| Abstrakt: |
High-affinity ouabain binding to Na/K-ATPase (sodium- and potassium-transport adenosine triphosphatase (EC 3.6.1.37)) requires phosphorylation of the α subunit of the enzyme either by ATP or by inorganic phosphate. For the native enzyme (α/β1), 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 α1 or the α3 subunit isoforms of Na/K-ATPase and a chimeric β subunit containing the transmembrane segment of the Na/K-ATPase β1 isoform and the external domain of the gastric H/K-ATPase β subunit (α/NHβ1 complexes). In the absence of Na, these complexes show a rate of ATP-dependent ouabain binding from 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 α/NHβ1 complexes requires phosphorylation of the protein. Buffer ions inhibit ouabain binding by α/NHβ1 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 α/NHβ1 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 α/NHβ1 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 β 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. |
