Molecular Mechanism of Cation Migration

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A preparation of pig kindney Na^+, K^+-ATPase showed changes in the fluorescenceenergy transfer between fluorescent probes in the alpha-subunit. The data obtained suggest that the fluorescence energy transfer from the BIPM to the FITC probe increased (as follows : NaE_1, E_1SP,E_2P) and decreased (as follows : E_2P,KE_2, NaE_1). Dynamic fluorescence changes which occurred without phosphorylation or Mg^<2+> seems to reflect change in the binding states of Na^+ and K^+ or process of the migraiton of these ions in the pump molecules.Phospholipase A_2 treatment strongly reduced the fluorescence intensity changes of the BIPM probe with only a slight reduction of the FITC probe in the a-chain of pig kidney Na^+, K^+-ATPase accompanying formaiton of phosphoenzymes. The data obteined suggest that PS or PI which have been shown to be prerequisite for the activity are also prerequisite for the appearance of dynamic BIPM fluorescence chage in the vicinity of Cys-964 which is supposed tobe present in the transmenbrance segment but not the FITC fuorescence change in that of Lys-501 to be present in the soluble domain.The Lys-480 in the alpha-subunits of Na^+, K^+-ATPase from pig kidneys was specifically modified with pyridoxal 5'-phosphate (PLP) or pyridoxal 5'-diphospho-5'-adenosine (AP_2PL) probes in the presence of NaCl. The data obtained suggest that PLP or AP_2PL probes at Lys-480 in the presence of Na^+ and Mg^<2+> do not affect the transphosphorylation from AcP to Asp-369 to form phpsphoenzymes but that they inhibit the transphosphorylation from the gamma-phosphoryl group of ATP and also ATP binding in the absence of Mg^<2+>.Paranitrophenylphosphate (pNPP) induced fluorescence changes in fluorescence isothiocyanate (FITC) -labeled Na^+, K^+-ATPase preparations. These data and others indicate that a much higher degree of oligomerization, rather than (alphabeta) _2, may be the functional unit of the enzyme in the membranes.