CHARACTERIZATION OF A NEW PHOTOAFFINITY DERIVATIVE OF OUABAIN - LABELING OF LARGE POLYPEPTIDE AND OF A PROTEOLIPID COMPONENT OF NA,K-ATPASE
CHARACTERIZATION OF A NEW PHOTOAFFINITY DERIVATIVE OF OUABAIN - LABELING OF LARGE POLYPEPTIDE AND OF A PROTEOLIPID COMPONENT OF NA,K-ATPASE
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DOI：
10.1021/bi00610a037
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发表时间：
1978-01-01
期刊：
影响因子：
2.9
通讯作者：
HOFFMAN, JF
中科院分区：
文献类型：
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作者：
FORBUSH, B;KAPLAN, JH;HOFFMAN, JF
摘要：我们合成了哇巴因的2-硝基-5-叠氮苯甲酰基(NAB)衍生物作为Na，K-ATPase的心苷结合位点的光亲和标记物，[~H]NAB-哇巴因与猪肾外髓提纯的Na，K-ATPase结合的位点数目与哇巴因相同(1.9nmol/mg)，亲和力大致相同(KD(哇巴因)/KD(NAB-哇巴因)[1.6])，并且哇巴因在这些位点上与Na，K-ATPase完全竞争。NAB-哇巴因的结合和抑制在黑暗中是可逆的，但在紫外光(310-370 nm)下30-40%的结合和抑制是不可逆的，这种结合和抑制通过对三氯乙酸、有机溶剂和热变性的稳定性被证明是共价的。实验前NaB-哇巴因的光解或酶与哇巴因的预先孵育可阻止共价标记。关于钠
Bliss Forbush III, Jack H. Kaplan, and Joseph F. Hoffman* abstract: We have synthesized 2-nitro-5-azidobenzoyl (NAB) derivatives of ouabain as photoaffinity labels of the cardiac glycoside binding site of Na, K-ATPase.[3H] NAB-ouabain was found to bind to the same number of sites on Na, K-ATPase (purified from pig kidney outer medulla) as ouabain (1.9 nmol/mg), with approximately the same affinity (Kd (ouabain)/Kd (NAB-ouabain)« 1.6), and ouabain was fully competitive with NAB-ouabain at these sites. NAB-ouabain binding and inhibition were reversible in the dark, but on exposure to ultraviolet light (310-370 nm) 30-40% of the binding and inhibition became irreversible; thisbinding was shown to be covalent by stability to trichloroacetic acid, organic solvents, and heat denaturation. Covalent labeling was prevented by photolysis of NAB-ouabain prior to the experiment, or by prior incubation of the enzyrhe with ouabain. On sodium