Enhanced Catalytic Constant for Glutathione S-Transferase (Atrazine) Activity in an Atrazine-Resistant Abutilon theophrasti Biotype

Glutathione S-transferases (GST, EC 2.5.1.18) were purified from leaves of atrazine-resistant andsusceptible velvetleaf (Abutilon theophrasti Medic.) biotypes using a protocol involving DEAE anion-exchange, S-hexylglutathione affinity, and Superose 12 gel-filtration chromatography. This protocol resulted in greater than 500-fold purification of GST activity with atrazine [GST (atrazine) activity] from both biotypes. There were no differences in the amount of dimeric GST (55,000M;r) from resistant and susceptible biotypes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified GST dimers from both biotypes indicated the presence of three GST subunits with M;rof 27,000, 26,000, and 25,000. GST subunits in both resistant and susceptible biotypes were glycosylated as revealed by probing Western blots with concanavalin A-biotin/avidinalkaline phosphatase. Native isoelectric focusing of the purified GSTs indicated three major isoforms in both biotypes with p I values of 4.1, 4.3, and 4.4. For both resistant and susceptible biotypes, analysis of initial-velocity GST(atrazine) activity of the purified GST fraction indicated a sequential, random, rapid-equilibrium, Bi Bi kinetic mechanism. Kinetic analysis of GST(atrazine) activity from resistant and susceptible biotypes indicated no significant difference in K;mvalues for GSH and atrazine. However, the catalytic constant (kcat) was approximately threefold greater for GST(atrazine) activity from the resistant biotype compared to the susceptible biotype. Inhibition constants (K;ivalues) for the glutathione-atrazine conjugate were approximately 1.5-fold higher for GST(atrazine) activity from the resistant biotype compared to the susceptible biotype. Accelerated atrazine detoxification via glutathione conjugation in the resistant biotype is primarily due to enhanced kcatfor GST(atrazine) activity.