Purification and characterization of two forms of glyoxalase II from the liver and brain of wistar rats

Abstract

Glyoxalase II ( S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) was purified to homogeneity and separated into two forms ( α, p I = 8.0; β p I = 7.4) from both liver and brain of wistar rats by column isoelectric focusing. These forms were also found to have different electrophoretic mobilities. No significant differences were found between the α and β forms from either source in the relative molecular mass (about 24 000) or in K m values using three substrates. The temperature-inactivation profiles were also similar, the two forms being stable up to 50°C. Chemical modification studies with phenylglyoxal suggest that these enzyme forms probably contain arginine residues near the active site. Inactivation of α and β forms by diethylpyrocarbonate and by photooxidation with methylene blue, and protection by S- d- mandeloylglutathione , a slowly reacting substrate, suggest the presence of histidine at the active site. The α and β forms show different half-line values in inactivation by histidine reagents, which may be due to a difference in the active-site structures of these enzymes. The results probably indicate distincts structures (sequences) for α and β forms.