Ishiguro S, Shirakawa H, Kean E L
Biochim Biophys Acta. 1985 Feb 14;812(3):752-66. doi: 10.1016/0005-2736(85)90269-x.
Rhodopsin-containing liposomes may provide a model for investigating the interaction of intrinsic membrane glycoproteins in biological systems. As part of the characterization of this preparation, the surface orientation of the carbohydrates of rhodopsin, assembled from purified bovine rhodopsin and egg phosphatidylcholine was examined, and is the topic of this report. The major tool used in these studies was the interaction with the carbohydrate-specific reagents, plant lectins. Two techniques were used: lectin-mediated aggregation of the liposomes, as measured by light scattering; the binding of 125I-labeled succinylated concanavalin A, and Scatchard analysis as a measure of affinity. The preparation most extensively examined had a mole ratio of rhodopsin:phospholipid of 1:100. Among a variety of lectins which were examined, only concanavalin A, succinylated concanavalin A, and wheat germ agglutinin were able to mediate the aggregation of rhodopsin-containing liposomes, as expected. The aggregation with concanavalin A was prevented by the presence of sugars having the alpha-D-glucopyranosyl configuration, and that brought about with wheat germ agglutinin, by N-acetylglucosamine (GlcNAc). In addition, the aggregation with concanavalin A was reversed with methyl alpha-D-mannoside, and with wheat germ agglutinin, by GlcNAc, suggesting that membrane fusion did not take place. On a molar basis, wheat germ agglutinin brought about a greatly reduced extent of aggregation as compared to concanavalin A, suggesting the relative inaccessibility of GlcNAc residues in the liposomes as compared to mannose. The initial rate of the aggregation, however, were similar with either lectin. The first-order rate constants were unaffected by wide variation in the concentrations of concanavalin A and wheat germ agglutinin, and by variation in the mole ratios of rhodopsin in the liposomes from 0.2 to 19 moles per 100 moles of egg lecithin. Rhodopsin-liposomes were also prepared from a total lipid extract of rod outer segments instead of egg lecithin. Similar kinetic properties were exhibited by this preparation as were obtained with the liposome prepared with the purified phospholipid. Scatchard analysis of the binding of 125I-labeled succinylated concanavalin A by rhodopsin liposomes indicated the presence of a single class of binding site as the preferred fit, with an apparent Kd of 2.8 X 10(-7) M. The binding was destroyed or extensively interfered with by trypsinization and by periodate treatment.
含视紫红质的脂质体可为研究生物系统中内在膜糖蛋白的相互作用提供一个模型。作为该制剂特性表征的一部分，研究了由纯化的牛视紫红质和卵磷脂组装而成的视紫红质碳水化合物的表面取向，这也是本报告的主题。这些研究中使用的主要工具是与碳水化合物特异性试剂植物凝集素的相互作用。采用了两种技术：通过光散射测量凝集素介导的脂质体聚集；125I标记的琥珀酰化伴刀豆球蛋白A的结合，以及用Scatchard分析作为亲和力的度量。研究最广泛的制剂中视紫红质与磷脂的摩尔比为1:100。在所研究的多种凝集素中，正如预期的那样，只有伴刀豆球蛋白A、琥珀酰化伴刀豆球蛋白A和麦胚凝集素能够介导含视紫红质脂质体的聚集。伴刀豆球蛋白A引起的聚集可被具有α-D-吡喃葡萄糖基构型的糖类阻止，而麦胚凝集素引起的聚集可被N-乙酰葡糖胺（GlcNAc）阻止。此外，伴刀豆球蛋白A引起的聚集可被α-D-甘露糖苷逆转，麦胚凝集素引起的聚集可被GlcNAc逆转，这表明没有发生膜融合。以摩尔为基础，与伴刀豆球蛋白A相比，麦胚凝集素引起的聚集程度大大降低，这表明与甘露糖相比，脂质体中GlcNAc残基相对难以接近。然而，两种凝集素的初始聚集速率相似。一级速率常数不受伴刀豆球蛋白A和麦胚凝集素浓度的广泛变化以及脂质体中视紫红质与每100摩尔卵磷脂的摩尔比从0.2到19摩尔变化的影响。视紫红质脂质体也由杆状外段的总脂质提取物而非卵磷脂制备。该制剂表现出与用纯化磷脂制备的脂质体相似的动力学性质。对视紫红质脂质体结合125I标记的琥珀酰化伴刀豆球蛋白A的Scatchard分析表明，作为最佳拟合存在一类单一的结合位点，表观解离常数为2.8×10(-7)M。这种结合可被胰蛋白酶处理和高碘酸盐处理破坏或广泛干扰。
