Characterization and partial purification of the human receptor for the heat-stable enterotoxin.
The receptor for the Escherichia coli heat-stable enterotoxin has been characterized and partially purified from the T84 human colonic cell line. Using a novel mutant heat-stable enterotoxin peptide as a radioligand (the C-terminal tyrosine residue is replaced by phenylalanine in the mutant), a single class of high-affinity receptor sites was detected in T84 cells, with a Kd of 0.1 nM, similar in affinity to the receptor described in human intestinal tissue. The receptor was solubilised from T84 cell membranes and affinity cross-linking of the solubilised preparation indicated that a single species of M(r) 160,000 served as the receptor. Freshly solubilised preparations of the receptor retained heat-stable enterotoxin-activable guanylyl cyclase activity. Purification of the receptor was achieved through sequential affinity chromatography on GTP--epoxy-Sepharose and wheat-germ-agglutinin columns resulting in purification of the receptor by 3000 fold. The heat-stable enterotoxin-binding characteristics of the receptor were unchanged during the purification and silver staining of the purified receptor preparation indicated a band of M(r) 160,000, which was specifically cross-linked to the 125I-labeled mutant peptide. The purified receptor retained guanylyl cyclase activity, but the activity was not stimulated on addition of human heat-stable enterotoxin, suggesting that accessory structural factors may be involved in the activation of the guanylyl cyclase/receptor.
Visweswariah
S S
SS
Astra Research Centre, Malleswaram, Bangalore, India.
Ramachandran
V
V
Ramamohan
S
S
Das
G
G
Ramachandran
J
J
D001427:Bacterial Toxins / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D003110:Colonic Neoplasms; D003432:Cross-Linking Reagents; D006152:Cyclic GMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D011869:Radioligand Assay; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D014407:Tumor Cells, Cultured
D001427:Bacterial Toxins; D003432:Cross-Linking Reagents; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D018000:Receptors, Peptide; C034151:heat stable toxin (E coli); D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D006152:Cyclic GMP