Lee J A, Fortes P A
Biochemistry. 1985 Jan 15;24(2):322-30. doi: 10.1021/bi00323a013.
Sodium plus potassium activated adenosinetriphosphatase [(Na,K)ATPase] is composed of a catalytic subunit (alpha) and a glycoprotein subunit (beta) of unknown function. A method has been developed to label the beta subunit of purified dog kidney (Na,K)ATPase with fluorescent probes. The method consists of oxidation of beta-subunit oligosaccharides, reaction of the resulting aldehydes with fluorescent hydrazides, and reduction of the hydrazones and unreacted aldehydes with NaBH4. Two oxidation methods were compared. Simultaneous treatment with neuraminidase and galactose oxidase did not inhibit significantly (Na,K)ATPase activity and allowed insertion of up to 11 mol of probe per mol of beta. In contrast, oxidation of (Na,K)ATPase oligosaccharides with periodate resulted in 50-80% inhibition of the (Na,K)ATPase activity with low or undetectable labeling. Eleven commercial probes and two novel hydrazides were tested for labeling of (Na,K)ATPase treated with galactose oxidase and neuraminidase. Eight probes did not label (Na,-K)ATPase but labeled red cell ghosts oxidized with periodate. Four probes labeled beta specifically but either adsorbed to the membrane tightly, or cross-linked the beta subunits, or formed unstable adducts. Lucifer yellow CH labeled beta specifically without membrane adsorption. Labeling stoichiometries from 1 to 11 mol of lucifer yellow CH per mol of beta were obtained without inhibition of (Na,K)ATPase activity and without significant alteration of the anthroylouabain binding capacity or its association and dissociation kinetics. Anthroylouabain specifically bound to the lucifer-labeled (Na,K)ATPase had a decreased quantum yield, probably due to resonance energy transfer. This suggests that the sites of lucifer attachment on beta are within energy transfer distance from the cardiac glycoside site on alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
钠钾激活的三磷酸腺苷酶[（Na，K）ATP酶]由一个催化亚基（α）和一个功能未知的糖蛋白亚基（β）组成。已开发出一种用荧光探针标记纯化的犬肾（Na，K）ATP酶β亚基的方法。该方法包括β亚基寡糖的氧化、所得醛与荧光酰肼的反应，以及用硼氢化钠还原腙和未反应的醛。比较了两种氧化方法。同时用神经氨酸酶和半乳糖氧化酶处理不会显著抑制（Na，K）ATP酶活性，并且每摩尔β可插入多达11摩尔的探针。相比之下，用过碘酸盐氧化（Na，K）ATP酶寡糖会导致（Na，K）ATP酶活性受到50 - 80%的抑制，且标记率低或无法检测到。测试了11种商业探针和两种新型酰肼对用半乳糖氧化酶和神经氨酸酶处理的（Na，K）ATP酶的标记情况。8种探针未标记（Na，-K）ATP酶，但标记了用过碘酸盐氧化的红细胞血影。4种探针特异性标记β，但要么紧密吸附在膜上，要么使β亚基交联，要么形成不稳定的加合物。荧光黄CH特异性标记β且不吸附在膜上。每摩尔β获得1至11摩尔荧光黄CH的标记化学计量比，同时（Na，K）ATP酶活性未受抑制，且蒽基哇巴因结合能力及其结合和解离动力学未发生显著改变。特异性结合荧光素标记的（Na，K）ATP酶的蒽基哇巴因量子产率降低，可能是由于共振能量转移。这表明荧光素在β上的附着位点与α上强心苷位点的能量转移距离内。（摘要截短于250字）
