D.S. Frear

N-demethylation of substituted 3-(phenyl)-1-methylureas: Isolation and characterization of a microsomal mixed function oxidase from cotton

Abstract A microsomal, mixed function oxidase that successively N -demethylates substituted 3-(phenyl)-1,1-dimethylurea substrates was isolated from etiolated cotton seedling hypocotyl extracts by differential and density gradient centrifugation. Active enzyme preparations were also isolated from leaves of cotton, plantain, buckwheat, wild buckwheat and broadbeans. The enzyme is located in the microsomal fraction of plant extracts and requires molecular oxygen and either NADPH or NADH as cofactors. The formation of 1 mole of formaldehyde for each mole of substrate demethylated was demonstrated with 14 C methyl-labeled substrate. The enzyme appears to be specific for substituted 3-(phenyl)-1-methylurea compounds, and the apparent K m values for three of these substrates were determined. Inhibition of the enzyme by carbon monoxide, ionic detergents, sulfhydryl reagents, chelating agents and electron acceptors, together with the demonstrated presence of a b 5 cytochrome and an active NADPH-cytochrome c reductase, are discussed as indirect evidence for a microsomal electron transport system in plants similar to those reported for animals. Several differences between the cotton N -demethylase and similar animal systems were also noted. These differences included a greater instability and a decreased sensitivity toward the insecticidal synergists 2,2-diethylaminoethyl-2,2-diphenylpentanoate (SKF 525-A) and 2-(3,4-methylenedioxyphenoxy)-3,6,9-trioxoundecane (Sesamex). The inhibition of N -demethylase activity by several N-methylcarbamates was investigated and the apparent K i value for the competitive inhibition by 1-naphthylmethylcarbamate was determined.

Biosynthesis of S-(4-ethylamino-6-isopropylamino- 2-s-triazino) glutathione: Partial purification and properties of a glutathione S-transferase from corn☆

Abstract A soluble glutathione S-transferase from corn leaves was purified 7·6-fold by differential centrifugation, ammonium sulfate fractionation and gel filtration. Active enzyme preparations were also isolated from leaves of sorghum, sugarcane, Johnson grass and Sudan grass. The enzyme catalyzed the conjugation of several substituted 2-chloro-s-triazine herbicides with reduced glutathione. The formation of 1 mole of chloride ion for every mole of glutathione conjugate produced was demonstrated with 14C and 36Cl-labeled 2-chloro- 4-ethylamino-6-isopropylamino-s-triazine. A rapid ion exchange assay system for following the rate of 14C-labeled s-triazine conjugate formation was developed. The pH optimum for 2-chloro-4-ethylamino-6- isopropylamino-s-triazine conjugate formation was between 6·6 and 6·8. Enzyme specificity for reduced glutathione was demonstrated. Specificity and inhibition studies with substituted s-triazines indicated that a chlorine atom in the 2-position and N-alkyl side-chains in the 4 and 6 positions were required for enzyme activity. The apparent Km values for 2-chloro-4-ethylamino-6-isopropylamino-s-triazine and reduced glutathione were 3·7 × 10−5 M and 2·4 × 10−3 M, respectively. Inhibition studies demonstrated a competitive inhibition with 2-methylmercapto-4,6-bis-isopropylamino-s-triazine (Ki = 2·8 × 10−5 M) and with sulfobromophthalein (Ki = 2·7 × 10−5 M). Since this appears to be the first report of glutathione S-transferase activity in plants, the similarities and differences between the corn leaf enzyme and previously reported animal glutathione S-transferase systems were discussed. The role of this enzyme system in the rapid in vivo detoxification and selectivity of substituted 2-chloro-s-triazine herbicides was also discussed.

The metabolism of 3,4-dichloropropionanilide in plants. Partial purification and properties of an aryl acylamidase from rice☆

Abstract An aryl acylamidase (aryl-acylamine amidohydrolase, EC 3.5.1.a) from rice, which hydrolyzes 3,4-dichloropropionanilide, has been partially purified and characterized. The distribution of the enzyme in rice and barnyard grass tissues was determined. The enzyme displayed a broad specificity for chlorinated ring-substituted propionanilide analogs, but was specific for 3,4-dichloropropionanalide when compared with several alkyl substituted analogs. The enzyme was inhibited by sulfhydryl reagents and was strongly inhibited at 1·0 × 10 −6 M by the insecticidal carbamates 1-naphthylmethylcarbamate,4-benzothienyl methylcarbamate, 2-chloro-4,5-xylyl methylcarbamate, 2,4,5-trimethylphenyl methylcarbamate, 3,4,5-trimethylphenyl methylcarbamate and 4-(methylthiol) 3,5-xylyl methylcarbamate. The partially purified enzyme had a broad pH optimum between 7·5 and 7·9, with an apparent K m of 2·93 × 10 −3 M for 3,4-dichloropropionanilide. The K i for 1-naphthyl methylcarbamate was 1·51 × 10 −8 M with 3,4-dichloropropionanilide as the substrate. The significance of the enzyme distribution in the resistant and susceptible species and the inhibition of the rice enzyme by insecticidal carbamates is discussed.

