Simultaneous binding of phosphate and TNP-ADP to FITC-modified NA+,K(+)-ATPase.
Simultaneous binding of phosphate and TNP-ADP to FITC-modified NA+,K(+)-ATPase.
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磷酸盐和 TNP-ADP 同时与 FITC 修饰的 NA ,K( )-ATP 酶结合。
DOI：
10.1021/bi00088a011
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Farley,RA
中科院分区：
文献类型：
--
作者：
Scheiner-Bobis,G;Antonipillai,J;Farley,RA
摘要：Na+，K+-ATP酶的ATP水解速率与ATP浓度的双倒数图不是线性的，可能反映了两个不同的ATP结合位点，也可能反映了一个单一的ATP结合位点，其对核苷酸的亲和力在高亲和力和低亲和力状态之间交替变化。为了确定多个核苷酸或核苷酸类似物是否能同时与Na ~+，K ~+-ATP酶结合，测定了核苷酸对磷酸对硝基苯酯水解和异硫氰酸荧光素（FITC）修饰的Na ~+，K ~+-ATP酶去磷酸化速率的影响。当ATP浓度高达8.3 mM时，FITC可阻断Na+，K+-ATP酶上ATP的高亲和力结合位点，抑制ATP的水解。未修饰的Na+，K+-ATP酶的p-硝基苯磷酸酶活性被低浓度的ATP（10-100 µ）和其他核苷酸所刺激，而在较高的核苷酸浓度下被抑制。相比之下，在ATP浓度小于100 µ时，FITC修饰的Na+，K+-ATP酶对磷酸对硝基苯酯的水解没有影响。当ATP浓度大于100 µ时，FITC修饰的Na+，K+-ATP酶对磷酸对硝基苯酯的水解受到抑制。这些结果表明，在FITC修饰的Na ~+，K ~+-ATP酶中，高亲和力位点的ATP效应消失，而低亲和力位点的ATP效应仍存在。在未修饰的Na ~+，K ~+-ATP酶中，无机磷和Mg ~（2+）形成的磷酸酶的去磷酸化速率被ATP抑制。虽然ATP在浓度高达1 mM时不影响FITC修饰的Na+，K+-ATP酶的去磷酸化速率，但TNP-ADP抑制FITC修饰的酶的去磷酸化。TNPADP效应的Kd为35 µ，显著高于TNP-ADP与未修饰Na+，K+-ATP酶结合的测量Kd（Moczydlowski & Fortes，1982）。这些结果表明，磷酸盐、TNP-ADP和FITC可以同时与Na+，K+-ATP酶结合，并且可以反映该酶上存在高亲和力和低亲和力的核苷酸位点。（Na+，K+-ATP酶，1个钠泵）主动地将钠离子和钾离子逆着每个离子的电化学电势梯度穿过细胞膜（Skou，1988; Glynn，1990）。该吸能过程所需的能量由腺苷S '-三磷酸（ATP）提供，其在泵循环期间由泵水解。已经提出了几种不同的动力学模型来描述
Revised Manuscript Received July 7, 1993· abstract: Double-reciprocal plots of the rate of ATP hydrolysis by Na+, K+-ATPase versus ATP concentration are not linear, and may reflect either two distinct binding sites for ATP or a single ATP binding sitewhose affinity for the nucleotide alternates between high-affinity and low-affinity states. In order to determine whether multiple nucleotides or nucleotide analogs can bind simultaneously to Na,+, K+-ATPase, the effects of nucleotides on the hydrolysis of p-nitrophenyl phosphateand on the dephosphorylation rate of Na+, K+-ATPase modified by fluorescein S'-isothiocyanate (FITC) were measured. FITC blocks the high-affinity binding site for ATP on the Na+, K+-ATPase and inhibits ATP hydrolysis at ATP concentrations as high as 8.3 mM. The hydrolysis ofp-nitrophenyl phosphate and phosphoenzyme formation from inorganic phosphate and Mg2+ were not affected by FITC modification. Thep-nitrophenylphosphatase activity of unmodified Na+, K+-ATPase was stimulated by low concentrations of ATP (10—100 µ) and other nucleotides, and was inhibited at higher nucleotide concentrations. In contrast, there was no effect on p-nitrophenyl phosphate hydrolysis by FITC-modified Na+, K+-ATPase at ATP concentrations less than 100 µ. The hydrolysis of p-nitrophenyl phosphate by FITC-modified Na+, K+-ATPase was inhibited at ATP concentrations greater than 100 µ. These observations demonstrate that theeffects of ATP acting at high-affinity sites are absent in FITC-modified Na+, K+-ATPase but the effects of ATP acting at low-affinity sites are still observed. In unmodified Na+, K+-ATPase, the rate of dephosphorylation of the phosphoenzyme formedfrom inorganic phosphate and Mg2+ was inhibited by ATP. Although ATP at concentrations up to 1 mM did not affect the rate of dephosphorylation of the FITC-modified Na+, K+-ATPase, TNP-ADP inhibited the dephosphorylation of FITC-modified enzyme. The for the TNPADP effect is 35 µ, considerably higher than the measured Kd for TNP-ADP binding to unmodified Na+, K+-ATPase (Moczydlowski & Fortes, 1982). These results demonstrate that it is possible to simultaneously bind phosphate, TNP-ADP, and FITC to the Na+, K+-ATPase, and may reflect the presence of both high-affinity and low-affinity nucleotide sites on the enzyme.Sodium-and potassium-activated adenosine-S'-triphos-phatase (Na+, K+-ATPase, 1 sodium pump) actively transports sodium ions and potassium ions across cell membranes against electrochemical potential gradients for each ion (Skou, 1988; Glynn, 1990). The energy required for this endergonic process is provided by adenosine S'-triphosphate (ATP), which is hydrolyzed by the pump during the pump cycle. Several different kinetic models have been proposed to describe the