Molecular Mechanism of Cation Migration

猪肾Na^+， K^+- atp酶的制备表明α -亚基荧光探针之间的荧光能量转移发生了变化。所得数据表明，BIPM向FITC探针传递的荧光能量增加（如下：NaE_1， E_1SP,E_2P），减少（如下：E_2P，KE_2, NaE_1）。在没有磷酸化或Mg^<2+>的情况下发生的动态荧光变化似乎反映了Na^+和K^+结合态的变化或这些离子在泵分子中的迁移过程。磷脂酶A_2处理显著降低了BIPM探针的荧光强度变化，仅轻微降低了猪肾Na^+， K^+- atp酶a链上的FITC探针伴随磷酸化酶的形成。得到的数据表明，已被证明是活性的先决条件的PS或PI也是Cys-964附近出现BIPM动态荧光变化的先决条件，Cys-964应该存在于跨膜段，而Lys-501的FITC荧光变化不存在于可溶性结构域。在NaCl存在下，用吡哆醛5′-磷酸（PLP）或吡哆醛5′-二磷酸-5′-腺苷（AP_2PL）探针特异性修饰猪肾Na^+， K^+- atp酶α -亚基中的Lys-480。结果表明，当Na^+和Mg^<2+>存在时，PLP或AP_2PL探针在Lys-480不影响AcP到Asp-369的转磷酸化形成磷酸酶，但它们抑制ATP的γ -磷酸基的转磷酸化以及在没有Mg^<2+>存在时ATP的结合。副硝基苯基磷酸（pNPP）诱导荧光异硫氰酸酯（FITC）标记的Na^+， K^+- atp酶制剂的荧光变化。这些数据和其他数据表明，在膜中酶的功能单位可能是低聚程度高得多的，而不是（α β） _2。

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.