Affinity purification of functional receptors for Escherichia coli heat-stable enterotoxin from rat intestine.
Active receptors for Escherichia coli heat-stable enterotoxin (ST) were partially purified by ligand-affinity chromatography. The affinity column was prepared by coupling ST to biotin derivatized with an extended N-hydroxysuccinylated spacer arm prior to binding to monomeric avidin immobilized on agarose. Detergent extracts of rat intestinal mucosa membranes were quantitatively depleted of ST binding activity when chromatographed on this affinity matrix. Biotinylated ST-receptor complexes were eluted from affinity columns with 2 mM biotin and these complexes quantitatively dissociated with bile salts. Using this technique, functional ST receptors were purified maximally about 2000-fold, with about 3% of the total activity in crude extracts recovered in these purified preparations. Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis and silver staining demonstrated a major protein subunit of 74 kDa. Affinity cross-linking of these preparations to 125I-ST demonstrated specific labeling predominantly of the 74-kDa subunit. In addition, lower amounts of labeled ST were incorporated into subunits of 164 and 45 kDa, confirming the heterogeneous nature of ST receptors. Purified receptors bound ST in a concentration-dependent fashion, with an IC50 of 10(-9) M. These studies demonstrate that ligand-affinity chromatography can be employed to purify ST receptors. The availability of purified receptors will facilitate further studies of mechanisms underlying ST-induced intestinal secretion.
Hugues
M
M
Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Crane
M R
MR
Thomas
B R
BR
Robertson
D
D
Gazzano
H
H
O'Hanley
P
P
Waldman
S A
SA
D000818:Animals; D001427:Bacterial Toxins / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000302:isolation & purification*; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase; D007413:Intestinal Mucosa / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008970:Molecular Weight; D051381:Rats; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D008024:Ligands; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C034151:heat stable toxin (E coli); D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled