Purification and characterization of a nylon-degrading enzyme

A nylon-degrading enzyme found in the extracellular medium of a ligninolytic culture of the white rot fungus strain IZU-154 was purified by ion-exchange chromatography, gel filtration chromatography, and hydrophobic chromatography. The characteristics of the purified protein (i.e., molecular weight, absorption spectrum, and requirements for 2,6-dimethoxyphenol oxidation) were identical to those of manganese peroxidase, which was previously characterized as a key enzyme in the ligninolytic systems of many white rot fungi, and this result led us to conclude that nylon degradation is catalyzed by manganese peroxidase. However, the reaction mechanism for nylon degradation differed significantly from the reaction mechanism reported for manganese peroxidase. The nylon-degrading activity did not depend on exogenous H2O2 but nevertheless was inhibited by catalase, and superoxide dismutase inhibited the nylon-degrading activity strongly. These features are identical to those of the peroxidase-oxidase reaction catalyzed by horseradish peroxidase. In addition, alpha-hydroxy acids which are known to accelerate the manganese peroxidase reaction inhibited the nylon-degrading activity strongly. Degradation of nylon-6 fiber was also investigated. Drastic and regular erosion in the nylon surface was observed, suggesting that nylon is degraded to soluble oligomers and that nylon is degraded selectively.     

Biochemical characterization of basilase, a fibrinolytic enzyme from Crotalus basiliscus basiliscus

Snake venoms, especially from the Crotalidae family, contain a variety of enzymes that prevent blood coagulation by virtue of their fibrinolytic enzymes. Nineteen snake venoms were screened for fibrinolytic activity and the highest activity was found in the venom of Crotalus basiliscus basiliscus venom. The active principle, basilase, was isolated, purified, and found to have fibrinolytic and fibrinogenolytic activity. It had a molecular weight of 22,000 and 1 mol of zinc per mole of protein associated with it. The proteolytic activity of the enzyme against dimethyl casein was inhibited by ethylenediaminetetraacetic acid and alpha 2-macroglobulin. It did not inactivate alpha 2-macroglobulin. Basilase did not have any of the following activities: thrombin-like, factor X-like, protein C activating, or urokinase-like. It caused neither hemorrhage nor platelet aggregation. In spite of its proteolytic activity, basilase did not hydrolyze the membranes of platelets. Basilase had 24% alpha-helix, 31% beta-sheet, 25% turns, and 20% unordered structure, as determined by Fourier Transform Infrared spectroscopy. Basilase is an enzyme that hydrolyzes fibrin directly without activation of plasminogen.     

Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme

A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising Q-Sepharose, hydroxyapatite, and phenyl-Superose chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min-1 (mg protein)-1. The dismutase obeyed Michaelis-Menten kinetics. The Vmax and Km calculated for chlorite were 2,200 U (mg protein)-1 and 170 microM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole. Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase displayed maxima for activity at pH 6.0 and 30 degrees C.     

日本刺沙蚕纤溶酶的酶学性质及其分类

目的:探讨日本刺沙蚕纤溶酶(NJF)的最适反应温度和最适反应pH 值以及金属阳离子和蛋白酶抑制剂对酶活性的影响,确定NJF的酶学分类.方法:采用偶氮酪蛋白(azocasein)水解法测定NJF的最适温度及最适pH 值;利用10种金属阳离子(Na+、K+、Cu2+、Ca2+、Zn2+、Co2+、Fe2+、Fe3+、Al3+和Hg2+)确定金属离子对NJF酶活性的影响;采用9种蛋白酶抑制剂(PMSF、EDTA、EGTA、β-巯基乙醇、苯甲脒、碘乙酸、抑肽酶、胃酶抑素A和大豆胰蛋白酶抑制剂)确定NJF的蛋白酶分类.结果:NJF具有较宽的热稳定性及pH稳定性范围,在40～80℃及pH7～11范围内有较好的稳定性,NJF酶最适温度为60℃,最适pH值为9;Hg2+能够强烈地抑制NJF的酶活性,Cu2+具有较弱的抑制作用,而其他金属阳离子对酶活性无明显抑制作用;NJF的酶活性能被典型的丝氨酸蛋白酶抑制剂PMSF完全抑制.结论:NJF是一种典型的丝氨酸蛋白酶.     