Acifluorfen metabolism in soybean: Diphenylether bond cleavage and the formation of homoglutathione, cysteine, and glucose conjugates

Abstract Metabolism of the substituted diphenylether herbicide, acifluorfen [sodium 5-(2-chloro-4-trifluoromethylphenoxy)-2-nitrobenzoate], was studied in excised leaf tissues of soybean [ Glycine max (L.) Merr. ‘Evans’]. Studies with [ chlorophenyl - 14 C]- and [ nitrophenyl - 14 C]acifluorfen showed that the diphenylether bond was rapidly cleaved. From 85 to 95% of the absorbed [ 14 C]acifluorfen was metabolized in less than 24 hr. Major polar metabolites were isolated and purified by solvent partitioning, adsorption, thin layer, and high-performance liquid chromatography. The major [ chlorophenyl - 14 C]-labeled metabolite was identified as a malonyl-β- d -glucoside (I) of 2-chloro-4-trifluoromethylphenol. Major [ nitrophenyl - 14 C]-labeled metabolites were identified as a homoglutathione conjugate [ S -(3-carboxy-4-nitrophenyl) γ-glutamyl-cysteinyl-β-alanine] (II), and a cysteine conjugate [ S -(3-carboxy-4-nitrophenyl)cysteine] (III).

Aryl hydroxylation of diclofop by a cytochrome P450 dependent monooxygenase from wheat

Abstract In tolerant wheat, diclofop metabolism in vivo was rapid (33% in 6 hr) and was sensitive to cytochrome P450 inhibitors (CO, tetcyclasis, and 1-aminobenzotriazole). Microsomal fractions from etiolated wheat seedling shoots were shown to support the in vitro hydroxylation of diclofop in the presence of molecular oxygen and NADPH. Enzyme activity was strongly inhibited by tetcyclasis, but showed less sensitivity to 1-aminobenzotriazole and CO. The major enzymatic reaction product in vitro was isolated and identified by HPLC and electron impact mass spectrometry as the NIH shift product, 2-[4-(2,5-dichloro-4-hydroxyphenoxy)phenoxy]propanoic acid. The enzyme had a pH optimum of 7.4, a Vmax of 5.7 nmol/mg protein · h, and an apparent Km of 41.6 μM.

Metribuzin metabolism in tomato: Isolation and identification of N-glucoside conjugates☆☆☆

Abstract Metribuzin [4-amino-6- tert -butyl-3-(methylthio)-1,2,4-triazin-5(4H)-one] metabolism was studied in tomato ( Lycopersicon esculentum Mill. “Sheyenne”). Pulse-treatment studies with seedlings and excised leaves showed that [5- 14 C]metribuzin was rapidly absorbed, translocated (acropetal), and metabolized to more polar products. Foliar tissues of 19-day-old seedlings metabolized 96% of the root-absorbed [ 14 C]metribuzin in 120 hr. Excised mature leaves metabolized 85–90% of the petiole-absorbed [ 14 C]metrubuzin in 48 hr. Polar metabolites were isolated by solvent partitioning, and purified by adsorption, thin-layer, and high-performance liquid chromatography. A minor intermediate metabolite (I) was identified as the polar β- d -( N -glucoside) conjugate of metribuzin. The biosynthesis of (I) was demonstrated with a partially purified UDP-glucose: metribuzin N -glucosyltransferase from tomato leaves. A possible correlation between foliar UDP-glucose: metribuzin N -glucosyltransferase activity levels and differences in the tolerance of selected tomato seedling cultivars to metribuzin was suggested. The major polar metabolite (II) was identified as the malonyl β- d -( N -glucoside) conjugate of metribuzin.

Alternate pathways of metribuzin metabolism in soybean: Formation of N-glucoside and homoglutathione conjugates☆☆☆

