Porins of Vibrio cholerae: Purification and characterization of OmpU
Porins of Vibrio cholerae: Purification and characterization of OmpU
复制标题
DOI：
10.1128/jb.178.2.524-530.1996
复制
发表时间：
1996-01-01
影响因子：
3.2
通讯作者：
Das, J
中科院分区：
文献类型：
--
作者：
Chakrabarti, SR;Chaudhuri, K;Das, J
霍乱弧菌高毒素株569B的三种外膜蛋白分子量分别为40、38和27 kDa，其合成均受生长介质渗透压的调节。40 kDa蛋白OmpT和38 kDa蛋白OmpU的成孔能力已被脂质体证实，而27 kDa蛋白OmpX虽然受渗透调节，但不是孔蛋白。在含1.0%氯化钠的条件下，外膜蛋白占总外膜蛋白的30%，在不含盐的情况下，占总外膜蛋白的60%。OmpU是一种酸性蛋白质，是一个38 kDa单体单位的同源三聚体，其二级结构主要含有一个β-折叠，每个单体单位伴随着三到四个钙离子，钙离子的去除不可逆转地扰乱了蛋白质的结构和成孔能力，OmpU的孔径为1.6 nm，OmpU通道的比活性是大肠杆菌porin OmpF的2-3倍，OmpU的合成与生长介质的渗透压相似，OmpT的孔径小于OmpU，类似于大肠杆菌的OMPC几种限制性内切酶消化霍乱弧菌基因组DNA的Southern杂交。ColiOmpF作为探针，没有发现ompU和OmpF基因之间的核苷酸序列同源性，OmpU也与OmpF、抗OmpF抗血清没有抗原性相关，但与45 kDa霍乱弧菌外膜蛋白OMPS发生交叉反应，其合成受生长介质中麦芽糖的存在调节，OmpU与兔和人血凝集。这种毒素受体调节的蛋白是霍乱弧菌可能的毒力决定因素之一(V.L.Miller和J.J.Mekalanos，J.细菌素，170：2575-2583,1988)。
Three outer membrane proteins with molecular masses of 40, 38, and 27 kDa of the hypertoxinogenic strain 569B of Vibrio cholerae have been purified to homogeneity, The synthesis of all the three proteins is regulated by the osmolarity of the growth medium. The pore-forming ability of the 40-kDa protein, OmpT, and the 38-kDa protein, OmpU, has been demonstrated by using liposomes, in which these proteins were embedded, The 27-kDa protein, OmpX, though osmoregulated, is not a porin. OmpU constitutes 30% of the total outer membrane protein when grown in the presence of 1.0% NaCl in the growth medium and 60% in the absence of NaCl. OmpU is an acidic protein and is a homotrimer of 38-kDa monomeric units, Its secondary structure contains predominantly a beta-sheet, and three to four Ca2+ ions are associated with each monomeric unit, Removal of Ca2+ irreversibly disrupts the structure and pore-forming ability of the protein, The pore size of OmpU is 1.6 nm, and the specific activity of the OmpU channel is two- to threefold higher than that of Escherichia coli porin OmpF, synthesis of which resembles that of OmpU with respect to the osmolarity of the growth medium, The pore size of OmpT, which is analogous to OmpC of E. coli, is smaller than that of OmpU, Southern blot hybridization of V. cholerae genomic DNA digested with several restriction endonucleases with nick-translated. coli ompF as the probe revealed no nucleotide sequence homolog between the ompU and ompF genes, OmpU is also not antigenically related to OmpF, Anti-OmpF antiserum, however, cross-reacted with the 45-kDa V, cholerae outer membrane protein, OmpS, the synthesis of which is regulated by the presence of maltose in the growth medium, OmpU hemagglutinated with rabbit and human blood. This toxR-regulated protein is one of the possible virulence determinants in V. cholerae (V. L. Miller and J. J. Mekalanos, J. Bacteriol, 170:2575-2583, 1988).