Interaction of glutathione transferase from horse erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.
7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with two thiol groups of the dimeric horse erythrocyte glutathione transferase at pH 5.0, with strong inactivation reversible on dithiothreitol treatment. The inactivation kinetic follows a biphasic pattern, similar to that caused by other thiol reagents as recently reported. Both S-methylglutathione and 1-chloro-2,4-dinitrobenzene protect the enzyme from inactivation. Analysis of the reactive SH group-containing peptide gives the sequence Ala-Ser-Cys-Leu-Tyr, identical with that of the peptide that contains the reactive cysteine 47 of the human placental transferase. In the presence of glutathione, the enzyme is not inactivated by this reagent, but it catalyzes its conjugation to glutathione. At higher pH values, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with 2 tyrosines/dimer and lysines, as well as with cysteines. Reaction with lysine seems essentially without effect on activity; whether the reactive tyrosines are important for activity could not be determined using this reagent only. However, 2 tyrosines among the 4 that are nitrated by tetranitro-methane are important for activity.
Del Boccio
G
G
Institute of Biochemical Sciences, University of Chieti G. D'Annunzio, Italy.
Pennelli
A
A
Whitehead
E P
EP
Lo Bello
M
M
Petruzzelli
R
R
Federici
G
G
Ricci
G
G
D009327:4-Chloro-7-nitrobenzofurazan / Q000737:chemistry*; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004912:Erythrocytes / Q000201:enzymology*; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000097:blood* / Q000737:chemistry; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis; D013439:Sulfhydryl Reagents; D013774:Tetranitromethane / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D013439:Sulfhydryl Reagents; D014443:Tyrosine; D005982:Glutathione Transferase; D009327:4-Chloro-7-nitrobenzofurazan; D005978:Glutathione; D013774:Tetranitromethane; D008239:Lysine; D003545:Cysteine; D004229:Dithiothreitol