TISSUE-SPECIFIC DISTRIBUTION AND MODULATORY ROLE OF THE GAMMA-SUBUNITOF THE NA,K-ATPASE

The Na,K-ATPase comprises a catalytic alpha subunit and a glycosylated beta subunit. Another membrane polypeptide, gamma, first described by Forbush et al. (Forbush, B., III, Kaplan, J. H., and Hoffman, J. F. ( 1978) Biochemistry 17, 3667-3676) associates with alpha and beta in pu rified kidney enzyme preparations. In this study, we have used a polyc lonal anti-gamma antiserum to define the tissue specificity and topolo gy of gamma and to address the question of whether gamma has a functio nal role. The trypsin sensitivity of the amino terminus of the gamma s ubunit in intact right-side-out pig kidney microsomes has confirmed th at it is a type I membrane protein with an extracellular amino terminu s. Western blot analysis shows that gamma subunit protein is present o nly in membranes from kidney tubules (rat, dog, pig) and not those fro m axolemma, heart, red blood cells, kidney glomeruli, cultured glomeru lar cells, a,transfected HeLa cells, all derived from the same (rat) s pecies, nor from three cultured cell lines derived from tubules of the kidney, namely NRK-52E (rat), LLC-PK (pig), or MDCK (dog). To gain in sight into gamma function, the effects of the anti-gamma serum on the kinetic behavior of rat kidney sodium pumps was examined. The followin g evidence suggests that gamma stabilizes E-1 conformation(s) of the e nzyme and that anti-gamma counteracts this effect: (i) anti-gamma inhi bits Na,K-ATPase, and the inhibition increases at acidic pH under whic h condition the E-2(K) --> E-1 phase of the reaction sequence becomes more rate-limiting, (ii) the oligomycin-stimulated increase in the lev el of phosphoenzyme was greater in the presence of anti-gamma indicati ng that the antibody shifts the E-1 <----><----> E2P equilibria toward E2P, and (iii) when the Na+-ATPase reaction is assayed with the Na+ c oncentration reduced to levels (less than or equal to 2 mM) which limi t the rate of the E-1 --> --> E2P transition, anti-gamma is stimulator y. These observations taken together with evidence that the pig gamma subunit, which migrates as a doublet on polyacrylamide gels, is sensit ive to digestion by trypsin, and that Rb+ ions partially protect it ag ainst this effect, indicate that the gamma subunit is a tissue-specifi c regulator which shifts the steady-state equilibria toward E-1. Accor dingly, binding of anti-gamma disrupts alpha beta-gamma interactions a nd counteracts these modulatory effects of the gamma subunit.