Enhanced Catalytic Constant for GlutathioneS-Transferase (Atrazine) Activity in an Atrazine-ResistantAbutilon theophrastiBiotype

Abstract Glutathione S -transferases (GST, EC 2.5.1.18) were purified from leaves of atrazine-resistant and-susceptible 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 (55,000 M ; 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 ; r of 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/avidin-alkaline 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 ; m values for GSH and atrazine. However, the catalytic constant ( k cat ) was approximately threefold greater for GST(atrazine) activity from the resistant biotype compared to the susceptible biotype. Inhibition constants ( K ; i values) 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 k cat for GST(atrazine) activity.