Ikigai H, Akatsuka A, Tsujiyama H, Nakae T, Shimamura T
Department of Microbiology and Immunology, Showa University School of Medicine, Tokyo, Japan.
Infect Immun. 1996 Aug;64(8):2968-73. doi: 10.1128/iai.64.8.2968-2973.1996.
El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cholerae O1 spontaneously assembled oligomeric aggregates on the membranes of rabbit erythrocyte ghosts and liposomes. Membrane-associated oligomers were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting into two to nine bands with apparent molecular masses of 170 to 350 kDa. ETH assembled oligomers on a liposomal membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone. Cholesterol could be replaced with diosgenin or ergosterol but not with 5alpha-cholestane-3-one, suggesting that sterol is essential for the oligomerization. The treatment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium. Because dextrin 20 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysis, this hemolysis is likely to be caused by pore formation on the membrane. The pore size(s) estimated from osmotic protection assays was in the range of 1.2 to 1.6 nm. The pore formed on a rabbit erythrocyte membrane was confirmed morphologically by electron microscopy. Thus, we provide evidence that ETH damages the target by the assembly of hemolysin oligomers and pore formation on the membrane.
源自霍乱弧菌O1的埃尔托溶血素（ETH；分子量65 kDa）在兔红细胞血影和脂质体膜上自发组装成寡聚聚集体。膜相关寡聚体通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹法分离为两到九条带，表观分子量为170至350 kDa。ETH在由磷脂酰胆碱和胆固醇组成的脂质体膜上组装寡聚体，但不在仅由磷脂酰胆碱组成的膜上组装。胆固醇可以被薯蓣皂苷元或麦角固醇取代，但不能被5α-胆甾烷-3-酮取代，这表明固醇对于寡聚化至关重要。用ETH处理羧基荧光素包封的脂质体导致羧基荧光素迅速释放到培养基中。由于糊精20（分子量900 Da）对ETH介导的溶血具有渗透保护作用，这种溶血可能是由膜上形成孔道引起的。通过渗透保护试验估计的孔径范围为1.2至1.6 nm。通过电子显微镜从形态学上证实了在兔红细胞膜上形成的孔道。因此，我们提供了证据表明ETH通过溶血素寡聚体的组装和膜上孔道的形成来损伤靶标。
