Identification and characterization of porins in Pseudomonas aeruginosa.
Identification and characterization of porins in Pseudomonas aeruginosa.
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发表时间：
1991-01
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通讯作者：
Hiroshi NikaidoS;Kishiko NikaidoQ;S. Harayama
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作者：
Hiroshi NikaidoS;Kishiko NikaidoQ;S. Harayama
早期的研究表明，铜绿假单胞菌外膜中的主要孔蛋白是蛋白F(Protein F，OprF)，它产生的通道比大肠杆菌孔蛋白产生的通道更宽。相反，Yoshihara和Nakae((1989)J.Biol)。化学。264,6297-6301)报道，根据L-阿拉伯糖的通量测量，蛋白F没有成孔活性，并且由蛋白C、D和E产生的铜绿假单胞菌外膜的通道比大肠杆菌的孔蛋白通道窄得多。在这项研究中，我们尽可能地遵循Yoshihara和Nakae的蛋白质纯化方案，发现蛋白质F具有与蛋白质D1、D2和E2相似的成孔活性。此外，蛋白脂体重组分析最终表明，由蛋白F形成的通道以及由未分离的外膜形成的通道允许四糖水苏糖以显著的速度扩散，表明这些通道比大肠杆菌孔蛋白通道大得多。在对Yoshihara和Nakae的研究中，蛋白F似乎在纯化过程中被无意中灭活了。我们进一步提出了一个假设，解决了铜绿假单胞菌大直径通道的存在和外膜低通透性之间的明显冲突。
Earlier studies have shown that the major porin species in Pseudomonas aeruginosa outer membrane is protein F (OprF), which produces channels wider than those produced by Escherichia coli porins. In contrast, Yoshihara and Nakae ((1989) J. Biol. Chem. 264, 6297-6301) reported that protein F has no pore-forming activity as measured by the flux of L-arabinose, and that the channels in P. aeruginosa outer membrane, being produced by proteins C, "D," and "E," are much narrower than E. coli porin channels. In this study, we followed the protein purification scheme of Yoshihara and Nakae as closely as possible, and found that protein F had a specific activity for pore formation similar to that of proteins D1, D2, and E2. Furthermore, proteoliposome reconstitution assays showed conclusively that the channels formed by protein F, as well as by unfractionated outer membranes, allowed the diffusion of a tetrasaccharide, stachyose, at a significant rate, indicating that these channels are much larger than E. coli porin channels. It appears likely that in the study of Yoshihara and Nakae protein F was inadvertently inactivated during purification. We further suggest a hypothesis that resolves the apparent conflict between the presence of large diameter channels and the low permeability of the outer membrane in P. aeruginosa.