Conjugation of chlorodinitrobenzene with reduced glutathione in the absence and presence of glutathione transferase from larvae of the southern armyworm, Spodoptera eridania

Abstract The reaction of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione in the absence and presence of glutathione transferases from larvae of the southern armyworm, Spodoptera eridania , produced the conjugate 2,4-dinitrophenyl S -glutathione with identical uv absorption spectra. The extinction coefficient was 10.63 m M −1 cm −1 at 340 nm (λ max ). Kinetics of spontaneous conjugation in 0.1 M Tris-HCl buffer (pH 8.0) at 30°C indicated that a reversible complex between CDNB and glutathione preceded the formation of the stable conjugate. The kinetic parameters associated with these two steps were 2.9 × 10 −2 M and 0.22 min −1 for the dissociation equilibrium constant ( K d ) and the first order rate constant of conjugation ( k c ), respectively. Glutathione transferase activity was present almost exclusively in the cytosolic fractions of the gut, head capsule, integument, and fat body of the last instar larva of the southern armyworm. Among the above tissue preparations, the head capsule and gut showed the highest activity per unit protein. Hemolymph glutathione transferase activity was approximately double the activity of the head and gut. The transferase activity from the hemolymph and gut exhibited identical K m values for CDNB and glutathione. This, in addition to a high correlation between the activity of the hemolymph and gut in individual larvae, argues that the two activities are due to the same enzyme(s) and are regulated by similar factors.