Abstract Metribuzin [4-amino-6- tert -butyl-3(methylthio)-1,2,4-triazin-5(4H)-one] metabolism was studied in soybean [ Glycine max (L.) Merr. Tracy]. Pulse treatment studies with seedlings and excised mature leaves showed that [5- 14 C]metribuzin was absorbed rapidly and translocated acropetally. In seedlings, >97% of the root-absorbed 14 C was present in foliar tissues after 24 hr. In excised leaves, 50–60% of the absorbed 14 C remained as metribuzin 48 hr after pulse treatment, 12–20% was present as polar metabolites, and 20–30% was present as an insoluble residue. Metabolites were isolated by solvent partitioning, and were purified by adsorption, ion-exchange, thin-layer, and high-performance liquid chromatography. The major metabolite (I) was identified as a homoglutathione conjugate, 4-amino-6- tert -butyl-3- S -(γ-glutamyl-cysteinyl-β-alanine)-1,2,4-triazin-5(4H)-one. Metabolite identification was confirmed by qualitative analysis of amino acid hydrolysis products, fast atom bombardment (FAB), and chemical ionization (CI) mass spectrometry, and by comparison with a reference glutathione conjugate synthesized in vitro with a hepatic microsomal oxidase system from rat. Minor metabolites were identified as an intermediate N -glucoside conjugate (II), a malonyl N -glucoside conjugate (III), and 4-malonylamido-6- tert -butyl-1,2,4-triazin-3,5(2H,4H)-dione ( N -malonyl DK, IV) by CI and FAB mass spectrometry. Alternative pathways of metribuzin metabolism are proposed.

Induced microsomal oxidation of diclofop, triasulfuron, chlorsulfuron, and linuron in wheat

Abstract Microsomal fractions from shoot tissues of etiolated wheat seedlings catalyzed the oxidation of diclofop, chlorsulfuron, triasulfuron, chlortoluron, and linuron. Microsomal oxidation products of chlorsulfuron, triasulfuron, and linuron were isolated and identified by mass spectrometry and cochromatography with reference standards. Oxidation was dependent on NADPH and molecular oxygen and was inhibited by CO in the presence of oxygen. Triasulfuron hydroxylation was inhibited to varying degrees by other known inhibitors of cytochrome P-450 enzymes and by several different postemergence herbicides. Enzyme activity was increased 2- to 3-fold by the removal of endogenous inhibitors and stimulated an additional 5- to 20-fold by the treatment of germinating seedlings with naphthalic anhydride, ethanol, or phenobarbital. In contrast to marked increases in monooxygenase activities following induction, microsomal cytochrome P-450 levels and NADPH cytochrome c reductase activities were not increased to a significant extent. Ethanol and phenobarbital were more effective than naphthalic anhydride as inducers of microsomal hydroxylase activity. The combined effect of naphthalic anhydride and ethanol as inducers of diclofop and triasulfuron hydroxylases was additive. Apparent Km values for triasulfuron, chlorsulfuron, and diclofop with constitutive and induced microsomal hydroxylases were compared. Differences in the response of herbicide monooxygenases to selected inhibitors, inducers, and substrates support the hypothesis that wheat microsomes contain a number of distinct cytochrome P-450-dependent monooxygenases with different substrate specificities and kinetic properties. These enzymes serve as important factors in the tolerance and selectivity of a broad spectrum of herbicides used in wheat production systems.

New metabolites of monuron in excised cotton leaves

Abstract Two new metabolites of 3-(4-chlorophenyl)-1-dimethylurea (monuron) have been isolated from cotton leaves. They have been identified as β- d -glucosides of 3-(4-chlorophenyl)-1-hydroxymethyl-1-methylurea and 3-(4-chlorophenyl)-1-hydroxymethylurea. After 24 hr, both O-glucosides constitute 20–25% of the methanol soluble monuron metabolites present in treated leaf tissues. The isolation and identification of these polar monuron metabolites provides direct evidence for the formation of N-hydroxymethyl intermediates in the oxidative N-demethylation of substituted phenylurea herbicides by higher plants. The significance of reactive N-hydroxymethyl intermediates, particularly 3-(4-chlorophenyl)-1-hydroxymethylurea, in the formation of β- d -glucosides and other polar, unknown methanol soluble and insoluble monuron residues in higher plants is discussed.

Herbicide metabolism in plants—I : Purification and properties of UDP-glucose: Arylamine N-glucosyl-transferase from soybean

Abstract A “soluble” enzyme system from soybean, which catalyzes the biosynthesis of N -glucosylarylamines, has been purified 20-fold by differential centrifugation, ammonium sulfate fractionation, gel filtration, and cellulose ion exchange chromatography. The enzyme was specific for the nucleotide glucosyl donors uridinediphosphate glucose (UDPG) and thymidinediphosphate glucose (TDPG), but exhibited a broad specificity toward acceptor arylamines. A number of substituted anilines and aminobenzoic acids were studied as acceptor arylamines including 2,5-dichloro-3-aminobenzoic acid (amiben) and 3,4-dichloroaniline, a metabolite of 3,4-dichloropropionanilide (propanil) in higher plants. The partially purified enzyme was found to have an optimum pH of 7·5 with 3,4-dichloroaniline as the arylamine acceptor and was inhibited by sulfhydryl reagents. The Km constants for UDPG and 3,4-dichloroaniline were 1·88 × 10 −3 M and 5·63 × 10 −4 M respectively. The Ki constant for uridinediphosphate (UDP) was 4·84 × 10 −4 M.