|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1996 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1995 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1994 : ¥1,000,000 (Direct Cost : ¥1,000,000)
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.