Duschl A, McCloskey M A, Lanyi J K
Department of Physiology and Biophysics, University of California, Irvine 92717.
J Biol Chem. 1988 Nov 15;263(32):17016-22.
A one-step purification method for halorhodopsin was developed. Functional proteoliposomes were prepared from this preparation using cholate, which is removed by dialysis in the presence of asolectin or the polar halobacterial lipids. Light-induced outward directed transport of chloride by halorhodopsin was followed by measuring passive proton efflux in the presence of uncoupler; initial rates and extents amounted to significant fractions of values obtained for halorhodopsin-containing cell envelope vesicles. The transport activity was much higher when cholate rather than octyl glucoside was used in the reconstitution. Since CD spectra in cholate but not in octyl glucoside showed band-splitting in the visible region, suggestive of exciton interaction between halorhodopsin monomers, the reconstitution may depend on an aggregate state of the halorhodopsin. The rate constants for three thermal steps in the halorhodopsin photocycle were greatly reduced in the detergent-solubilized samples, but they increased in the proteoliposomes to values similar to those for halorhodopsin in cell envelope vesicles. Thus, the reconstitution yields halorhodopsin with both photochemical and transport activities restored. Freeze-fracture electron micrographs of the proteoliposomes showed unilammellar liposomes with numerous particles of 100-150 A diameter at the fracture faces. These should correspond to halorhodopsin aggregates, formed in the bilayer in an apparently concentration-dependent manner.
开发了一种嗜盐视紫红质的一步纯化方法。使用胆酸盐从该制剂中制备功能性蛋白脂质体，在大豆卵磷脂或极性嗜盐菌脂质存在下通过透析去除胆酸盐。通过在解偶联剂存在下测量被动质子外流来跟踪嗜盐视紫红质光诱导的氯离子外向转运；初始速率和程度相当于从含嗜盐视紫红质的细胞膜囊泡获得的值的很大一部分。在重组过程中使用胆酸盐而非辛基葡糖苷时，转运活性要高得多。由于在胆酸盐中而非辛基葡糖苷中的圆二色光谱在可见光区域显示出谱带分裂，这表明嗜盐视紫红质单体之间存在激子相互作用，因此重组可能取决于嗜盐视紫红质的聚集状态。在去污剂溶解的样品中，嗜盐视紫红质光循环中三个热步骤的速率常数大大降低，但在蛋白脂质体中它们增加到与细胞膜囊泡中嗜盐视紫红质的值相似。因此，重组产生了光化学和转运活性均得以恢复的嗜盐视紫红质。蛋白脂质体的冷冻断裂电子显微照片显示，在断裂面上有单层脂质体，其直径为100 - 150埃的颗粒众多。这些颗粒应该对应于以明显的浓度依赖性方式在双层中形成的嗜盐视紫红质聚集体。
