Purification of OmpU from Vibrio cholerae classical strain 569B: evidence for the formation of large cation-selective ion-permeable channels by OmpU.
The outer membrane of the classical Vibrio cholerae strain 569B was isolated by sucrose density centrifugation. The simple treatment of the isolated outer membrane or the cell envelopes with different detergents allowed the purification of two outer membrane proteins, the 38 kDa OmpU and the 25 kDa OmpV. Furthermore, a 35 kDa outer membrane protein (probably the 35 kDa OmpA-like protein) was purified by two-fold treatment of the cell envelope with 2% SDS solution. A subsequent wash of the SDS-pellet with 2% Genapol buffer yielded in the 38 kDa OmpU protein, which formed SDS-resistant oligomers (66 kDa). The Genapol pellet contained OmpV. Reconstitution experiments with lipid bilayer membranes demonstrated that OmpU was a channel-forming component, whereas OmpV had a small channel-forming ability if any. The OmpU channels appeared to be large and water-filled and had a single-channel conductance of about 2 nS in 1 M KCl for the monomer in a trimer, which means that they have a larger cross-section than enterobacterial porins. The channels showed rapid switching between open and closed configuration. They were slightly cation-selective, which suggests that they contain an excess of negatively charged amino groups.
Benz
R
R
Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Germany.
Maier
E
E
Chakraborty
T
T
D018829:Adhesins, Bacterial / Q000302:isolation & purification* / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D002412:Cations / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004352:Drug Resistance, Microbial; D004755:Enterobacteriaceae / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000302:isolation & purification* / Q000502:physiology; D017136:Ion Transport; D008970:Molecular Weight; D018272:Porins / Q000302:isolation & purification* / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D013045:Species Specificity; D014734:Vibrio cholerae / Q000737:chemistry* / Q000502:physiology
D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D002412:Cations; D007473:Ion Channels; C096349:OmpU protein, Vibrio cholerae; D018272:Porins; D011188:Potassium