Molecular structural analysis of membrane proteins incorporated into liposomes or expressed transitionally in the endoplasmic reticulum of transfected COS cells

为了在亚纳米水平上分析大鼠肝细胞内质网（ER）膜结合蛋白的结构，采用原子力显微镜和冷冻断裂复制法检测细胞色素P-450 (P-450) IIB和P-450还原酶在COS细胞内质网脂质体中结合或过渡表达，然后用电镜观察。在转染P-450 IIB2基因的COS细胞中，P-450 IIB的表达在实验中是多种多样的，内质网中掺入的分子数量是不可控的。因此，这一制度对于目前的目的是不够的。另外，去浊剂法（Mimms et al., 1981）、冻融法（Pick, 1981）和冻融透析法（Oku & MacDonald, 1983）制备的脂质体也获得了良好的结果；获得了模仿内质网膜的大单层脂质体。因此，三种方法制备的脂质体适合于内质网膜与膜结合蛋白之间相互作用的分析。通过蛋白脂质体与脂质体的融合可以控制蛋白质的掺入数量。然后，为了检验内质网中细胞色素P-450 （P-450）分子密度与内质网中分子分布之间的关系，将从大鼠肝脏中纯化的P-450 IIB2分子掺入由磷脂组成与内质网相似的磷脂混合物制成的脂质体中，通过洗涤剂去除和冷冻解冻。将掺入的脂质体用蒸馏水爆炸，并检测所产生的平面膜碎片。当大量P-450分子掺入时，许多颗粒分散在膜碎片中。如果掺入的P-450分子减少，则颗粒减少并形成簇。

To analyze the structure of membrane-bound proteins in the endoplasmic reticulum (ER) of rat hepatocytes at subnanomicrometer level, cytochrome P-450 (P-450) IIB and P-450 reductase incorporated into liposomes or expressed transitionally in the ER of COS cells were examined by atomic force microscopy and freeze-fracture replica method followed by electron microscopy. Expression of P-450 IIB in COS cells transfected P-450 IIB2 gene was various among experiments, and the number of incorporated molecules in ER was uncontrollable. Thus, this system was inadequate for the present purpose. Alternatively, liposomes made by the detergent eliminating method (Mimms et al., 1981), the freeze-thawing method (Pick, 1981) and the freeze-thawing-dialysis method (Oku & MacDonald, 1983) gave good results ; large unilamellar liposomes that mimic the membrane of the endoplasmic reticulum were obtained. Liposomes produced by the three methods are thus suitable for the analysis of the interaction between the endoplasmic reticulum membrane and membrane-bound proteins. The number of incorporated proteins could be controlled by fusing proteoliposomes with liposomes. Then, to examine the relationship between the density of cytochrome P-450 (P-450) molecules in the ER and distribution of the molecules in ER,P-450 IIB2 molecules purified from rat livers were incorporated into liposomes made from the mixture of phospholipids in which the phospholipid composition was similar to that in ER by the detergent elimination followed by freeze-thawing. The incorporated liposomes were exploded with distillled water and the resulting planar membrane fragments were examined. When large amount of P-450 molecules were incorporated, many particles were scattered in the membrane fragments. If the incorporated P-450 molecules was decreased, the particles decreased and formed clusters.