Purification and partial characterization of glyoxalase I from bovine brain.
Glyoxalase I was purified to homogeneity from bovine brain using affinity chromatography on S-hexylglutathione-Sepharose 6B with a yield of 22%. The enzyme was a dimer (44,000 Daltons) composed of, apparently, identical subunits (22,000 Daltons), as shown by SDS electrophoresis, and contained one mole of Zn2+/monomer. The active site metal ion, Zn2+, was removed by dialysis against EDTA, but the activity of the apoenzyme obtained was not completely restored after addition of Co2+ and Zn2+ (<25%), while a recovery of 50% was obtained after addition of Mg2+. The enzyme was inhibited by S-bromobenzylglutathione and S-p-nitrobenzylglutathione with a Ki value of 21 microM and 32 microM, respectively. The highest dissociation constant observed for the brain enzyme with respect to that reported for human erythrocytes, or other mammalian forms of enzyme could be related to a tissue-specific dependence of the glyoxalase I activity.
Lupidi
G
G
Dept. of Biology M.C.A., University of Camerino, Italy. lupidi@cambio.unicam.it
Venardi
G
G
Bollettini
M
M
Marmocchi
F
F
Rotilio
G
G
D000818:Animals; D001051:Apoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002413:Cations, Divalent / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D007791:Lactoylglutathione Lyase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D013997:Time Factors; D015003:Yeasts / Q000201:enzymology
D001051:Apoenzymes; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D007791:Lactoylglutathione Lyase; D008274:Magnesium