McDonnel A, Staehelin L A
J Cell Biol. 1980 Jan;84(1):40-56. doi: 10.1083/jcb.84.1.40.
A highly purified chlorophyll a/b light-harvesting complex (chl a/b LHC; chl a/b ratio 1.2) was obtained from Triton-solubilized chloroplast membranes of pea and barley according to the method of Burke et al. (1978, Arch. Biochem. Biophys. 187: 252--263). Gel electrophoresis of the cation-precipitated chl a/b LHC from peas reveals the presence of four polypeptides in the 23- to 28-kdalton size range. Three of these peptides appear to be identical to those derived from re-electrophoresed CPII and CPII* bands. In freeze-fracture replicas, the cation-precipitated chl a/b LHC appears as a semicrystalline aggregate of membranous sheets containing closely spaced granules. Upon removal of the cations by dialysis, the aggregates break up into their constituent membranous sheets without changing their granular substructure. These membranous sheets can be resolubilized in 1.5% Triton X-100, and the chl a/b LHC particles then reconstituted into soybean lecithin liposomes. Freeze-fracture micrographs of the reconstituted chl a/b LHC vesicles suspended in a low salt medium reveal randomly dispersed approximately 80-A particles on both concave and convex fracture faces as well as some crystalline particle arrays, presumably resulting from incompletely solubilized fragments of the membranous sheets. Based on the approximately 80-A diameter of the particles, and on the assumption that one freeze-fracture particle represents the structural unit of one chl a/b LHC aggregate, a theoretical mol wt of approximately 200 kdalton has been calculated for the chl a/b LHC. Deep-etching and negative-staining techniques reveal that the chl a/b LHC particles are also exposed on the surface of the bilayer membranes. Addition of greater than or equal to 2 mM MgCl2 or greater than or equal to 60 mM NaCl to the reconstituted vesicles leads to their aggregation and, with divalent cations, to the formation of extensive membrane stacks. At the same time, the chl a/b LHC particles become clustered into the adhering membrane regions. Under these conditions the particles in adjacent membranes usually become precisely aligned. Evidence is presented to aupport the hypothesis that adhesion between the chl a/b LHC particles is mediated by hydrophobic interactions, and that the cations are needed to neutralize surface charges on the particles.
根据伯克等人（1978年，《生物化学与生物物理学文献》187卷：252 - 263页）的方法，从豌豆和大麦经曲拉通增溶的叶绿体膜中获得了高度纯化的叶绿素a/b捕光复合物（chl a/b LHC；叶绿素a/b比例为1.2）。对豌豆中阳离子沉淀的chl a/b LHC进行凝胶电泳分析，结果显示在23至28千道尔顿大小范围内存在四种多肽。其中三种多肽似乎与从再电泳的CPII和CPII*条带中得到的多肽相同。在冷冻蚀刻复制品中，阳离子沉淀的chl a/b LHC呈现为包含紧密排列颗粒的膜状片层的半结晶聚集体。通过透析去除阳离子后，聚集体分解成其组成的膜状片层，而颗粒亚结构不变。这些膜状片层可在1.5%曲拉通X - 100中重新溶解，然后将chl a/b LHC颗粒重组成大豆卵磷脂脂质体。悬浮在低盐培养基中的重组chl a/b LHC囊泡的冷冻蚀刻显微照片显示，在凹面和凸面断裂面上均有随机分布的直径约80埃的颗粒以及一些晶体颗粒阵列，推测这是由膜状片层未完全溶解的片段导致的。基于颗粒直径约80埃，并假设一个冷冻蚀刻颗粒代表一个chl a/b LHC聚集体的结构单元，已计算出chl a/b LHC的理论分子量约为200千道尔顿。深度蚀刻和负染色技术表明，chl a/b LHC颗粒也暴露在双层膜表面。向重组囊泡中加入大于或等于2 mM的MgCl2或大于或等于60 mM的NaCl会导致它们聚集，并且在二价阳离子存在下会形成广泛的膜堆叠。同时，chl a/b LHC颗粒聚集成附着的膜区域。在这些条件下，相邻膜中的颗粒通常会精确对齐。有证据支持以下假设：chl a/b LHC颗粒之间的粘附是由疏水相互作用介导的，并且需要阳离子来中和颗粒表面电荷。
