Alan G. Clark

Evidence that DDT-dehydrochlorinase from the house fly is a glutathione S-transferase

Abstract DDT-dehydrochlorinase has been isolated in a highly purified form by a procedure involving affinity chromatography, gel-permeation chromatography, and preparative isoelectrofocusing. At least two protein species appeared to possess DDT-dehydrochlorinase activity; the principal one of these was purified by a factor of 660-fold. This appeared to be dimeric with subunits of molecular weight of 23,000 and 25,000. Another protein with this activity appeared to consist of two identical subunits of M r 25,000. The protein with greatest activity was isoelectric at pH 7.1. It was found to be homogeneous on analytical gel electrophoresis in both the presence and absence of SDS. The same protein generated a number of minor protein bands on analytical electrofocusing in polyacrylamide gels, but there is evidence that these bands may be artifactual. Both purified forms of the enzyme possessed substantial glutathione S -transferase activity with both CDNB and DCNB. An acidic protein, a dimer of subunits of M r 23,000 had substantial GSH transferase activity with CDNB as substrate, but had no DDT-dehydrochlorinase activity.

The comparative enzymology of the glutathione S-transferases from non-vertebrate organisms

CONTENTS

Characterization of multiple glutathione transferases from the house fly, Musca domestica (L)

Abstract Glutathione transferases have been purified to a high degree of homogeneity from three strains of house fly by a procedure involving affinity chromatography on glutathione-sulfobromophthalein conjugate immobilized on Sepharose 4B, followed by preparative isoelectrofocusing. The affinity chromatography yielded purifications of between about 10- and 100-fold, depending on the strain and the substrate with which activity was measured. Each strain was shown to possess several proteins with glutathione S-transferase activity which fell into two clearly defined groups. The first group, of relatively low isoelectric point, showed activity with CDNB but little with DCNB, p-nitrobenzylchloride, or 1,2-epoxy-3-(p-nitrophenoxy)propane, whereas the second group, of higher isoelectric points, showed substantial activity with all substrates tested. Studies on the subunit structure of these enzymes demonstrated the existence of three different sized subunits of Mr 20,000, 22,000, and 23,500. From the experimental evidence recorded here, the existence of at least three functionally different glutathione transferases is inferred.

The Identification and Structural Characterization of C7orf24 as γ-Glutamyl Cyclotransferase AN ESSENTIAL ENZYME IN THE γ-GLUTAMYL CYCLE

The hypothetical protein C7orf24 has been implicated as a cancer marker with a potential role in cell proliferation. We have identified C7orf24 as γ-glutamyl cyclotransferase (GGCT) that catalyzes the formation of 5-oxoproline (pyroglutamic acid) from γ-glutamyl dipeptides and potentially plays a significant role in glutathione homeostasis. In the present study we have identified the first cDNA clones encoding a γ-glutamyl cyclotransferase. The GGCT gene is located on chromosome 7p14-15 and consists of four exons that span 8 kb. The primary sequence is 188 amino acids in length and is unlike any protein of known function. We crystallized functional recombinant γ-glutamyl cyclotransferase and determined its structure at 1.7 Å resolution. The enzyme is a dimer of 20,994-Da subunits. The topology of GGCT is unrelated to other enzymes associated with cyclotransferase-like activity. The fold was originally classified as “BtrG-like,” a small family that only includes structures of hypothetical proteins from Mus musculus, Escherichia coli, Pyrococcus horikoshii, and Arabidopsis thaliana. Since this is the first member of this family with a defined function, we propose to refer to this structure as the γ-glutamyl cyclotransferase fold. We have identified a potential active site pocket that contains a highly conserved glutamic acid (Glu98) and propose that it acts as a general acid/base in the reaction mechanism. Mutation of Glu98 to Ala or Gln completely inactivates the enzyme without altering the overall fold.

The purification by affinity chromatography of a glutathione S-transferase from larvae of Galleria mellonella.

Abstract Glutathione-sulphobromophthalein conjugate, covalently linked to an agarose matrix, acts as an affinity chromatographic medium for a glutathione S-transferase from Galleria mellonella . In a three step preparation, an enzyme of 98% purity is obtained, in 30% yield.

Heterogeneity of the glutathione transferase genes encoding enzymes responsible for insecticide degradation in the housefly.

One of the four glutathione-S-transferases (GST) that is overproduced in the insecticide-resistant Cornell-R strain of the housefly (Musca domestica) produces an activity that degrades the insecticide dimethyl parathion and conjugates glutathione to lindane. In earlier work, it was shown that the resistant Cornell-R carries an amplification, probably a duplication, of one or more of its GST loci and that this amplification is directly related to resistance. Using polymerase chain reaction (PCR) amplification with genomic DNA, multiple copies of the gene encoding the parathion-degrading activity (called MdGst-3) were subcloned from both the ancestral, insecticide-susceptible strain BPM and from the insecticide-resistant Cornell-R. In BPM, three different MdGst-3 genes were identified while in Cornell-R, 12 different MdGst-3 sequences were found that, though closely related to ancestral genes, had diverged by a few nucleotides. This diversity in MdGst-3 genomic sequences in Cornell-R is reflected in the expressed sequences, as sampled through a cDNA bank. Population heterozygosity cannot account for these multiple GST genes. We suggest that selection for resistance to insecticides has resulted in not only amplification of the MdGst-3 genes but also in the divergence of sequence between the amplified copies.

