为了持续的视力,光活化视紫质(Rho*)必须经历水解和释放的全反式视网膜,产生视觉周期的底物和可用于11-顺式视网膜再生的apo-视蛋白。对于视紫红质在其天然膜环境中的这种水解的动力学尚待描述。我们开发了一种由异丙醇/硼氢化物同时变性和生色团捕获组成的方法,接着是详尽的蛋白质消化,完全提取,和液相色谱-质谱联用。使用我们的方法,我们追踪了Rho*水解,随后与释放的全反式视网膜形成N-视黄亚甲基-磷脂酰乙醇胺(N-ret-PE)加合物,并将全反式视黄醛还原为全反式视黄醇。我们发现,与通常用于研究膜蛋白的洗涤剂胶束相比,天然膜中的水解发生得更快。天然膜中水解的活化能测定为17.7±2.4kcal/mol。我们的数据支持的解释,视紫红质的信号状态,是经历水解和释放其全反式视网膜的主要物种。在没有NADPH的情况下,游离的全反式视黄醛与磷脂酰乙醇胺(PE)反应,形成大量的N-ret-PE(在生理pH值下约占全部全反式视网膜的40%),比Rho*水解快一个数量级的速率。然而,通过NADPH依赖性地将全反式视黄醛还原为全反式视黄醇,N-ret-PE形成被高度减弱。N-ret-PE形成和全反式-视网膜还原均不影响Rho*的水解速率。我们的研究提供了Rho*的水解和全反式视网膜的释放及其重新进入视觉周期的全面图片,改变可导致严重视网膜病变的过程。
For sustained vision, photoactivated rhodopsin (Rho*) must undergo hydrolysis and release of all-trans-retinal, producing substrate for the visual cycle and apo-opsin available for regeneration with 11-cis-retinal. The kinetics of this hydrolysis has yet to be described for rhodopsin in its native membrane environment. We developed a method consisting of simultaneous denaturation and
chromophore trapping by isopropanol/borohydride, followed by exhaustive protein digestion, complete extraction, and liquid chromatography-mass spectrometry. Using our method, we tracked Rho* hydrolysis, the subsequent formation of N-retinylidene-phosphatidylethanolamine (N-ret-PE) adducts with the released all-trans-retinal, and the reduction of all-trans-retinal to all-trans-retinol. We found that hydrolysis occurred faster in native membranes than in detergent micelles typically used to study membrane proteins. The activation energy of the hydrolysis in native membranes was determined to be 17.7 ± 2.4 kcal/mol. Our data support the interpretation that metarhodopsin II, the signaling state of rhodopsin, is the primary species undergoing hydrolysis and release of its all-trans-retinal. In the absence of NADPH, free all-trans-retinal reacts with phosphatidylethanolamine (PE), forming a substantial amount of N-ret-PE (∼40% of total all-trans-retinal at physiological pH), at a rate that is an order of magnitude faster than Rho* hydrolysis. However, N-ret-PE formation was highly attenuated by NADPH-dependent reduction of all-trans-retinal to all-trans-retinol. Neither N-ret-PE formation nor all-trans-retinal reduction affected the rate of hydrolysis of Rho*. Our study provides a comprehensive picture of the hydrolysis of Rho* and the release of all-trans-retinal and its reentry into the visual cycle, a process in which alteration can lead to severe retinopathies.