATPase. The sets of proteins revealed by each method in outer medulla of pig kidney were different. Proteins interacting with Na,K-ATPase that have molecular masses 10, 15, 70, 75, 105, 120, and 190 kD were found using the immunoprecipitation method. The chemical cross-linking method revealed proteins with molecular masses 25, 35, 40, 58, 68-70, and 86-88 kD. The protein overlay method revealed in the same tissue proteins with molecular masses 38, 42, 43, 60, 62, 66, 70, and 94 kD. Key words: Na,K-ATPase, protein–protein interactions, immunoprecipitation, chemical cross-linking, protein overlay