E2P PHOSPHOFORMS OF NA,K-ATPASE - I - COMPARISON OF PHOSPHOINTERMEDIATES FORMED FROM ATP AND P-I 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 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.