Glyoxalase II from Zea mays: properties and inhibition study of the enzyme purified by use of a new affinity ligand.
The synthesis of N-(p-nitrocarbobenzoxy)glutathione (N-pNCBG) is reported. N-pNCBG and glutathione (GSH) were coupled to Affi-gel 10 by a thioester linkage and resulted in very effective bound ligands for a fast purification of glyoxalase II from corn. The S-(N-pNCBG)-affinity column showed a glyoxalase II binding capacity of up to 2-fold higher than that of the glutathione-affinity column. A single form of glyoxalase II was evidenced by PAGE in both crude extracts and in the affinity purified enzyme. A 45% recovery of glyoxalase II activity (purification, approx. 433-fold) was obtained for both matrices by a single chromatography. The purified glyoxalase is an acidic protein (pI 4.5) of about 26,000 relative molecular mass. Substrate studies for the corn glyoxalase II show, among possible substrates tested, that S-D-lactyl-glutathione is the preferred substrate. An inhibition study was performed with methyl-, propyl-, hexyl-, p-nitrobenzyl-, p-chlorophenacyl-, carbobenzoxy-, and p-nitrocarbobenzoxy-S-glutathione. Methyl-S-glutathione did not inhibit corn glyoxalase II; the others were found to be linear competitive inhibitors. The derivatives containing a thioether bond are weaker inhibitors than those containing a thioester bond or a carbonyl group. p-Nitrobenzyl-S-glutathione is the weakest inhibitor; the carbobenzoxy-S-derivatives are stronger inhibitors than the p-chlorophenacyl S-derivative.
Norton
S J
SJ
Department of Biochemistry, University of North Texas, Denton.
Principato
G B
GB
Talesa
V
V
Lupattelli
M
M
Rosi
G
G
D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol / Q000201:enzymology; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology*; D000099301:Hydroxyacylglutathione Hydrolase
D005978:Glutathione; D013869:Thiolester Hydrolases; D000099301:Hydroxyacylglutathione Hydrolase