Purification and characterization of a new enzyme, N-alkylglycine oxidase from Cladosporium sp. G-10

A new enzyme, N-alkylglycine oxidase, was isolated from a soil mold, Cladosporium sp. G-10. This protein, which was purified to near homogeneity by ammonium sulfate precipitation followed by successive column chromatography on phenyl-Sepharose, DEAE-Sepharose and Sephadex G-200, was a single polypeptide with a molecular mass of 52,000. In the presence of O2 and H2O, this enzyme acted on some N-alkylglycine derivatives, such as N epsilon-carboxymethyllysine, N-carboxymethyl-6-aminocaproic acid, sarcosine and N-ethylglycine, and produced corresponding N-alkylamine, glyoxylic acid and H2O2. This enzyme had optimum activity at 30 degrees C, pH 8-10, and was most inhibited by ZnSO4, pCMB, iodoacetic acid, and SDS.     

Purification and characterization of the acid soluble 26-kDa polypeptide from soybean seeds

Whey proteins from soybean seeds of Japanese varieties were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Among 11 varieties of soybean, three green and one black soybeans lacked a 26-kDa band that was found in all yellow soybeans. In this paper, the 26-kDa protein was named AS26k (acid soluble 26-kDa protein) temporarily. The AS26k protein was purified from Glycine max cv. Nattosyoryu, which is yellow soybean, through four purification steps: 30-35% saturated ammonium sulfate fractionation, ion exchange chromatography on S Sepharose Fast Flow, gel filtration on Sephadex G-100, and hydrophobic chromatography on phenyl Sepharose CL-4B. Purified AS26k was cleaved with V8 proteinase from Staphylococcus aureus or CNBr. The cleaved polypeptide contained two typical dehydrin motif sequences: DEYGNPV and (M)DKIKEKLPG, and a 19 amino acids sequence similar to a pea dehydrin. Native AS26k had a molecular mass of 32 kDa on gel filtration and a pl of 7.2 on two-dimensional PAGE. Similarly to other dehydrins and late embryogenesis abundant (LEA) proteins, AS26k was rich in hydrophilic amino acids, and highly heat stable. These results showed that AS26k was a dehydrin, a group II LEA protein in soybean seeds.     

Purification and characterization of a cytosolic thyroid-hormone-binding protein (CTBP) in Xenopus liver

A variety of cytosolic thyroid-hormone-binding proteins with different characteristics have previously been reported. Here, we first describe the thyroid-hormone-binding characteristics of adult Xenopus liver cytosol, then a novel procedure for purifying cytosolic thyroid-hormone-binding protein (CTBP) from Xenopus liver (xCTBP). The procedure consists of combining preparative isoelectrofocusing, FPLC cation-exchange chromatography, HPLC hydrophobic-interaction chromatography and ultraviolet light cross-linking of 125I-labeled 3,3'5-triiodo-L-thyronine (T3). The isolated xCTBP thus prepared retained all the characteristics of the major thyroid- hormone-(TH)-binding component of the unfractionated cytosol. It is a monomeric protein of approximately 59 kDa with an isoelectric point of 7.0 +/- 0.1, binds T3 with a higher affinity than its analogs with a Kd of approximately 9 nM, and is sensitive to sulfhydryl agents but not to NADPH. In several respects, xCTBP differs from most CTBP-like preparations from other sources described hitherto. Microsequencing of a 23-amino-acid peptide generated from xCTBP by cyanogen bromide digestion revealed 92-100% identity of a 23-amino-acid sequence of several mammalian (amino acids 236-258) and avian (amino acids 245-267) cytosolic aldehyde dehydrogenases (ALDH); xCTBP also exhibited significant similarity of amino acid composition with rat ALDH. This novel finding of sequence identity between a CTBP and ALDH, and the diversity of CTBPs from different sources, suggest that a variety of cytosolic proteins, depending on the species and tissue, can function as thyroid-hormone-binding proteins.     

