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 mu M and 1.5 mu M, r espectively. Time course inactivation of GSTP1-1 by two pesticides was prevented by 3 mM of hexylglutathione, but not by methylglutathione. The fact that the inactivated enzyme recovered all the 5,5'-dithio-bis (2-nitrobenzoic acid) titrable thiol groups, with concomitant recovery of all its original activity after treatment with 100 mM dithiothreit ol, suggested that captan and captafol were able to induce the formati on of disulfide bonds. That the inactivation of GSTP1-1 by captan and captafol involves the formation of disulfide bonds between the four cy steinil groups of the enzymes was confirmed by the SDS-PAGE experiment s on nondenaturant conditions. In fact, on SDS PAGE, GSTP1-1 as well a s the cys47ala, cys101ala, and cys47ala/cys101ala GSTP1-1 mutants trea ted with captan and captafol showed several extra bands, with apparent molecular masses higher and lower than the molecular mass of native G STP1-1 (23.5 kDa), indicating that both intra- and inter-subunit disul fide bonds were formed. These extra bands returned to the native 23.5 kDa band with concomitant restoration of activity when treated with di thiothreitol.