Abstract

High-affinity ouabain binding to Na+/K(+)-ATPase (sodium- and potassium-transport adenosine triphosphatase (EC 3.6.1.37)) requires phosphorylation of the alpha subunit of the enzyme either by ATP or by inorganic phosphate. For the native enzyme (alpha/beta 1), the ATP-dependent reaction proceeds about 4-fold more slowly in the absence of Na+ than when saturating concentrations of Na+ are present. Hybrid pumps were formed from either the alpha 1 or the alpha 3 subunit isoforms of Na+/K(+)-ATPase and a chimeric beta subunit containing the transmembrane segment of the Na+/K(+)-ATPase beta 1 isoform and the external domain of the gastric H+/K(+)-ATPase beta subunit (alpha/NH beta 1 complexes). In the absence of Na+, these complexes show a rate of ATP-dependent ouabain binding from approximately 75-100% of the rate seen in the presence of Na+ depending on buffer conditions. Nonhydrolyzable nucleotides or treatment 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 presence or absence of Na+ is similar, suggesting that protons are probably not substituting for Na+. Hybrid alpha/NH beta 1 pumps also show slightly higher apparent affinities (2-3-fold) for ATP, Na+, and ouabain; however, these are not sufficient to account for the increase in ouabain binding in the absence of Na+. In contrast to phosphoenzyme formation and ouabain binding by alpha/NH beta 1 complexes in the absence of Na+, ATPase activity, measured as release of phosphate from ATP, requires Na+. These data suggest that the transition from E1P to E2P during the catalytic cycle does not occur when the sodium binding sites are not occupied. Thus, the chimeric beta subunit reduces or eliminates the role of Na+ in phosphoenzyme formation from ATP, but Na+ binding or release by the enzyme is still required for ATP hydrolysis and release of phosphate.

Highlights

  • From the +Department of Physiology and Biophysics and Wepartment of Biochemistry, University of Southern California School of Medicine, Los Angeles, California 90033

  • ATP-dependent Binding of fH10uabain-During the transport of Na" and K+, Na+fK+-ATPase undergoes a reaction sequence that includes Na " binding at the intracellular face of the enzyme, ATP hydrolysis, Na" transport across the plasma membrane, K+ binding at the extracellular face of the enzyme, and its transport to the cytoplasm of the cell (Fig. lA) [35, 36]

  • The 0' subunit of Na+/K+-ATPase shows extensive homology to the larger family of P-type cation transport ATPases, and numerous studies have identified sites within the 0' subunit that are involved in ATP binding and hydrolysis

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Summary

Introduction

From the +Department of Physiology and Biophysics and Wepartment of Biochemistry, University of Southern California School of Medicine, Los Angeles, California 90033. Buffer ions inhibit ouabain binding by cx/NHf31 in the absence of Na" rather than promote ouabain binding, indicating that they are not substituting for sodium ions in the phosphorylation reaction. In contrast to phosphoenzyme formation and ouabain binding by cx/NHf31 complexes in the absence of Na", ATPase activity, measured as release of phosphate from ATP, requires Na+. These data suggest that the transition from ElP to E2P during the catalytic cycle does not occur when the sodium binding sites are not occupied.

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