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.

Some properties of a glutathione S-transferase from the larvae of Galleria mellonella

Abstract The glutathione transferase from Galleria mellonela has an estimated molecular weight of 41,000. It appears to be composed of two identical subunits of 25,000 molecular weight. Its substrate specificity resembles that of the glutathione S-transferase B from rat liver. It has no detectable activity with the insecticides DDT, methyl parathion or lindane (γ-hexachlorocyclohexane). The kinetics are complex and resemble those reported for glutathione S-transferase A from rat liver, except that the enzymic activity is weakly inhibited by the product, chloride ion. Its susceptibility to inhibition by thiol reagents also resembles that of rat glutathione S-transferases. In general, the properties of this insect enzyme are very similar to those from rat liver.

A functional comparison of ovine and porcine trypsins.

Trypsin was isolated from ovine and porcine pancreas using affinity chromatography on immobilized p-aminobenzamidine. Molecular masses of the two proteins were 23900 and 23435 Da, determined by matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry. The purified trypsins were compared using the kinetic properties K(m) and k(cat) which were determined at pH 8.0 and between 25 and 55 degrees C. Comparison of the Michaelis constants for ovine and porcine trypsins toward N-alpha-benzoyl-arginine-p-nitroanilide (BapNA) indicated that ovine trypsin had higher affinity for this substrate than the porcine enzyme. The rates of the reactions catalysed by the two enzymes correlated strongly over the range of temperatures and substrate concentrations tested, as did the k(cat) values. The specific activity of ovine trypsin for BapNA was, on average, approximately 10% higher than that of the porcine enzyme over the range of conditions tested. Porcine trypsin was less susceptible to denaturation at low pH or high temperature than was ovine trypsin. Porcine and ovine trypsin produced seven identically sized fragments from auto-catalytic hydrolysis. Proposed regions of identity between ovine and porcine trypsins were I(54)-K(77), L(98)-R(107), S(134)-K(178) and N(209)-K(116). Hydrolysis of beta-lactoglobulin, egg white lysozyme or casein by ovine or porcine trypsin yielded virtually identical patterns of fragments although the rate at which fragments were produced, in the case of beta-lactoglobulin, differed between the two enzymes. On balance the two enzymes appear to be functionally identical in their action.

Analysis of reduced glutathione using a reaction with 2,4'-dichloro-1-(naphthyl-4-ethoxy)-s-triazine (EDTN)

A fluorescent adduct was formed between 2,4′-dichloro-l-(naphthyl-4-ethoxy)-s-triazine (EDTN) and reduced glutathione in a reaction at 37 °C and pH 9.2. This reaction was used as the basis of an assay for reduced glutathione. The fluorescence was examined at an excitation wavelength of 319 nm and an emission wavelength of 425 nm after extraction of residual unreacted EDTN with methylene dichloride and subsequent dilution of the aqueous phase with ethanol containing 0.01 percent Triton X-100. The reaction rate was low at pH 7 but was accelerated by addition of preparations containing the enzyme glutathione-S-transferase. The adduct gave a discrete peak using isocratic elution with HPLC on a Nova-pak C18 3 μm reverse phase column and a solvent system of methanol: 0.1 M phosphate buffer pH 6.3 (40∶60). An analytical concentration range of 24 to 240 μM reduced glutathione was obtained with an ultraviolet detection system but the concentration range was 7.5 to 75 μM when a fluorescence detection system was used. Adducts of other mercapturic acid pathway thiol compounds were not formed at 37 °C under the conditions used and hence did not interfere in the assay. They were formed by heating EDTN and the respective thiol compound at 60 °C for 30 min and they clearly separated from the reduced glutathione compound on HPLC analysis. A strong reaction was observed with digitonin while solutions of tyrosine, at 10 mM concentration, also reacted but these reactants are unlikely to interfere with reduced glutathione analysis in biological systems. When adduct formation was used to estimate reduced glutathione concentrations in some mammalian and plant tissues the reaction using 2,4′-dichloro-l-(naphthyl-4-ethoxy)-s-triazine and HPLC separation gave the same results as ano-phthaldialdehyde assay for liver and muscle but the HPLC method gave slightly lower values for other mammalian and plant tissues. The differences were attributed to other material in the tissue extracts which was fluorescing at the same wavelengths as the reduced glutathione adduct.