Rhodopsin-lipid associations in bovine rod outer segment membranes. Identification of immobilized lipid by spin-labels.
Rhodopsin-lipid associations in bovine rod outer segment membranes. Identification of immobilized lipid by spin-labels.
复制标题
牛视杆外节膜中的视紫红质-脂质结合。
作者：
A. Watts;I. Volotovski;D. Marsh
利用自旋标记技术研究了牛视杆细胞外节膜中视紫红质与脂质的相互作用。自旋标记的脂肪酸，甾醇，磷脂酰胆碱，磷脂酰乙醇胺，磷脂酰丝氨酸，磷脂酰甘油和磷脂酸分子都显示一个双组分光谱探测ROS膜时。其中一个光谱分量占总光谱强度的33-43%，并且是强固定的氮氧自由基自旋标记的特征。这种固定化的组分在-4至37 ℃下分解。剩余的67-57%的积分光谱强度具有与提取的ROS膜脂质的双层中的相同自旋标记的光谱非常相似的形式。对ROS固定区域的小选择性M许多重要的生物学功能由在脂质双层膜中组织的蛋白质执行（Sandemann，1978），因此理解这些蛋白质与它们的脂质之间的相互作用是特别重要的。由于各种原因，杆外节（ROS）膜非常适合于研究膜蛋白和脂质基质之间的相互作用。首先，视紫红质构成牛ROS膜的总膜蛋白的85-90%（Montal & Korenbrot，1976;奥布莱恩，1978; Daemen，1973; Papermaster等人，1976），因此任何蛋白质-脂质相互作用都很可能直接归因于视紫红质-脂质缔合。这种水平的单一蛋白富集通常通过重构、重组或特异性富集程序来实现，所述程序遭受蛋白聚集或变性或两者的危害以及去污剂去除的危害，去污剂去除通常是一个漫长且不完全的过程。ROS膜的分离无需使用去污剂或涉及脱脂或蛋白质损失的富集过程。其次，可获得的视紫红质的结构信息有限。这来自X射线衍射研究（Charbre，1975）、X射线散射（Sardet等人，1976）和中子散射（Osborne等人，1978）数据和超离心实验（刘易斯等人，1974），并且已经确定了一些尺寸信息（Sardet等人，1976; Osborne等人，1978年）。这些参数是有用的，当解释脂质和膜蛋白的结构方面的整体之间的相互作用的观察。第三，ROS膜特别令人感兴趣，因为视紫红质在其膜环境中负责视觉感知的主要步骤，导致从光吸收到神经兴奋。该过程涉及视紫红质的完整光解循环，包括再生，以及随后通过构象变化对内部递质的细胞质活性的调节，来自Max-Planck-Institut fur biophysikalische Chemie，Abteilung Spektroskopie，D-3400 Gottingen-Nikolausberg，联邦德国。1979年5月29日收到。I. D. V.是一个DFG交换津贴的Abteilung生物化学动力学的接受者。$永久地址：北京市朝阳区朝阳区朝阳路108号明斯克，U.S.S.R.膜由磷脂酰丝氨酸显示，而脂肪酸、磷脂酰胆碱、磷脂/乙醇胺和甾醇自旋标记物几乎均等地分配到这些区域中。选择性不仅仅是由于磷脂酰丝氨酸上的头基电荷，因为带负电荷的磷脂酰甘油和磷脂酸自旋标记物不显示增强的选择性。这些结果被解释在ROS膜中存在的两个群体的脂质，主要的一个是流体双层交换和周围的固定化的脂质，这是在直接接触视紫红质。根据有关视紫红质大小的现有信息，计算出固定化脂质足以在蛋白质周围形成单个完整的壳。在视紫红质中的位置（Saibil等，1976; Liebman等人，1974; Downer和Englander，1975; McDowell和威廉姆斯，1976; Ostroy，1977; Hubbell等人，1977年）。膜脂可能参与这两个阶段：在稳定的结构，视紫红质在各种状态的光解和再生周期，并在调节构象变化，导致发射机的响应。在目前的工作中，我们证明了在ROS膜中存在固定化的脂质组分，通过一些脂质自旋标记进行监测。这种固定化的脂质被揭示为自旋标记的ROS膜的独特的双组分ESR谱的一个组分，其本身的观察表明这两个组分存在的时间长于s。提取的ROS脂质双层的类似实验仅显示单组分光谱，这是脂质双层中经历各向异性运动的自旋标签的典型光谱。这种流体双层光谱非常类似于从自旋标记的ROS膜观察到的其他成分。有人建议，“固定化”的组件来自运动扰动双层脂质。从光谱减法和积分，它表明，固定的脂质的量是24个脂质分子/蛋白质，这可能是足以形成一个单一的外壳周围视紫红质，ROS膜的主要蛋白质。脂肪酸，甾醇，和一些磷脂自旋标记的实验表明，磷脂酰丝氨酸分子的固定区域的有限的偏好。ROS膜的组织，视紫红质周围的固定化脂质的组成，和视紫红质的膜内部分的大致大小进行了讨论。实验部分
Rhodopsin-lipid interactions have been studied in bovine rod outer segment (ROS) membranes by using spin-labels. Spin-labeled fatty acid, sterol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and phosphatidic acid molecules all display a two-component spectrum when probing ROS membranes. One of the spectral components represents 33-43% of the total spectral intensity and is characteristic of a strongly immobilized nitroxide spin-label. This immobilized component is resolved from -4 to 37 O C . The remaining 67-57% of the integrated spectral intensity has a very similar form to the spectra of the same spin-labels in bilayers of extracted ROS membrane lipid. A small selectivity for the immobilized regions of ROS M a n y important biological functions are performed by proteins organized in lipid bilayer membranes (Sandemann, 1978), and an understanding of the interactions between these proteins and their lipids is therefore of particular importance. Rod outer segment (ROS’) membranes are well suited to the study of the interactions between integral membrane proteins and the lipid matrix for a variety of reasons. Firstly, rhodopsin constitutes 85-90% of the total membrane protein of bovine ROS membranes (Montal & Korenbrot, 1976; O’Brien, 1978; Daemen, 1973; Papermaster et al., 1976), and therefore any protein-lipid interactions will, in all probability, be directly due to rhodopsin-lipid associations. Such a level of single protein enrichment is normally achieved by reconstitution, recombination, or specific enrichment procedures which suffer from the hazards of either protein aggregation or denaturation or both and of detergent removal, which is often a long and incomplete process. ROS membranes are isolated without the use of detergents or of enrichment processes