Purification of diphtheria toxin receptor from Vero cells.
Diphtheria toxin receptor has been solubilized from Vero cell membranes with octyl beta-D-glucoside. CRM197, the product of a mutated diphtheria toxin gene, was used for the identification of the receptor. The binding activity of the solubilized receptor was assayed by precipitating the receptor with acetone in the presence of phospholipids and carrier proteins. The solubilized receptor was purified by the combination of several chromatographic steps in the presence of the detergent, resulting in about a 10(6)-fold purification of the receptor. The purified receptor showed essentially a single band of 14.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When partially purified receptor fractions were subjected to ligand blotting analysis using 125I-CRM197 as the probe, the 14.5-kDa protein and a few minor protein bands were identified as diphtheria toxin-binding molecules. These results show clearly that the 14.5-kDa protein is the diphtheria toxin receptor, or at least the major diphtheria toxin-binding molecule. When partially purified receptor was applied to a Sephacryl S-300 column in the presence of detergent, the receptor was eluted in the fractions corresponding to the 60-90-kDa size range. This suggests that the protein forms a complex with itself or with another protein.
Mekada
E
E
Institute of Life Science, Kurume University, Fukuoka, Japan.
Senoh
H
H
Iwamoto
R
R
Okada
Y
Y
Uchida
T
T
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002853:Chromatography, Liquid; D003429:Cross Reactions; D004167:Diphtheria Toxin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000302:isolation & purification; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000302:isolation & purification*; D014709:Vero Cells
D001426:Bacterial Proteins; D004167:Diphtheria Toxin; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; C028581:CRM197 (non-toxic variant of diphtheria toxin)