Moleacular Mechanism of Cation Migration

制备的猪肾脏Na^+，K^+-ATP酶显示α亚基中荧光探针之间的荧光能量转移发生了变化。用N-（对-（2-苯并咪唑基）苯基）马来酰亚胺（BIPM）探针在Cys-964处激发（305 nm）和异硫氰酸荧光素（FITC）探针在Lys-501处激发（470 nm），在BIPM-FITC双标记酶中，伴随反应中间体的形成，在520 nm处产生不同的FITC荧光强度变化。这些数据表明，BIPM向FITC探针的荧光能量转移依次为：NaE_1、E_1P、E_2P; E_2P、KE_3、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^+结合状态的变化或这些离子在泵分子中的迁移过程。磷脂酶A_2处理强烈地降低了BIPM探针的荧光强度变化，而在猪肾Na^+的a链中的FITC探针仅轻微地降低，K^+-ATP酶伴随磷酸酶的形成。处理降低了两种花期变化率。两种探针的各向异性通过处理几乎没有改变。BIPM处理的酶的Na^+，K^+-ATP酶活性降低到15%。加入磷脂酰丝氨酸（PS）或磷脂酰肌醇（PI）后，BIPM酶的Na^+，K^+-ATP酶活性增加，BIPM-FITC酶的FITC荧光变化率增加，BIPM荧光变化幅度增大。这些数据表明，PS或PI已被证明是活性的先决条件，也是Cys-964活力中出现动态B IPM荧光变化的先决条件，Cys-964被认为存在于跨膜段拜特，而不是Lys-501中的FITC荧光变化到可溶性结构域。

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.