involving delipidation or protein loss. Secondly, a limited amount of structural information for rhodopsin is available. This has come from X-ray diffraction studies (Charbre, 1975), X-ray scattering (Sardet et al., 1976) and neutron-scattering (Osborne et al., 1978) data, and ultracentrifugation experiments (Lewis et al., 1974), and some dimensional information has been determined (Sardet et al., 1976; Osborne et al., 1978). Such parameters are useful when interpreting observations of the interactions between lipids and integral membrane proteins in structural terms. Thirdly, ROS membranes are particularly interesting since rhodopsin, within its membrane environment, is responsible for the primary step in visual perception, leading from light absorption to nerve excitation. This process involves both the full photolytic cycle of rhodopsin, including regeneration, and the consequent modulation of the cytoplasmic activity of an internal transmitter through conformational changes taking From the Max-Planck-Institut fur biophysikalische Chemie, Abteilung Spektroskopie, D-3400 Gottingen-Nikolausberg, Federal Republic of Germany. Received May 29, 1979. I.D.V. was the recipient of a DFG exchange stipend to the Abteilung Biochemische Kinetik. $Permanent address: Institute of Photobiology, Academy of Science of B.S.S.R., Minsk, U.S.S.R. membranes is shown by phosphatidylser‘ne, while the fatty acid, phosphatidylcholine, phosphatidy / ethanolamine, and sterol spin-labels partition almost equally into these regions. The selectivity is not solely due to the head-group charge on phosphatidylserine since the negatively charged phosphatidylglycerol and phosphatidic acid spin-labels do not display an enhanced selectivity. These results are interpreted in terms of two populations of lipid existing in ROS membranes, the major one being fluid bilayer in exchange with and surrounding the immobilized lipid which is in direct contact with rhodopsin. On the basis of available information on the size of rhodopsin, it is calculated that the immobilized lipid is sufficient to form a single complete shell around the protein. place in rhodopsin (Saibil et al., 1976; Liebman et al., 1974; Downer & Englander, 1975; McDowell & Williams, 1976; Ostroy, 1977; Hubbell et al., 1977). The membrane lipid could be involved in both of these stages: in stabilizing the structure of rhodopsin during the various states of the photolytic and regenerative cycles and in regulating the conformational changes leading to the transmitter response. In the present work, we demonstrate the existence of an immobilized lipid component in ROS membranes as monitored by a number of lipid spin-labels. This immobilized lipid is revealed as one component of a distinct two-component ESR spectrum of spin-labeled ROS membranes, the observation of which in itself demonstrates that the two components exist for longer than s. Similar experiments with bilayers of extracted ROS lipid show only a one-component spectrum, typical of the spin-labels undergoing anisotropic motion in a lipid bilayer. Such fluid bilayer spectra closely resemble the other components observed from spin-labeled ROS membranes. It is suggested that the “immobilized” component arises from motionally perturbed bilayer lipid. From spectral subtraction and integration it is shown that the amount of immobilized lipid is 24 lipid molecules/protein, which is probably sufficient to form a single shell around rhodopsin, the major protein of ROS membranes. Experiments with fatty acid, sterol, and a number of phospholipid spin-labels demonstrate a limited preference for the immobilized region by phosphatidylserine molecules. The organization in the ROS membrane, the composition of the immobilized lipids around rhodopsin, and the approximate size of the intramembranous portion of rhodopsin are discussed. Experimental Section