The carbohydrate moieties of the beta-subunit of Na+, K(+)-ATPase: their lateral motions and proximity to the cardiac glycoside site.
The carbohydrate moieties of the beta-subunit of Na+, K(+)-ATPase: their lateral motions and proximity to the cardiac glycoside site.
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Na , K( )-ATP 酶 β 亚基的碳水化合物部分：它们的横向运动和靠近强心苷位点。
DOI：
10.1016/s0006-3495(96)79562-0
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
BallJr,WJ
中科院分区：
文献类型：
--
作者：
Amler,E;Abbott,A;Malak,H;Lakowicz,J;BallJr,WJ
与哺乳动物Na+，K（+）-ATP酶的催化（α）亚基相关的β亚基是一种跨膜糖蛋白，具有三个位于细胞外的N-糖基化位点。虽然β似乎是必不可少的功能酶，β和它的糖的作用仍然未知。在这些研究中，稳态和动态荧光测量的荧光团荧光黄（LY）共价连接到β的碳水化合物链已经证明，绑定的探针是高度溶剂暴露，但限制在他们的扩散运动。此外，探针的β环境没有改变Na+或K+或哇巴因诱导的酶构象的变化，但二价阳离子和寡霉素除了引起LY荧光的适度变化。频域测量反映了与α上的强心苷受体位点结合的蒽酰哇巴因（AO）和碳水化合物连接的LY之间发生的Förster荧光能量转移（FET），表明它们非常接近（18 A）。在作为供体的LY和在酶的推定ATP结合位点结构域处共价结合的赤藓红-5-异硫氰酸酯之间进行的另外的FET测定表明，这两个区域分开约85 A的距离，并且该距离在二价阳离子结合时减小，并且在Na+E1->K+E2构象转变时增加。这些数据表明，一个模型的本地化的寡糖的末端部分，将它们放置在平均约18 A的AO结合位点和这个距离或更少的细胞外膜表面。
The beta-subunit associated with the catalytic (alpha) subunit of the mammalian Na+, K(+) -ATPase is a transmembrane glycoprotein with three extracellularly located N-glycosylation sites. Although beta appears to be essential for a functional enzyme, the role of beta and its sugars remains unknown. In these studies, steady-state and dynamic fluorescence measurements of the fluorophore lucifer yellow (LY) covalently linked to the carbohydrate chains of beta have demonstrated that the bound probes are highly solvent exposed but restricted in their diffusional motions. Furthermore, the probes' environments on beta were not altered by Na+ or K+ or ouabain-induced enzyme conformational changes, but both divalent cation and oligomycin addition evoked modest changes in LY fluorescence. Frequency domain measurements reflecting the Förster fluorescence energy transfer (FET) occurring between anthroylouabain (AO) bound to the cardiac glycoside receptor site on alpha and the carbohydrate-linked LY demonstrated their close proximity (18 A). Additional FET determinations made between LY as donor and erythrosin-5-isothiocyanate, covalently bound at the enzyme's putative ATP binding site domain, indicated that a distance of about 85 A separates these two regions and that this distance is reduced upon divalent cation binding and increased upon the Na+E1-->K+E2 conformational transition. These data suggest a model for the localization of the terminal moieties of the oligosaccharides that places them, on average, about 18 A from the AO binding site and this distance or less from the extracellular membrane surface.