Lipopolysaccharide tightly bound to porin monomers and trimers from Escherichia coli K-12.
Lipopolysaccharide (LPS) bound to isolated porin was detected on polyacrylamide gels by using a carbohydrate-specific silver stain and on Western blots by using anti-lipid A monoclonal antibodies. Porin was isolated from Escherichia coli JF733 (Ra chemotype) and D21f2 (Re chemotype). Isolated porin was separated from loosely associated LPS by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). Unheated porin traveled on gels as aggregates, presumably trimers, with an apparent molecular weight of 78,000 to 83,000. After heating to 100 degrees C for 2 min in SDS, the porin traveled as a monomer with a molecular weight of 36,000. The unheated, high-molecular-weight trimer band reacted in the gel with the carbohydrate-specific silver stain, while the heated monomer band showed no staining. In contrast, lipid A-specific monoclonal antibodies showed reactivity on Western blots to the 36,000-molecular-weight band but not to the trimer. Finally, both monomer and trimer bands were isolated from gels and rerun by SDS-PAGE. LPS was released from the trimer preparation when the sample was heated, but the monomer band that was formed by heating the trimer isolate still reacted with anti-lipid A antibodies. Quantitative Limulus amebocyte lysate analysis revealed an approximately equal molar ratio of LPS to protein in the electroeluted porin monomer. Thus, some but not all of the LPS could be released from trimer complexes by boiling in SDS. The isolated monomer did not release more LPS on boiling in SDS a second time but still had LPS tightly bound, as detected by lipid A-specific monoclonal antibodies.
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D007118:Immunoassay; D007124:Immunoenzyme Techniques; D007473:Ion Channels / Q000378:metabolism*; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000032:analysis / Q000378:metabolism*; D008970:Molecular Weight; D018272:Porins
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D007473:Ion Channels; D008070:Lipopolysaccharides; D018272:Porins