Esmann M, Marsh D
Max-Planck-Institut für biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, Federal Republic of Germany.
Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7606-9. doi: 10.1073/pnas.89.16.7606.
Diffusion-controlled Heisenberg spin exchange between spin-labeled Na+,K(+)-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting), EC 3.6.1.37] proteins has been studied by saturation transfer ESR spectroscopy in reconstituted membranes. Na+,K(+)-ATPase from the salt gland of Squalus acanthias was solubilized in a polyoxyethylene ether detergent, octa(ethylene glycol) dodecyl monoether. Part of the solubilized enzyme was covalently spin-labeled with a nitroxide derivative of indanedione and recombined with various proportions of the unlabeled enzyme while the native lipid/protein ratio was maintained. Purified membranes were then reconstituted from the various samples by precipitation with divalent ions. The reciprocal integrated intensities of the saturation transfer ESR spectra were found to increase linearly with the fraction of protein that was spin-labeled, and the gradient of the concentration dependence increased with increasing temperature over the range 4 degrees-25 degrees C. Comparison with theoretical analyses of the effects of weak Heisenberg spin exchange [Marsh, D. & Horváth, L. I. (1992) J. Magn. Reson. 97, 13-26] suggests that the effects on the saturation transfer ESR intensity are attributable to short-range diffusional collisions between the spin-labeled protein molecules. The effective value of the local translational diffusion coefficient is 1.8-2.9 microns2.s-1 at 15 degrees C, depending on the diffusion model used, which is much larger than the values obtained for the long-range diffusion coefficient in cells by photobleaching techniques. The temperature dependence of the translational diffusion is larger than expected but correlates with the anomalous temperature dependence of the rotational diffusion observed in the same system.
通过饱和转移电子自旋共振光谱法，在重构膜中研究了自旋标记的Na⁺,K⁺-ATP酶[ATP磷酸水解酶(Na⁺/K⁺转运), EC 3.6.1.37]蛋白之间的扩散控制海森堡自旋交换。将棘鲛盐腺中的Na⁺,K⁺-ATP酶溶解在聚氧乙烯醚洗涤剂八(乙二醇)十二烷基单醚中。部分溶解的酶与茚满二酮的氮氧化物衍生物进行共价自旋标记，并与不同比例的未标记酶重新组合，同时保持天然脂质/蛋白质比例。然后通过用二价离子沉淀从各种样品中重构纯化膜。发现饱和转移电子自旋共振光谱的倒数积分强度随自旋标记蛋白的比例线性增加，并且浓度依赖性梯度在4℃至25℃范围内随温度升高而增加。与弱海森堡自旋交换效应的理论分析[Marsh, D. & Horváth, L. I. (1992) J. Magn. Reson. 97, 13 - 26]比较表明，对饱和转移电子自旋共振强度的影响归因于自旋标记蛋白分子之间的短程扩散碰撞。根据所使用的扩散模型，在15℃时局部平动扩散系数的有效值为1.8 - 2.9微米²·秒⁻¹，这比通过光漂白技术在细胞中获得的长程扩散系数的值大得多。平动扩散的温度依赖性比预期的大，但与在同一系统中观察到的旋转扩散的异常温度依赖性相关。
