Chromium(III)ATP inactivating (Na++ K+)‐ATPase supports Na+‐Na+ and Rb+‐Rb+ exchanges in everted red blood cells but not Na+,K+ transport

Abstract

1. The chromium(III) complex of ATP, and MgATP complex analogue, inactivates (Na+ + K+)-ATPase by forming a stable chromo-phosphointermediate. The rate constant k2 of inactivation at 37.degree. C of the .beta.,.gamma.-bidentate of CrATP is enhanced by Na+ (Ko.5 = 1.08 mM), imidazole (K0.5 = 15 mM) and Mg2+ (K0.5 = 0.7 mM). These cations did not affect the dissociation constant of the enzyme-chromium-ATP complex. 2. The inactive chromophosphoenzyme is reactivated slowly by high concentrations of Na+ at 37.degree. C. The half-maximal effect on the reactivation was reached at 40 mM NaCl, when the maximally observable reactivation was studied. However, 126 mM NaCl was necessary to see the half-maximal effect on the apparent reactivation velocity constant. K+ ions hindered the reactivation with a Ki of 70 .mu.M. 3. Formation of the chromophosphoenzyme led to a reduction of the Rb+ binding sites and of the capacity to occlude Rb+. 4. The .beta.,.gamma.-bidentate of chromium(III)ATP (Kd - 8 .mu.M) had a higher affinity than the .alpha.,.beta.,.gamma.-tridentate of chromium(III)ATP (Kd = 44 .mu.M) or the cobalt tetramine complex of ATP (Kd = 500 .mu.M). The .beta.,.gamma.-bidentate of the chromium(III) complex of adenosine 5''-[.beta.,.gamma.-methylene]triphosphate also inactivated (Na+ + K+)ATPase. 5. Although CrATP could not support Na+,K+ exchange in everted vesicles prepared from human red blood cells, it supported the Na+-Na+ and Rb+-Rb+ exchange. 6. It is concluded that CrATP opens up Na+ and K+ channels by forming a relatively stable modified enzyme-CrATP complex. This stable complex is also formed in the presence of the chromium complex of adenosine 5''-[.beta.,.gamma.-methylene]triphosphate. Because the .beta.,.gamma.-bidentate of chromium ATP is recognized better than the .alpha.,.beta.,.gamma.-tridentate, it is concluded that the triphosphate site recognizes MgATP with a straight polyphosphate chain and that the Mg2+ resides between the .beta.- and the .gamma.-phosphorus. The enhancement of inactivation by Mg2+ and Na+ may be caused by conformational changes at the triphosphate site.