Identification of a model cardiac glycoside receptor: comparisons with Na+,K+-ATPase.
Identification of a model cardiac glycoside receptor: comparisons with Na+,K+-ATPase.
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模型强心苷受体的鉴定：与Na ,K -ATP酶的比较。
DOI：
10.1021/bi973037d
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
BallJr,WJ
中科院分区：
文献类型：
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作者：
Kasturi,R;Yuan,J;McLean,LR;Margolies,MN;BallJr,WJ
高亲和力抗地高辛单克隆抗体（mAb）的可用性提供了其作为表征受体结构和强心苷结合之间关系的模型的潜力。我们已经表征了强心苷哇巴因的荧光衍生物蒽酰哇巴因（AO）与mAb 26-10、45-20和40-50的结合[Mudgett-Hunter，M.，等人（1995）Mol. Immunol.22，477]和羔羊肾Na+，K+-ATP酶，通过监测所得AO荧光发射光谱、各向异性、寿命值和从蛋白质色氨酸（Trp）到AO的Förster共振能量转移（FRET）。这些数据表明，Na+，K+-ATP酶的AO结合位点附近的结构环境与mAb 26-10相似，但与mAb 45-20和40-50不同。AO与mAb 26-10的抗原结合片段（Fab）复合的模型使用已知的X射线晶体结构数据产生[Jeffrey，P.D.，等人（1993）Proc.Natl. Acad. Sci. U. S. A. 90，10310]显示重链Trp残基（Trp-H100）靠近（约3 μ m）蒽酰基部分。这与AO与mAb 26-10结合时观察到的能量转移一致，并表明作为抗体强心苷结合位点的一部分的Trp-H100是结合AO的荧光性质的主要决定因素。相反，生成的AO与Fab 40-50复合的模型[Jeffrey，P.D.，等人（1995）J. Mol. Biol.248，344]显示了重链Tyr残基（Tyr-H100），其是强心苷结合位点的一部分，位于距蒽酰基部分约10 nm处。最接近的Trp残基（H52和L35）位于距离蒽酰基部分约17 nm处，并且没有观察到FRET，尽管这些Trp残基足够接近以发生显著的FRET。AO与Na+，K+-ATP酶结合后的能量转移表明，存在一个完全淬灭或两个高度淬灭的酶Trp残基，分别来自蒽酰基部分的Δ 10和Δ 17。这些数据表明，参与荧光能量转移到AO的Na+，K+-ATP酶Trp残基可能是强心苷结合位点的一部分。
The availability of high-affinity anti-digoxin monoclonal antibodies (mAbs) offers the potential for their use as models for the characterization of the relationship between receptor structure and cardiac glycoside binding. We have characterized the binding of anthroylouabain (AO), a fluorescent derivative of the cardiac glycoside ouabain, to mAbs 26-10, 45-20, and 40-50 [Mudgett-Hunter, M., et al. (1995)Mol. Immunol.22, 477] and lamb kidney Na+,K+-ATPase by monitoring the resultant AO fluorescence emission spectra, anisotropy, lifetime values, and Förster resonance energy transfer (FRET) from protein tryptophan(s) (Trp) to AO. These data suggest that the structural environment in the vicinity of the AO-binding site of Na+,K+-ATPase is similar to that of mAb 26-10 but not mAbs 45-20 and 40-50. A model of AO complexed to the antigen binding fragment (Fab) of mAb 26-10 which was generated using known X-ray crystal structural data [Jeffrey, P. D., et al. (1993)Proc. Natl. Acad. Sci.U.S.A.90, 10310] shows a heavy chain Trp residue (Trp-H100) that is close (∼3 Å) to the anthroyl moiety. This is consistent with the energy transfer seen upon AO binding to mAb 26-10 and suggests that Trp-H100, which is part of the antibody's cardiac glycoside binding site, is a major determinant of the fluorescence properties of bound AO. In contrast, the generated model of AO complexed to Fab 40-50 [Jeffrey, P. D., et al. (1995)J. Mol. Biol.248, 344] shows a heavy chain Tyr residue (Tyr-H100) which is part of the cardiac glycoside binding site, located ∼10 Å from the anthroyl moiety. The closest Trp residues (H52 and L35) are located ∼17 Å from the anthroyl moiety, and no FRET is observed despite the fact that these Trp residues are close enough for significant FRET to occur. The energy transfer seen upon AO binding to Na+,K+-ATPase suggests the presence of one completely quenched or two highly quenched enzyme Trp residues ∼10 and ∼17 Å, respectively, from the anthroyl moiety. These data suggest that the Na+,K+-ATPase Trp residue(s) involved in fluorescence energy transfer to AO is likely to be part of the cardiac glycoside binding site.