Soluble pig intestinal cell membrane components with affinities for E. coli K88+ antigen.
Pig intestinal brush borders (BB) were radiolabeled by iodination using the lactoperoxidase-hydrogen peroxide procedure. The BB were then detergent solubilized, centrifuged to remove particulate material, and chromatographed on Sepharose CL-4B. The fractions were incubated with K88+ E. coli using an in vitro binding assay. Binding of the iodinated membranes to K88+ E. coli occurred throughout a wide range of molecular weight components, in excess of 690K daltons to near 25K daltons. The system utilizing intact K88+ E. coli and solubilized BB was shown to be saturable. Prior contact of K88+ E. coli with nonradiolabeled membranes or specific antibodies to K88+ pili inhibited binding of the radiolabeled BB. Simple sugars were tested for their ability to block binding of the labeled BB; partial inhibition occurred with galactose (17.9%), galactosamine (32%), glucose (10.6%), and N-acetylglucosamine (32%). Calcium enhanced binding with as little as 10 microM. A 10 x increase in binding occurred with 500 microM calcium. Affinity chromatography using K88+ pili coupled on agarose beads avidly bound the labeled BB. The receptor membranes were eluted with high molar concentrations of salt, however considerable degradation occurred. Despite low yields from the affinity system, receptor membranes with higher binding activities were recovered. Protein: glycoprotein ratios were 1:4. Elution with SDS and electrophoresis on 12.5% polyacrylamide gels in the presence of a reducing agent produced two major subunits 35--32K and 23K daltons. These components were recovered from the gels and retained their binding activity. This information suggests that the intestinal receptor responsible for binding of K88+ E. coli is a glycoprotein, that in the native state exists in multimeric forms.
D000818:Animals; D000942:Antigens, Bacterial; D000954:Antigens, Surface / Q000276:immunology*; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000276:immunology; D002846:Chromatography, Affinity; D002849:Chromatography, Gas; D004926:Escherichia coli / Q000276:immunology*; D029968:Escherichia coli Proteins; D036781:Fimbriae Proteins; D005779:Immunodiffusion; D007422:Intestines / Q000648:ultrastructure*; D008871:Microvilli / Q000276:immunology; D013552:Swine; D014020:Tissue Extracts / Q000276:immunology*
D000942:Antigens, Bacterial; D000954:Antigens, Surface; D029968:Escherichia coli Proteins; C026888:K88 antigen, E coli; D014020:Tissue Extracts; D036781:Fimbriae Proteins; D002118:Calcium