Nu. Fedosova et al., E2P PHOSPHOFORMS OF NA,K-ATPASE - I - COMPARISON OF PHOSPHOINTERMEDIATES FORMED FROM ATP AND P-I BY THEIR REACTIVITY TOWARD HYDROXYLAMINE AND VANADATE, Biochemistry (Easton), 37(39), 1998, pp. 13634-13642
The properties of Na,K-ATPase phosphoenzymes formed either from ATP in
the presence of Mg2+ and Na+ or from P-i in the absence of alkali cat
ions were investigated by biochemical methods and spectrofluorometry e
mploying the styryl dye RH421. We characterized the phosphoenzyme spec
ies by their reaction to N-methyl hydroxylamine, which attacks specifi
cally the protein-phosphate bond. We studied reactions of the phospho-
and dephospho-enzymes with vanadate, which is a transition-state anal
ogue of phosphate in this enzyme. On the basis of substantial differen
ces in the properties of the phosphoenzyme species formed either from
ATP or P-i, especially in their reaclivity to N-methyl hydroxylamine,
it is suggested that the two phosphoenzyme species are two subconforma
tions of the E2P phosphoform. Analysis of the RH421 fluorescence respo
nses under a variety of experimental conditions and comparing differen
t enzyme sources suggested that the increase of RH421 fluorescence ind
uced by inorganic phosphate in the absence of alkali cations is associ
ated with the formation of the covalent acyl-phosphate bond.