Inactivation of (Na+ + K+)-ATPase by Chromium(III) Complexes of Nucleotide Triphosphates

Abstract

(Na⁺+ K⁺)-ATPase from beef brain and pig kidney are slowly inactivated by chromium(III) complexes of nucleotide triphosphates in the absence of added univalent and divalent cations. The inactivation of (Na⁺+ K⁺)-ATPase activity was accompanied by a parallel decrease of the associated K⁺-activated p-nitrophenylphosphatase and a parallel loss of the capacity to form, Na⁺-dependently, a phosphointermediate from [γ-³²P]ATP. The kinetics of inactivation and of phosphorylation with [γ-³²P]CrATP and [α-³²P]CrATP are consistent with the assumption of the formation of a dissociable complex of CrATP with the enzyme (E) followed by phosphorylation of the enzyme: The dissociation constant of the CrATP complex of the pig kidney enzyme at 37°C was 43 μM. The inactivation rate constant (k₊₂= 0.033 min⁻¹) was in the range of the dissociation rate constant k_d of ADP from the enzyme of 0.011 min⁻¹. The phosphoenzyme was unreactive towards ADP as well as to K⁺. No hydrolysis of the native isolated phosphoenzyme was observed within 6 h under a variety of conditions, but high concentrations of Na⁺ reactivated it slowly. The capacity of the Cr-phosphoenzyme of 121 ± 18 pmol/unit enzyme is identical with the capacity of the unmodified enzyme to form, Na⁺-dependently, a phosphointermediate. The Cr-phospho-enzyme behaved after acid denaturation like an acylphosphate towards hydroxylamine, but the native phosphoenzyme was not affected by it. ATP protected the enzyme against the inactivation by CrATP (dissociation constant of the enzyme ATP complex = 2.5 μM) as well as low concentrations of K⁺. CrATP was a competitive inhibitor of (Na⁺+ K⁺)-ATPase. It is concluded that CrATP is slowly hydrolyzed at the ATP-binding site of (Na⁺+ K⁺)-ATPase and inactivates the enzyme by forming an almost non-reactive phosphoprotein at the site otherwise needed for the Na⁺-dependent protein kinase reaction as the phosphate acceptor site.