TAILIEUCHUNG - Báo cáo khoa học: The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel

OprM, the outer membrane component of the MexAB-OprM multidrug efflux pump ofPseudomonas aeruginosa, hasbeenassumed to facilitate the export of antibiotics across the outer membrane of this organism. Here we purified to homogeneity the OprM protein, reconstituted it into lipo-somemembranes, and tested its channel activitybyusing the liposome swellingassay. Itwas demonstrated thatOprMis a channel-forming protein and exhibits the channel property that amino acids diffuse more efficiently than saccharides | Eur. J. Biochem. 269 4738-4745 2002 FEBS 2002 doi The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel Eisaku Yoshihara Hideaki Maseda and Kohjiro Saito Department of Molecular Life Science School of Medicine Tokai University Isehara Japan OprM the outer membrane component of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa has been assumed to facilitate the export of antibiotics across the outer membrane of this organism. Here we purified to homogeneity the OprM protein reconstituted it into liposome membranes and tested its channel activity by using the liposome swelling assay. It was demonstrated that OprM is a channel-forming protein and exhibits the channel property that amino acids diffuse more efficiently than saccharides. However antibiotics showed no significant diffusion through the OprM channel in the liposome membrane suggesting that OprM functions as a gated channel. We reasoned that the protease treatment may cause the disturbance of the gate structure of OprM. Hence we treated OprM reconstituted in the membranes with a-chymotrypsin and examined its solute permeability. The results demon strated that the protease treatment caused the opening of an OprM channel through which antibiotics were able to diffuse. To elucidate which cleavage is intimately related to the opening we constructed mutant OprM proteins where the amino acid at the cleavage site was replaced with another amino acid. By examining the channel activity of these mutant proteins it was shown that the proteolysis at tyrosine 185 and tyrosine 196 of OprM caused the channel opening. Furthermore these residues were shown to face into the periplasmic space and interact with other component s . We considered the possible opening mechanism of the OprM channel based on the structure of TolC a homologue of OprM. Keywords multidrug efflux pump OprM channel gate P. aeruginosa. Pseudomonas .

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