John N. Smith

Glutathione S-transferases from the New Zealand grass grub, Costelytra zealandica Their isolation and characterization and the effect on their activity of endogenous factors

Abstract Isolation of glutathione S- transferase from the New Zealand grass grub, is complicated by the marked loss of activity from crude homogenates. This loss may be due to proteolysis or to modification by endogenous chemicals. The effect may be minimized by immediate fractionation with ammonium sulphate and by inclusion of 5mM glutathione in homogenates. Two enzymes species, isoelectric at pH 8.7 and 5.9 respectively, could be isolated by ammonium sulphate fractionation, affinity chromatography, anion exchange chromatography and chromatography on hydroxyl apatite. They had different substrate specificities and had differing subunit structure. The pI 8.7 enzyme appeared to be a homodimer of subunits of M r 23,700 and the pI 5.9 enzyme one of subunit M r 22,500. A third major enzyme species, isoelectric at pH 4.3 differed from the other two enzymes in having low affinity for the affinity matrix. This preparation was heterogeneous. The enzymically active species in this preparation had the same molecular weight as that of the pI 8.7 enzyme, had a very similar substrate specificity to the basic enzyme species and was characterized by kinetic parameters almost identical to those of the pI 8.7 enzyme.

Photometric determination of methyl parathion GSH S-methyl transferase

Abstract Two methods are described for the assay of the enzyme that transfers a methyl group from methyl parathion ( O,O -dimethyl- O , p -nitrophenyl phosphorothionate) to glutathione. A colorimetric assay depends on the measurement of alkali-soluble, chloroform-insoluble aromatic nitro compounds in the reaction mixture and a more rapid and sensitive direct spectrophotometric assay is described that uses the double wavelength Perkin-Elmer 356 Spectrophotometer. The utility of both methods has been demonstrated by the measurement of some kinetic constants and the distribution of the enzyme among some insects and vertebrates.

A continuous spectrophotometric assay for arylsulfatase activity, dependent on the formation of complexes between cupric ions and nitrocatechols.

Abstract The nitrocatechols 2-hydroxy-5-nitrophenol and 2-hydroxy-5-methyl-4-nitrophenol formed strongly colored complexes with cupric ions, the dissociation constants of these complexes being 1.61 and 1.74 × 10−4 m , respectively. Complex formation with 2-hydroxy-5-methyl-4-nitrophenol was independent of pH between pH 5.4 and 7.2. The aryl-sulfate sulfohydrolase (EC 3.1.6.1) from the New Zealand mollusk, Haliotis iris, was not inhibited by low concentrations of cupric ion so that the enzymatic hydrolysis of sulfate esters of the above nitrocatechols could be monitored continuously in the presence of cupric ions, by following the formation of the yellow complexes. Assay methods based on this phenomenon gave results identical with those obtained by the discontinuous method of alkaline development. Rate measurements were linearly related to enzyme concentration whichever assay method was used. At very high pH, cupric ions decreased the intensity of color of the nitrocatechol anions.

Benzoic acid conjugation in tuatara

Abstract The excretion and metabolism of orally administered [ 14 C]-labelled benzoic acid (100 mg/kg) was examined in the reptile Sphenedon punctatus (tuatara). The major excreted metabolite was chromatographically and electrophoretically identical with ornithuric acid. Conjugation with glycine or glucuronic acid was not detected. 7–21 percent of the dose was recovered from the urine and faeces, the bulk of the excreted radioactivity being eliminated in the first seven days. Free benzoic acid and conjugates were excreted in the first week but only conjugates could be detected in fauces collected at later intervals. These results are discussed in relation to the taxonomic position of tuatara.