Different crosslinking agents identify distinctly different putative Escherichia coli heat-stable enterotoxin rat intestinal cell receptor proteins.
The receptor for heat-stable enterotoxins (ST) produced by Escherichia coli and related organisms is located in the brush border region of intestinal villus cells. Heterobifunctional and homobifunctional crosslinkers were used to covalently couple 125I-ST to rat intestinal cell brush border membrane proteins. Experimental conditions during ligand binding and subsequent crosslinking significantly influence the efficiency of crosslinking, and the number of peptides specifically crosslinked to the 125I-ST. Multiple proteins efficiently coupled to 125I-ST with agents that can couple through the ST amino terminus. The crosslinker 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC), which can react with the carboxy terminus of the ST, covalently crosslinked 125I-ST to a single protein with an apparent Mr of 125,000-130,000, larger than the proteins identified using longer crosslinkers. Each of the proteins identified by crosslinking migrate with the same retention time on gel filtration after solubilization, with an approximate molecular size of 150,000-200,000.
Thompson
M R
MR
Department of Internal Medicine, University of Cincinnati, Ohio.
Giannella
R A
RA
D001386:Azides / Q000378:metabolism; D001427:Bacterial Toxins / Q000378:metabolism*; D002850:Chromatography, Gel / Q000379:methods; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004768:Enterotoxins / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000378:metabolism; D006162:Guanylate Cyclase; D007422:Intestines / Q000166:cytology / Q000648:ultrastructure*; D007457:Iodine Radioisotopes; D008871:Microvilli / Q000648:ultrastructure; D000077423:Polidocanol; D011092:Polyethylene Glycols; D011956:Receptors, Cell Surface / Q000378:metabolism*; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide; D012685:Sepharose; D013388:Succinimides / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D001386:Azides; D001427:Bacterial Toxins; D003432:Cross-Linking Reagents; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D013388:Succinimides; C034151:heat stable toxin (E coli); D000077423:Polidocanol; C062325:sulfosuccinimidyl 6-((4-azido-2-nitrophenyl)amino)hexanoate; D011092:Polyethylene Glycols; D014443:Tyrosine; C029534:hydroxysuccinimidyl-4-azidobenzoate; D012685:Sepharose; C049225:Superose 12; C035760:bis(sulfosuccinimidyl)suberate; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D005022:Ethyldimethylaminopropyl Carbodiimide