A Study of Glutathione S-Aryltransferase from Costelytra Zealandica

<p>An investigation has been made of the stability, purification and properties of Glutathione S-aryltransferase (Ec 2.5.1.13) from the grass-grub, Costelytra zealandica. The enzyme was found to be extremely unstable in crude homogenates of grass-grubs that had been stored frozen at -2O degrees C, but was considerably more stable in homogenates of live grass-grubs. The instability increased with increase of pH. Glutathione gave some protection against inactivation. Selective fractionation of crude homogenates with (NH4)2SO4 provided some evidence for the presence of an endogenous inhibitor of the enzyme. DEAE-cellulose chromatography and isoelectric focusing studies showed the presence of two major GSH S-aryltransferases with isoelectric points of 4.6 and 8.7. Both enzymes were present in the homogenate from a single, live, grass-grub. The molecular weight and optimum pH of each enzyme was identical within experimental error. A brief comparative study of GSH S-transferases showed the presence of GSH S-alkyl- and GSH s-alkene-transferase, but in only very small amounts compared with GSH S-aryltransferase. Differences in stability were demonstrated and some cross-specificity was indicated. Several inhibitor-substituted Sepharoses were prepared in an attempt to purify GSH s-aryltransferase by affinity chromatography. Although columns of the inhibitors removed the enzyme from solution an active enzyme could not be recovered. The effects of pH and temperature on the enzyme-catalysed reaction of GSH and 1, 2-dichloro-4-nitrobenzene (DCNB) were investigated in detail. Analysis of the variation of pKGSH with pH showed the presence of active site groups with pK approximately 9 involved in GSH binding. Calculation of the heat of ionization of these groups in the pI 8.7 enzyme, from the effect of temperature on their pK, suggested that the groups may be Lysine epsilon-NH2. Values for the enthalpy, free energy and entropy of GSH-binding to the pI 8.7 enzyme and of DCNB-binding to the enzyme-GSH complex were also obtained.</p>

The Comparative Metabolism of Some Substituted Phenyl-N-Methylcarbamate Insecticides

<p>1. The metabolism of the N-methylcarbamates of 3-tertbutylphenol; 3,5-ditertbutylphenol; and 2-isopropoxyphenol was investigated in insects and mammals. 2. The major degradative pathway in enzyme systems from insects and mice was oxidative. The major metabolites from tertbutyl substituted phenyl-N-methylcarbamates were N-hydroxymethyl derivatives and tertbutanol derivatives. Baygon yielded N-hydroxymethyl, ring hydroxyl and O-dealkyl derivatives as major metabolites. 3. The rates of oxidation of the three insecticides in each enzyme system were similar. 4. Oxidation was inhibited by piperonyl butoxide and Metopirone, apparent I50 for singly oxidised metabolites was 10-4 M, and for metabolites with two oxidations 10-5M. 5. Enzymic hydrolysis of carbamate insecticides required reduced cofactor in insect and mouse systems. Mouse blood did not effect hydrolysis. 6. A wide variation of oxidising ability was found in live insects. Musca domestica was most active, Tenebrio molitor and Costelytra zealandica were least active. 7. Insecticide synergists reduce insects' ability to oxidise Baygon to acetone. 8. Musca domestica and Lucilia sericata larvae oxidised carbamate insecticides slower than the adult forms. 9. Mice excrete 3-tertbutylphenyl-N-methylcarbamate as phenolic metabolites, with only minor oxidative products. 10. Different rates of metabolism among insects could account for the selective toxicity of aryl-N-methylcarbamates.</p>

A Study of Glutathione S-Aryltransferase from Costelytra Zealandica

<p>An investigation has been made of the stability, purification and properties of Glutathione S-aryltransferase (Ec 2.5.1.13) from the grass-grub, Costelytra zealandica. The enzyme was found to be extremely unstable in crude homogenates of grass-grubs that had been stored frozen at -2O degrees C, but was considerably more stable in homogenates of live grass-grubs. The instability increased with increase of pH. Glutathione gave some protection against inactivation. Selective fractionation of crude homogenates with (NH4)2SO4 provided some evidence for the presence of an endogenous inhibitor of the enzyme. DEAE-cellulose chromatography and isoelectric focusing studies showed the presence of two major GSH S-aryltransferases with isoelectric points of 4.6 and 8.7. Both enzymes were present in the homogenate from a single, live, grass-grub. The molecular weight and optimum pH of each enzyme was identical within experimental error. A brief comparative study of GSH S-transferases showed the presence of GSH S-alkyl- and GSH s-alkene-transferase, but in only very small amounts compared with GSH S-aryltransferase. Differences in stability were demonstrated and some cross-specificity was indicated. Several inhibitor-substituted Sepharoses were prepared in an attempt to purify GSH s-aryltransferase by affinity chromatography. Although columns of the inhibitors removed the enzyme from solution an active enzyme could not be recovered. The effects of pH and temperature on the enzyme-catalysed reaction of GSH and 1, 2-dichloro-4-nitrobenzene (DCNB) were investigated in detail. Analysis of the variation of pKGSH with pH showed the presence of active site groups with pK approximately 9 involved in GSH binding. Calculation of the heat of ionization of these groups in the pI 8.7 enzyme, from the effect of temperature on their pK, suggested that the groups may be Lysine epsilon-NH2. Values for the enthalpy, free energy and entropy of GSH-binding to the pI 8.7 enzyme and of DCNB-binding to the enzyme-GSH complex were also obtained.</p>

The Comparative Metabolism of Some Substituted Phenyl-N-Methylcarbamate Insecticides

<p>1. The metabolism of the N-methylcarbamates of 3-tertbutylphenol; 3,5-ditertbutylphenol; and 2-isopropoxyphenol was investigated in insects and mammals. 2. The major degradative pathway in enzyme systems from insects and mice was oxidative. The major metabolites from tertbutyl substituted phenyl-N-methylcarbamates were N-hydroxymethyl derivatives and tertbutanol derivatives. Baygon yielded N-hydroxymethyl, ring hydroxyl and O-dealkyl derivatives as major metabolites. 3. The rates of oxidation of the three insecticides in each enzyme system were similar. 4. Oxidation was inhibited by piperonyl butoxide and Metopirone, apparent I50 for singly oxidised metabolites was 10-4 M, and for metabolites with two oxidations 10-5M. 5. Enzymic hydrolysis of carbamate insecticides required reduced cofactor in insect and mouse systems. Mouse blood did not effect hydrolysis. 6. A wide variation of oxidising ability was found in live insects. Musca domestica was most active, Tenebrio molitor and Costelytra zealandica were least active. 7. Insecticide synergists reduce insects' ability to oxidise Baygon to acetone. 8. Musca domestica and Lucilia sericata larvae oxidised carbamate insecticides slower than the adult forms. 9. Mice excrete 3-tertbutylphenyl-N-methylcarbamate as phenolic metabolites, with only minor oxidative products. 10. Different rates of metabolism among insects could account for the selective toxicity of aryl-N-methylcarbamates.</p>