Interaction of glutathione transferase P1-1 with captan and captafol.
Glutathione transferase (GST, EC 2.5.1.18) P1-1 was strongly inhibited by captan and captafol in a time- and concentration-dependent manner. The IC50 values for captan and captafol were 5.8 microM and 1.5 microM, respectively. Time-course inactivation of GSTP1-1 by two pesticides was prevented by 3 microM of hexyl-glutathione, but not by methylglutathione. The fact that the inactivated enzyme recovered all the 5,5'-dithiobis(2-nitrobenzoic acid) titrable thiol groups, with concomitant recovery of all its original activity after treatment with 100 microM dithiothreitol, suggested that captan and captafol were able to induce the formation of disulfide bonds. That the inactivation of GSTP1-1 by captan and captafol involves the formation of disulfide bonds between the four cysteinil groups of the enzymes was confirmed by the SDS-PAGE experiments on nondenaturant conditions. In fact, on SDS-PAGE, GSTP1-1 as well as the cys47ala, cys101ala, and cys47ala/cys101ala GSTP1-1 mutants treated with captan and captafol showed several extra bands, with apparent molecular masses higher and lower than the molecular mass of native GSTP1-1 (23.5 kDa), indicating that both intra- and inter-subunit disulfide bonds were formed. These extra bands returned to the native 23.5 kDa band with concomitant restoration of activity when treated with dithiothreitol.
di Ilio
C
C
Istituto Di Scienze Biochimiche, Universita G. D'Annunzio, Chieti, Italy.
Sacchetta
P
P
Angelucci
S
S
Bucciarelli
T
T
Pennelli
A
A
Mazzetti
A P
AP
Lo Bello
M
M
Aceto
A
A
D002215:Captan / Q000031:analogs & derivatives* / Q000494:pharmacology*; D053138:Cyclohexenes; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D010084:Oxidation-Reduction
D053138:Cyclohexenes; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C001614:captafol; D005982:Glutathione Transferase; D002215:Captan