Purification and characterization of papaya glutamine cyclotransferase, a plant enzyme highly resistant to chemical, acid and thermal denaturation

Papaya glutamine cyclotransferase (PQC), present in the laticiferous cells of the tropical species Carica papaya, was purified near to homogeneity. Starting from the soluble fraction of the collected plant latex, a combination of ion-exchange chromatography on SP-Sepharose Fast Flow, hydrophobic interaction chromatography on Fractogel TSK Butyl-650 and affinity chromatography on immobilized trypsin provided a purification factor of 279 with an overall yield of 80%. In the course of the purification procedure, the two solvent accessible thiol functions located on the hydrophobic surface of the enzyme were converted into their S-methylthioderivatives. Papaya QC, a glycoprotein with a molecular mass of 33000 Da, contains a unique and highly basic polypeptide chain devoid of disulfide bridges as well as of covalently attached phosphate groups. Its absorption spectrum is dominated by the chromophores tyrosine which, nonetheless, do not contribute to the fluorescence emission of the plant enzyme. With a lambdamax of emission at 338 nm and a moderate susceptibility to be quenched by acrylamide, most of the tryptophyl residues of papaya QC appear to be sterically shielded by surrounding protein atoms. Fluorescence can thus be used to monitor unfolding of this enzyme. Preliminary experiments show that papaya QC is exceptionally resistant to chemical (guanidinium hydrochloride), acid and thermal denaturation. At first sight also, this enzyme exhibits high resistance to proteolysis by the papaya cysteine proteinases, yet present in great excess (around 100 mol of proteinases per mol of PQC) in the plant latex. Altogether, these results awaken much curiosity and interest to further investigate how the structure of this plant enzyme is specified.     

Purification and characterization of a novel peptidase (IImes) from mesquite (Prosopis velutina) pollen

Although the mesquite plant (Prosopis velutina) is not as widely distributed as some other allergenic species, its pollen can induce serious pollinosis in areas where it is localized. We previously isolated and characterized a peptidase from mesquite pollen with trypsin-like specificity (peptidase Imes) (Matheson, N., Schmidt, J., and Travis, J. (1995) Am. J. Respir. Cell Mol. Biol. 12, 441-448). Now we have characterized a second enzyme with specificity for hydrophobic residues (mesquite pollen peptidase IImes). This enzyme has a molecular mass near 92 kDa and activity that was not affected by reducing or chelating agents but was inhibited by specific synthetic serine proteinase inhibitors and the aminopeptidase inhibitor bestatin. However, it was not inhibited by human plasma proteinase inhibitors, nor did it inactivate any of those tested. The enzyme possessed amidolytic activity against p-nitroanilide substrates most effectively after alanine residues and also displayed aminopeptidase activity against non-p-nitroanilide peptides with a preference for phenylalanine. This specificity for hydrophobic amino acid residues was corroborated by inhibition studies with chloromethyl ketone and organophosphonate inhibitors. More interesting from a physiological point of view is that the bioactive peptides, angiotensins I and II and vasoactive intestinal peptide, were also hydrolyzed rapidly, indicating an ability of peptidase IImes to act also as an oligopeptidase. Because these bioactive peptides play a role in the inflammatory responses in allergic asthma, our data suggest that the purified mesquite pollen peptidase IImes may be involved in the degradation of neuro- and vasoactive peptides during pollen-initiated allergic reactions.     

Purification, properties, and N-terminal amino acid sequence of a kallikrein-like enzyme from the venom of Lachesis muta rhombeata (Bushmaster)

An analytical procedure was developed to measure bromate residues in baked goods using a sequence of clean-up procedures followed by high performance liquid chromatography (HPLC) with a post-column reaction for oxidants. Deionized water was used to extract bromate from bread samples. The extract was treated with a C-18 solid phase extraction column to remove lipids, a cation exchange column with the silver cation to remove chloride, and an ultrafiltration membrane to remove proteins. Further treatment of the extract with the sodium form of a propylsulphonic acid ion exchange column was necessary to remove the silver that leached from the silver column. The method had a detection limit of 3 ng/g in baked goods. Recoveries of bromate from breads ranged from 73 to 86% at a fortified bromate level of 5-100 ng/g. Pullman-type white bread, produced by a sponge and dough method, was prepared in our laboratory for measurement of residual bromate. The dough was scaled in three different weights at different specific volumes (3.8, 4.1, 4.3), and samples of each of the three weights were baked for six different baking times ranging from 24 to 34 min. When bromate at a level of 25 mg/kg was added to flour, no residual bromate was detected in any of the samples, regardless of weight and baking time.     

