title: Characterization of glyoxalase I purified from pig erythrocytes by affinity chromatography.
abstract: Glyoxalase I (EC 4.4.1.5) was purified about 10000-fold from pig erythrocytes in a yield of approx. 20%. The purification included affinity chromatography on S-hexylglutathione coupled to Sepharose 4B. The purified enzyme normally contained two catalytically active components which were resolved by polyacrylamide-gel electrophoresis. After treatment with reduced glutathione only one component was found. The two components were also demonstrable after isoelectric focusing or DEAE-cellulose chromatography and could also in these cases be fused into one species by preincubation with reduced glutathione. It is proposed that the most acidic form of glyoxalase I is a mixed disulphide with glutathione. Except for these interconvertible forms, the purified enzyme was homogeneous, as judged by disc electrophoresis and sodium dodecyl sulphate/polyacrylamidegel electrophoresis. The molecule is a dimer (48000 daltons), composed of apparently identical subunits (24000 daltons). The isoelectric point was 4.8 at 4 degrees C. The amino acid composition was consistent with the low isoelectric point. The enzyme contained about two thiol groups per enzyme molecule. EDTA inactivated the enzyme and bivalent metal ions could restore fully or partially the catalytic activity; Mg(2+) and Mn(2+) gave highest activity. It is proposed that a major biological function of glyoxalase I is the detoxification of methylglyoxal formed by enterobacteria in the alimentary canal.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity; D004912:Erythrocytes / Q000201:enzymology*; D005978:Glutathione; D007525:Isoelectric Focusing; D007791:Lactoylglutathione Lyase / Q000097:blood / Q000302:isolation & purification; D008190:Lyases / Q000097:blood / Q000302:isolation & purification; D008970:Molecular Weight; D013552:Swine
chemical_list: D000596:Amino Acids; D008190:Lyases; D007791:Lactoylglutathione Lyase; D005978:Glutathione