THE INFLUENCE OF BETA-SUBUNIT STRUCTURE ON THE INTERACTION OF NA+ K+-ATPASE COMPLEXES WITH NA+ - A CHIMERIC BETA-SUBUNIT REDUCES THE NA+ DEPENDENCE OF PHOSPHOENZYME FORMATION FROM ATP/

High-affinity ouabain binding to Na+/K+-ATPase (sodium- and potassium- transport adenosine triphosphatase (EC 3.6.1.37)) requires phosphoryla tion of the alpha subunit of the enzyme either by ATP or by inorganic phosphate, For the native enzyme (alpha/beta 1), the ATP-dependent rea ction proceeds about 4-fold more slowly in the absence of Na+ than whe n saturating concentrations of Na+ are present, Hybrid pumps were form ed from either the alpha 1 or the alpha 3 subunit isoforms of Na+/K+-A TPase and a chimeric beta subunit containing the transmembrane segment of the Na+/K+-ATPase beta 1 isoform and the external domain of the ga stric H+/R(+)-ATPase beta subunit (alpha/NH beta 1 complexes), In the absence of Na+, these complexes show a rate of ATP-dependent ouabain b inding from similar to 75-100% of the rate seen in the presence of Na depending on buffer conditions, Nonhydrolyzable nucleotides or treatm ent of ATP with apyrase abolishes ouabain binding, demonstrating that ouabain binding to alpha/NH beta 1 complexes requires phosphorylation of the protein, Buffer ions inhibit ouabain binding by alpha/NH beta 1 in the absence of Na+ rather than promote ouabain binding, indicating that they are not substituting for sodium ions in the phosphorylation reaction, The pH dependence of ATP-dependent ouabain binding in the p resence or absence of Na+ is similar, suggesting that protons are prob ably not substituting for Na+, Hybrid alpha/NH beta 1 pumps also show slightly higher apparent affinities (2-3-fold) for ATP, Na+, and ouaba in; however, these are not sufficient to account for the increase in o uabain binding in the absence of Na+. In contrast to phosphoenzyme for mation and ouabain binding by alpha/NH beta 1 complexes in the absence of Na+. ATPase activity, measured as release of phosphate from ATP, r equires Na+. These data suggest that the transition from E(1)P to E(2) P during the catalytic cycle does not occur when the sodium binding si tes are not occupied, Thus, the chimeric beta subunit reduces or elimi nates the role of Na+ in phosphoenzyme formation from ATP, but Na+ bin ding or release by the enzyme is still required for ATP hydrolysis and release of phosphate.