Different Rates of Metabolism of Two Chloroacetanilide Herbicides in Pioneer 3320 Corn

Abstract

ABSTRACrThe in vivo rates of uptake and detoxification of alachlor and metolachlor were determined using Pioneer corn 3320 seedlings. Equal amounts of the radiolabeled herbicides were applied to etiolated coleoptiles and, at various intervals after treatment, the unabsorbed radioactivity was removed and quantified. Analysis of 80% methanol extracts by reverse phase liquid chromatography showed no significant differences in the rate of uptake of metolachlor and alachlor. However, the rate of glutathione conjugation of alachlor in vivo was two-to threefold greater than the rate for metolachlor at 2 and 4 hours after herbicide application.Since the initial step in detoxification is conjuption of the chloroacetanilide to glutathione, the activities of the enzymes responsible for conjuption, the glutathione-S-transferases (GST) were also analyzed in vitro, using crude extracts and the purified GST enzymes. The specific activities of the extracts were consistent with the results in vivo. Using alachlor as a substrate, the specific activity for glutathione conjugation was almost threefold higher than that for metolachlor. Kinetic analysis of purified GST III indicates that the enzyme has a higher affinity for alachlor (K,,,app = 1.69 millimolar) than for metolachlor (K",app = 8.9 millimolar).

Isolation and Characterization of GlutathioneS-Transferase Isozymes from Sorghum1

“…These subunits, by exhibiting high affinity for chloroacetanilide herbicides, would allow for detoxification of micromolar concentrations of these herbicides, which are inhibitory to plant growth (Deal and Hess, 1980;Fuerst and Gronwald, 1986;Fuerst et al, 1991). These K m values contrast sharply with the apparent K m for metolachlor of 8.9 mm for the GST III/III (formerly GST III) isozyme found in maize coleoptiles (O'Connell et al, 1988).…”

“…In all previous determinations of kinetic parameters for purified plant GSTs, initial-velocity data have been analyzed using the standard Michaelis-Menton equation for a unireaction (O'Connell et al, 1988;Williamson and Beverley, 1988;Singhal et al, 1991;Irzyk and Fuerst, 1993;Droog et al, 1995;Flury et al, 1995). However, kinetic models describing bireactant mechanisms are better suited for the determination of kinetic constants for GSTs, which utilize two substrates.…”

Barley Aleurone Cells Contain Two Types of Vacuoles: Characterization of Lytic Organelles by Use of Fluorescent Probes

“…Test compounds were as follows: 25 M BCECF, 25 M taurocholate, 25 M N-ethylmaleimideglutathione (NEM-GS), 25 M MCB-glutathione (MCB-GS), 1 mM vanadate, 1 mM probenecid, and/or 50 mM ammonium acetate. Substrates requiring conjugation to glutathione (NEM and MCB) before uptake were prepared as described by O'Connell et al (1988), using DMSO as the solvent for the compound. Uptake into isolated vacuoles was initiated by the addition of 8 L of isolated vacuoles and allowed to proceed for 30 min.…”

“…Images were captured on Ektachrome 160T film by using a 5-sec exposure. The glutathione conjugate of ZFR-CMAC was prepared as described above (O'Connell et al, 1988), using ethanol as a solvent for ZFR-CMAC.…”

Degradation of glutathione S‐conjugates by a carboxypeptidase in the plant vacuole

“…All other GS-conjugates were synthesized as described earlier [7]. The assays were incubated at 25°C for 1 h, and the reaction was stopped by addition of 300 l.tl cold acetone.…”