E2P phosphoforms of Na,K-ATPase. I. Comparison of phosphointermediates formed from ATP and Pi by their reactivity toward hydroxylamine and vanadate.
The properties of Na,K-ATPase phosphoenzymes formed either from ATP in the presence of Mg2+ and Na+ or from Pi in the absence of alkali cations were investigated by biochemical methods and spectrofluorometry employing the styryl dye RH421. We characterized the phosphoenzyme species by their reaction to N-methyl hydroxylamine, which attacks specifically the protein-phosphate bond. We studied reactions of the phospho- and dephospho-enzymes with vanadate, which is a transition-state analogue of phosphate in this enzyme. On the basis of substantial differences in the properties of the phosphoenzyme species formed either from ATP or Pi, especially in their reactivity to N-methyl hydroxylamine, it is suggested that the two phosphoenzyme species are two subconformations of the E2P phosphoform. Analysis of the RH421 fluorescence responses under a variety of experimental conditions and comparing different enzyme sources suggested that the increase of RH421 fluorescence induced by inorganic phosphate in the absence of alkali cations is associated with the formation of the covalent acyl-phosphate bond.
Fedosova
N U
NU
Department of Biophysics, University of Aarhus, Denmark. nf@biophys.au.dk
Cornelius
F
F
Klodos
I
I
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D004789:Enzyme Activation / Q000187:drug effects; D005456:Fluorescent Dyes; D006898:Hydroxylamines / Q000378:metabolism* / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism*; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011726:Pyridinium Compounds / Q000378:metabolism; D012754:Sharks; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013343:Styrenes / Q000378:metabolism; D013552:Swine; D014638:Vanadates / Q000378:metabolism* / Q000494:pharmacology
D005456:Fluorescent Dyes; D006898:Hydroxylamines; D010710:Phosphates; D010750:Phosphoproteins; D011726:Pyridinium Compounds; D013343:Styrenes; C070534:RH 421; D014638:Vanadates; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; C018338:N-methylhydroxylamine