Purification and characterization of urotensin II from the brain of a teleost (trout, Oncorhynchus mykiss) and an elasmobranch (skate, Raja rhina)

Peptides related to urotensin II have been isolated in pure form from an extract of whole brain of a teleost, the rainbow trout (Oncorhynchus mykiss) and of an elasmobranch, the longnose skate (Raja rhina). The primary structure of the trout peptide [Gly-Gly-Asn-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val] is similar to that of urotensin II peptides isolated from the urophyses of other teleost fish. For example, trout urotensin II contains only one amino acid substitution (Thr4-->Ser) compared with urotensin II beta 1 isolated from the urophysis of the carp. The primary structure of the skate peptide [Asn-Asn-Phe-Ser-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val] is the same as urotensin II isolated from the caudal spinal cord region of the dogfish Scyliorhinus canicula. The data provide chemical evidence to support the conclusion of earlier morphological studies [Yulis, C. R., and Lederis, K. (1988) Gen. Comp. Endocrinol. 70, 301-311) that certain species of fish possess an extensive extraurophyseal distribution of urotensin II-immunoreactive neurons.     

Purification, characterization, and cloning of a eubacterial 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme involved in biosynthesis of terpenoids

The eubacterial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) was purified 3,000-fold from Streptomyces sp. strain CL190 to apparent homogeneity with an overall yield of 2.1%. The purification procedure consisted of (NH4)2SO4 precipitation, heat treatment and anion exchange, hydrophobic interaction, and affinity chromatographies. The molecular mass of the enzyme was estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 100 to 105 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of around 7.2, with apparent Km values of 62 microM for NADPH and 7.7 microM for HMG-CoA. A gene from CL190 responsible for HMG-CoA reductase was cloned by the colony hybridization method with an oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The amino acid sequence of the CL190 HMG-CoA reductase revealed several limited motifs which were highly conserved and common to the eucaryotic and archaebacterial enzymes. These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural conformation and/or catalytic properties of the enzyme.     

Purification and characterization of human liver-specific membrane lipoprotein (LSP)

A liver-specific lipoprotein (LSP) which is present in 105,000 g supernatants of human liver has previously been shown to produce a lesion resembling chronic active hepatitis in rabbits immunized with human liver fractions containing this lipoprotein. In addition, it has been implicated as the principal target antigen involved in the liver cell cytotoxicity exhibited in vitro by lymphocytes from patients with chronic active hepatitis. The organ-specificity and species cross-reactivity of LSP is confirmed. Although very labile, is has been prepared in a stable form by gel filtration of Sepharose 6B in a Tris buffer containing 1 mm disodium EDTA. LSP is also stable in a borate buffer containing EDTA but is unstable in a number of other buffer systems tested. When prepared by this method it contains approximately 2% albumin as the only detectable contaminant. Gel filtration studies on the apoprotein of LSP revealed that it has an apparent mol. wt of between 4 X 10(6) and 20 X 10(6). SDA-polyacrylamide gel electrophoresis indicated that the lipoprotein may be composed of up to thirteen sub-units of different molecular sizes.     

