The long-term of this research program is to analyse at a molecular level the mechanism of action of a commonly occurring E. coli heat- stable enterotoxin abbreviated ST I. This enterotoxin is an important worldwide cause of diarrhea in human and domestic animals. The project's specific aim is to identify and purify St I binding components from intestinal cells of neonatal calves. Since this enterotoxin is a common cause of diarrhea in newborn calves, this animal model represents an excellent source of intestinal tissue for isolating large quantities of enterocytes bearing the receptor(10II-1012 cells per intestine). The initial receptor solubilization attempts carried out on rat intestinal cells will be used as guidelines for defining optimal solubilization conditions for receptor components present on calf enterocytes. Using a radiobinding assay adapted for detecting detergent- solubilized membrane components that bind a radioiodinated analogue of the enterotoxin, we plan to find detergents and purification conditions that can maintain the receptor's ability to bind ST I. Affinity chromatography will be attempted by coupling a synthetic analogue of ST I to an agarose matrix or by derivatizing analogues of ST I with a biotin group, in an effort to use the avidin-biotin interaction to fish out the St I-receptor complex. Attempts would then be made to chemically characterize the receptor and each of its possible subunits. Finally, the preparation of antibodies to purified St I-binding components (monoclonal antibodies) will also be initiated to insure the development of alternate methods of affinity purification and for screening for translated gene products during our future attempt to clone the gene coding for the receptor and its components. This program will lead to an understanding of the structure of the receptor which may in turn help define its true physiological role as well as to shed light on how the ST I-receptor binding event activates a novel form of particulate guanylate cyclase.
| Carpick, B W; Gariepy, J (1993) The Escherichia coli heat-stable enterotoxin is a long-lived superagonist of guanylin. Infect Immun 61:4710-5 |
| Li, Y; Luo, L Z; Snyder, R M et al. (1988) Expression of the M gene of vesicular stomatitis virus cloned in various vaccinia virus vectors. J Virol 62:776-82 |
