Recovery of intestinal membrane binding sites for K88 E. coli from pig mucosal organ cultures

Summary

Putative receptors for K88+ E. coli from piglet intestinal epithelium were released into the organ culture medium and were demonstrated by direct binding with K88+ E. coli through the utilization of an in vitro binding procedure or by immunoprecipitation with K88 antigen.

Incorporation of ¹⁴C-glucosamine by newborn to day old and 3-week to 6-week old piglet jejunal and ileal mucosa, in organ culture, occurred throughout the 24 hr culture period. Uptake in both age groups and both areas of the intestine was similar with a somewhat greater incorporation by the older age group.

Secretion of ¹⁴C-glucosamine-labeled components into the culture medium was demonstrated by gel filtration of the concentrated medium. Some large molecular weight components eluted in the void volume in excess of 2 x 10⁶ daltons. A second peak of activity was spread from approximately 690K to 25K daltons. All eluted fractions demonstrated binding to K88+ E. coli.

Antibodies to purified brush borders from susceptible pigs produced prominent precipitation bands following double diffusion with concentrated organ culture media which confirmed that the organ culture media contained labeled proteins of brush border origin.

Immunoprecipitation of the intestinal mucosal organ culture media with K88+ pili and pilus antisera, followed by electrophoresis with SDS and reduced conditions, demonstrated a subunit of approximately 35K daltons.