Purification and characterization of an interleukin-1beta-converting enzyme family protease that activates cysteine protease P32 (CPP32)

CPP32, a member of the interleukin-1beta-converting enzyme (ICE) family of cysteine proteases, cleaves poly(ADP-ribose) polymerase and sterol regulatory element binding proteins during apoptosis. CPP32 normally exists in the cytosol as a 32-kDa inactive precursor and only becomes activated when cells are undergoing apoptosis. The activation is a proteolytic event that generates a p20/p11 heterodimer. We report here the identification, purification, and characterization of a hamster CPP32-activating protease (CAP) that cleaves and activates CPP32. The biochemical properties of CAP suggest that it is another member of the ICE family of proteases. Purified CAP consists of two prominent polypeptides of 19 and 13 kDa. Protein sequencing revealed that CAP is derived from the hamster homolog of Mch2alpha, a member of the ICE family recently identified based on the sequence conservation among the ICE family members. CAP activity is inhibited by CrmA, a cowpox virus protein that prevents host cell apoptosis. CAP itself is also activated through proteolytic cleavage. These data are consistent with the idea that the activation of the ICE family of proteases during apoptosis proceeds through a cascade of proteolytic events.     

Purification and characterization of peroxidase from Phellinus igniarius (author''s transl)

A Peroxidase (EC 1.11.1.7) of the basidiomycet Phellinus igniarius was derived from mycel and a medium containing glucose and extract of yeast by using various methods of preparation. The enzyme resists extreme conditions (pH, temperature salt concentration). Its optimum pH for activities is in the acid range. Two isoenzymes were found. The molecular weight, isoelectric point, Michaelis-Menten constant, indolacetic acid oxidase activity and spectral and analytical properties of this peroxidase were determined. It is assumed that the enzyme has an intracellular as well as an extracellular field of activity.     

Purification and characterization of a 16-kDa cysteine proteinase of Gymnophalloides seoi (Gymnophallidae) metacercariae

Gymnophalloides seoi, a new human intestinal trematode transmitted by oysters, is highly prevalent in southwestern coastal areas of Korea. The aim of this preliminary study was to acquire an understanding of the pathogenesis of G. seoi infection, and, to this end, we followed 2 consecutive steps, Sephacryl S-300 HR and CM-Trisacryl M chromatography, to purifiy a 16-kDa proteinase from crude extract of G. seoi metacercariae. Enzyme activities were completely inhibited by L-trans-epoxysuccinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid, cysteine proteinase inhibitors, strongly suggesting that the enzyme is a cysteine proteinase. Activity of the proteinase was maximal at pH 5.0 in 0.1 M of buffer and potentiated in the presence of 5 mM dithiothreitol. The proteinase showed differential abilities to hydrolyze macromolecules and immunoglobulins; it completely degraded extracellular matrix proteins such as collagen and fibronectin during overnight incubation but digested hemoglobin very slowly. The proteinase also cleaved human immunoglobulins IgG2a and sIgA; heavy chains were more susceptible than light chains to its digestive activity. The proteinase showed no antigenicity on both enzyme-linked immunosorbent assay and immunoblots, however. Our results strongly suggest that the cysteine proteinase of G. seoi metacercariae is potentially significant in nutrient uptake and evasion by the worm of the host immune response.     

Purification and characterization of Streptococcus mutans glucosyltransferase (GtfC) expressed in Escherichia coli

Streptococcus mutans constitutively expresses three glucosyltransferases, i.e., GtfB, GtfC, and GtfD; which synthesize glucan polymers from sucrose. To obtain individual GTF without complexing with one another, a purification strategy was developed to recover recombinant GTF expressed from Escherichia coli. The recombinant GtfC was aggregated and associated with the insoluble fraction in E. coli homogenates. GtfC was solublized with the 8M urea, renatured to its biologically active form by serial dialysis against sodium phosphate buffer, and subsequently purified to homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography. The GtfC enzyme preparation was purified 16.3-fold and the molecular weight was estimated to be 140 kDa. GtfC synthesized water insoluble glucan in a primer independent manner and its enzymatic activities could be enhanced by dextran. Purified GtfC had a pH optimum of 6.5, a K(m) of 9.26 mM for sucrose and a pI of 5.5. Distinct from the previous reports, results from this study offers an alternative for the purification of the recombinant GTFs free from any detergent contamination to make it more suitable for utilization in vivo.     

