Kutchai H, Mahaney J E, Geddis L M, Thomas D D
Department of Molecular Physiology & Biological Physics, University of Virginia, Charlottesville 22908.
Biochemistry. 1994 Nov 15;33(45):13208-22. doi: 10.1021/bi00249a007.
Hexanol at 7 degrees C stimulates the activity of the Ca-ATPase of sarcoplasmic reticulum (SR). Time-resolved phosphorescence spectroscopy studies of SR whose Ca-ATPase is covalently labeled with erythrosin isothiocyanate (ERITC) indicate that at 7 degrees C hexanol (1) cause a concentration-dependent increase in the rate of decay of phosphorescence anisotropy, (2) causes larger oligomers of Ca-ATPase to dissociate into smaller oligomers, and (3) increases the rotational mobility of Ca-ATPase in all its oligomeric states. Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled stearic acid (SASL) in SR suggests that at 7 degrees C hexanol diminishes the fraction of SR lipids in the boundary lipid domain and disorders and fluidizes both the boundary lipid and the unrestricted lipid domain. In protein-free liposomes of extracted SR lipids hexanol increases fluidity and decreases order to a greater extent near the center of the lipid bilayer than near the polar head groups. At 25 degrees C hexanol has biphasic effects on Ca-ATPase activity: at 10 and 20 mM hexanol increases activity, but at 30 mM and especially at 40 mM there is inhibition of Ca-ATPase activity. The influence of hexanol at 25 degrees C on the oligomeric state of Ca-ATPase is also biphasic. At 10 and 20 mM, hexanol promotes the dissociation of larger oligomers into smaller ones, whereas at higher concentrations, 30 and 40 mM, hexanol causes larger oligomers to be formed from smaller ones. Lidocaine at 25 degrees C inhibits Ca-ATPase activity and causes dramatic slowing of the decay of phosphorescence anisotropy of ERITC-labeled SR by causing the formation of larger oligomers of Ca-ATPase from smaller ones. In protein-free liposomes of SR lipids at 25 degrees C, lidocaine disorders and fluidizes the acyl chains near the center of the bilayer (as did hexanol), but has opposite effects near the polar head groups. The opposite effects of hexanol and lidocaine on the oligomeric state of the SR Ca-ATPase provide a new molecular explanation for the opposite effects of hexanol and lidocaine on the activity of the Ca-ATPase. We conclude that the biphasic effects of hexanol on the activity of Ca-ATPase can be accounted for by biphasic effects of hexanol on the oligomeric state of the Ca-ATPase. This study supports the view that anesthetics can alter interactions between membrane proteins.
7摄氏度下的己醇可刺激肌浆网（SR）的Ca - ATP酶活性。对其Ca - ATP酶用异硫氰酸藻红蛋白（ERITC）进行共价标记的SR进行时间分辨磷光光谱研究表明，在7摄氏度时，己醇（1）导致磷光各向异性衰减速率呈浓度依赖性增加，（2）使Ca - ATP酶的较大寡聚体解离成较小寡聚体，（3）增加处于所有寡聚状态的Ca - ATP酶的旋转流动性。对SR中自旋标记硬脂酸（SASL）进行电子顺磁共振（EPR）光谱分析表明，在7摄氏度时，己醇减少了边界脂质域中SR脂质的比例，并使边界脂质和非限制脂质域紊乱且流动性增加。在提取的SR脂质的无蛋白脂质体中，己醇在脂质双层中心附近比在极性头部基团附近更大程度地增加流动性并降低有序性。在25摄氏度时，己醇对Ca - ATP酶活性有双相效应：在10和20 mM时己醇增加活性，但在30 mM尤其是40 mM时存在对Ca - ATP酶活性的抑制。25摄氏度时己醇对Ca - ATP酶寡聚状态的影响也是双相的。在10和20 mM时，己醇促进较大寡聚体解离成较小寡聚体，而在较高浓度30和40 mM时，己醇使较小寡聚体形成较大寡聚体。25摄氏度时利多卡因抑制Ca - ATP酶活性，并通过使较小的Ca - ATP酶形成较大寡聚体导致ERITC标记的SR的磷光各向异性衰减显著减慢。在25摄氏度时，在SR脂质的无蛋白脂质体中，利多卡因使双层中心附近的酰基链紊乱并增加流动性（如同己醇），但在极性头部基团附近有相反作用。己醇和利多卡因对SR Ca - ATP酶寡聚状态的相反作用为己醇和利多卡因对Ca - ATP酶活性的相反作用提供了新的分子解释。我们得出结论，己醇对Ca - ATP酶活性的双相效应可由己醇对Ca - ATP酶寡聚状态的双相效应来解释。本研究支持麻醉剂可改变膜蛋白之间相互作用的观点。
