Moleacular Mechanism of Cation Migration

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A preparation of pig kindney Na^+, K^+-ATPase showed changes in the fluorescence energy transfer between fluorescent probes in the alpha-subunits. Excitation (305 nm) of the N-(p-(2-benzimidazolyl)phenyl) maleimide(BIPM) probe at Cys-964, and excitation (470 nm) of the fluorescein isothiocyanate(FITC) probe at Lys-501 gave different FITC fluorescene intensity changes at 520 nm in BIPM-FITC doubly labeled anzyme accompanying formatin of reactino intermediates. These data suggest that the fluorescence energy transfer from the BIPM to the FITC probe increased (as follows NaE_1, E_1P,E_2P) and decreased (as follows : E_2P,KE_3, NAE_1). Dynamic fluorescence changes which occurred without phosphorylation or Mg^<2+> seems to reflectchange in the binding states of Na^+ and K^+ or process of the migration of these ions in the pump molecules.Phopholipase A_2 treatment strongly reduced the fluorescence intensity changes of the BIPM probe with only a slight reductin of the FITC probe in the a-chain of pig kidney Na^+, K^+-ATPase accompanying formatin of phosphoenzymes. The treatment reduced both rate of florescence changes. The anisotropy of both probes were little changed by the treatment. The treatment reduced the Na^+, K^+-ATPase activity of BIPM treated enzyme to 15%. The addition of phosphatidyl serine(PS) or phosphatidyl inositol(PI) increased the extent of the BIPM fluorescence change accompanying the increase in the Na^+, K^+-ATPase activity of BIPM enzyme and the rate of FITC fluorescence changes the BIPM-FITC enzyme. These data suggest that PS or PI which have been shown to be prerequisite for the activity are also prerequisite for the apearance of dynamic B IPM fluorescence change in the viinity of Cys-964 which is supposed to be present in the transmenbrance segment byt not for the FITC fluorescence change in that of Lys-501 to by present in the soluble domain.