Purification of detergent-solubilized form and membrane-binding domain of rat gamma-glutamyltransferase by immuno-affinity and hydrophobic chromatography.
A new method to purify papain- or detergent-solubilized form (papain or detergent form) of gamma-glutamyltransferase from rat hepatomas as well as from rat kidney by immuno-affinity column chromatography is presented. The antibody-column was prepared by coupling the anti-kidney papain form antibody, which had been purified by using a kidney papain form-Sepharose column, to CNBr-activated Sepharose 4B. The enzyme bound to the antibody-column was eluted with 0.04 M NH4OH. By this method, detergent forms were purified 300 and 1600-fold in approx. 50% yields from rat kidney and rat ascites hepatoma AH 13, respectively, and the papain form was also purified 16 000-fold in a similar yield from primary hepatoma which has a very low activity of this enzyme. Preparations thus obtained apparently did not contain any peptide other than heavy and light subunit peptides of this enzyme on SDS-polyacrylamide gel electrophoresis. The detergent form of kidney enzyme was preferentially absorbed to a hydrophobic column of aminooctyl-Sepharose, while the papain form was not, suggesting that the detergent form might be adsorbed to the column through hydrophobic interaction of the membrane-binding domain. The domain peptide was also purified by the hydrophobic column after release from the detergent form by papain treatment. The molecular weight of the peptide was estimated to be about 16 000 on SDS-polyacrylamide gel electrophoresis. On double immunodiffusion, the domain peptide reacted with anti-detergent form antibody but not with anti-papain form antibody. The domain-specific antibody was also purified from the anti-detergent form antibody.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D003902:Detergents; D007120:Immunochemistry; D007668:Kidney / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification*; D010206:Papain; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D005723:gamma-Glutamyltransferase / Q000302:isolation & purification*
D003902:Detergents; D008565:Membrane Proteins; D005723:gamma-Glutamyltransferase; D010206:Papain