Purification, characterization, sequence determination, and mass spectrometric analysis of a trypsin inhibitor from seeds of the Brazilian tree Dipteryx alata (Leguminosae)

Dipteryx alata trypsin inhibitor (DATI) has been purified and completely sequenced. It showed homology to members of the Bowman-Birk family of inhibitors. The last step of DATI purification by RP-HPLC (narrow-borc C18 column) suggested the existence of some isoforms of the inhibitor due to the presence of a cluster of very close peaks in the chromatogram. By using electrospray ionization mass spectrometry (ESIMS) and laser desorption mass spectrometry (LDIMS), the identification of DATI isoforms was made possible. From the ESIMS data, the following molecular masses were found: 6803.22 +/- 0.92 for isoform a; 6890.94 +/- 0.73 for b; 6977.58 +/- 0.39 for c; 7065.07 +/- 0.67 for d; 7151.42 +/- 0.86 for e; and 7291.70 +/- 0.43 for f. Similar masses were found when using LDIMS. Isoform b was the most abundant and its molecular mass matched the molecular mass of 6893 calculated from the sequence of DATI. The mass differences between a and b, b and c, c and d, and d and e were equal to 87, which corresponds to Ser. Isoform a might not have the N-terminal Ser present in isoform b, while the other additional Ser residues might comprise a row localized in the C- or N-terminal. The appearance of all these isoforms could result from posttranslational N- and C-terminal processing.     

Purification from Bothrops lanceolatus (fer de lance) venom of a fibrino(geno)lytic enzyme with esterolytic activity

Bothrops lanceolatus venom has high caseinolytic, phospholipasic, esterolytic and hemorrhagic activities. In spite of having no coagulant effect on plasma, this venom contains a thrombin-like enzyme. Using gel filtration and ion-exchange chromatographies, we have purified an esterolytic fraction (F-II-1a) from this venom with a protein yield of 4% and a 58% recovery in enzyme activity. SDS-PAGE in the presence of beta-mercaptoethanol showed that the enzyme is a single chain polypeptide with a MW=38,100. Immunodiffusion and immunoelectrophoresis of fraction F-II-1a against serum from horses immunized with B. lanceolatus venom and against rabbit antiserum prepared using fraction F-II-1a both showed a single immunoprecipitin line. The Km and Vmax values for TAME hydrolysis were 0.85 mM and 38.6 micromol/min/mg, respectively. The esterolytic activity was completely inhibited by PMSF (10 mM) but not by EDTA (20 mM). Fraction F-II-1a hydrolyzed the alpha and beta chains of fibrinogen. Degradation of the alpha chain occurred within 10 min while that of the beta-chain was slower. The enzyme had no effect on the gamma-chain even after 4 h of hydrolysis.     

Purification and characterization of hepatic glutathione transferases from an insectivorous marsupial, the brown antechinus (Antechinus stuartii)

1. Five unique glutathione transferase isoenzymes were purified from the hepatic cytosol of an insectivorous marsupial, the brown antechinus. The purified GSTs were characterized by structural and catalytic properties including apparent molecular weight and isoelectric point, specificity towards model substrates, kinetic parameters, sensitivity to inhibitors and cross-reactivity with antisera raised against human GSTs. 2. An alpha class GST, Antechinus GST 1-1, predominated in the hepatic cytosol, representing 71% of the total GST purified. The substrate specificity of Antechinus GST 1-1 was similar to that of other alpha class GSTs, particularly with respect to its high activity with cumene hydroperoxide. The mu class was represented by three GST isoenzymes, Antechinus GST 3-3, GST 3-4 and GST 4-4. These isoenzymes represented 8, 2 and 10% of the total GST purified respectively. A single GST, Antechinus GST 22, belonged to the pi class of GSTs and represented 12% of the total GST purified. The hepatic GST isoenzyme ratio (by class) observed in the brown antechinus was more similar to that observed in the human than in rat. 3. A previous study investigating a herbivorous marsupial, the brushtail possum (Trichosurus vulpecula) also identified a predominant hepatic GST belonging to the alpha class and displaying peroxidase activity. The evolutionary conservation of a similar predominant GST isoenzyme in these marsupials suggests that they play an important role in the detoxication metabolism of these unique mammals.