Characterization of conformational changes in (Na,K) ATPase labeled with fluorescein at the active site
Characterization of conformational changes in (Na,K) ATPase labeled with fluorescein at the active site
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活性位点荧光素标记的 (Na,K) ATP 酶构象变化的表征
DOI：
10.1007/bf00744678
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发表时间：
1980
影响因子：
3
通讯作者：
S. Karlish
中科院分区：
文献类型：
--
作者：
S. Karlish
本文研究了异硫氰酸荧光素(FITC)与(Na，K)ATPase在ATP结合区或附近的构象变化。每个ATPase分子结合一个或两个荧光素基团。在标记的酶中，(Na，K)ATPase活性、来自ATP的磷酸化和核苷酸结合被取消，但来自无机磷的磷酸化或K-磷酸酶活性仅部分失活。荧光素基团只被掺入(Na，K)ATPase的96kD催化链中，在与FITC孵育过程中存在ATP可防止掺入和抑制酶活性。在胰酶处理标记的膜时，荧光素首先出现在58kD的片段中，最后释放到介质中。荧光素标记的(Na，K)ATPase对无阳离子或富Na+介质中的E_1或E_1·Na构象在K~+(或Tl~+、Rb~+、Cs~+、NH_4~+)同种异构体转化为E_2·(K)的过程中有较大的荧光猝灭(15~20%)。阳离子滴定表明，K+和Na+离子竞争在一个结合部位，分别稳定E_1·Na或E_2·(K)，K_K≈为0.23 mm，K_a≈为1.2 mm。E_2·(K)→E_1·Na的构象转换速率很慢，k=0.3秒−_1，但与先前的经验相反[7，8]ATP不能刺激这一速率。E_1+K+→E_2·(K)的跃迁速率随K~+浓度的增加而急剧增加，并呈现饱和行为，由此推算出K_(Max)≈286s−_1和K_k≈_(74 Mm)。这一数据支持并推广了先前的观点，即K+离子最初结合在低亲和力(可能是细胞质导向的)位置的E1态，被沿着E2·(K)方向稳定的构象变化(KC≈1000)捕获到闭塞形式的E2·(K)。此外，还提出在分离的(Na，K)ATPase的胞质表面上至少存在两个K+结合位点，但亲和力的巨大差异使荧光滴定不可能检测到多个结合位点。除了K+外，各种配体还能产生荧光猝灭或E2形式的标记的(Na，K)ATPase。其中包括镁加无机磷酸盐，不含或不含K+离子(E2P或E2P·K)或含哇巴因(E2-哇巴因或E2P·哇巴因)。Na+离子可拮抗这些效应。收集的数据支持这样的观点，即可能存在许多亚种的E1和E2形式(无论是磷酸化的还是非磷酸化的)，结合或封闭了不同数量的Na+和/或K+离子，每个亚种都具有催化反应和/或运输阳离子的特有能力。讨论了荧光素标记酶的构象变化与(Na，K)ATPase亚基结构之间的关系，特别是结合ATP水解酶的“半位点”模型。
Conformational changes have been studied in (Na,K) ATPase labeled at or near the ATP binding region with fluorescein following incubation with fluorescein isothiocyanate (FITC). One or two fluorescein groups are bound per ATPase molecule. (Na,K) ATPase activity, phosphorylation from ATP, and nucleotide binding are abolished in labeled enzyme, but phosphorylation from inorganic phosphate or K-phosphatase activity are only partially inactivated. The fluorescein groups are incorporated only into the 96 KD catalytic chain of the (Na,K) ATPase, and presence of ATP during the incubation with FITC protects against the incorporation and inhibition of enzymic activity. Upon trypsin treatment of labeled membranes the fluorescein appears first in a 58 KD fragment and eventually is released into the medium. The fluorescein-labeled (Na,K) ATPase shows a large quenching of fluorescence (15–20%) on conversion of the E1 or E1 · Na conformation in cation-free or Na+-rich media to the E2 · (K) form in K+ (or congeners Tl+, Rb+, Cs+, NH4+) rich media. Cation titrations suggest that K+ and Na+ ions compete at a single binding site and stabilize E1 · Na or E2 · (K) respectively;KK≈0.23 mM,KNa≈1.2 mM. The rate of the conformational transition E2 · (K) → E1 · Na is slow,k=0.3 sec−1, but contrary to previous experience [7, 8] ATP does not stimulate this rate. The rate of the transitions E1 + K+ → E2 · (K) rises sharply with K+ concentration and shows saturation behavior, from which akmax≈286 sec−1 andKk≈74 mM are deduced. The data support and extend the previous suggestion that K+ ions bound initially at a low-affinity (probably cytoplasm oriented) site in state E1 are trapped in the occluded form E2 · (K) by the conformational change poised far (Kc≈1000) in the direction of E2 · (K). It is proposed in addition that at least two binding sites for K+ exist at the cytoplasmic surface of isolated (Na,K) ATPase in state E1 but a large difference in affinities precludes detection in fluorescence titrations of more than one site. A variety of ligands in addition to K+ produce fluorescence-quenched or E2 forms of the labeled (Na,K) ATPase. These include Mg2+ plus inorganic phosphate, without or with K+ ions (E2P or E2P · K) or with ouabain (E2-ouabain or E2P · ouabain). Na+ ions antagonize these effects. The collected data support the notion that there may be many subspecies of the E1 and E2 forms (either phosphorylated or nonphosphorylated) with different numbers of Na+ and/or K+ ions bound or occluded, each subspecies having a characteristic ability to catalyze reactions and/or transport cations. The relationship between the conformational changes in fluorescein-labeled enzyme and the subunit structure of the (Na,K) ATPase is discussed with particular reference to “half of the site” models for ATP hydrolysis.