Glutathione S-transferase from the diamond back moth (Plutella xylostella linnaeus)

Glutathione S-transferase was present in all the developmental stages of Plutella xylostella. The enzyme levels increased rapidly and reached a maximum at the pupal stage and then declined towards adulthood. The resistant strain was found to contain between 3–4 times more glutathione S-transferase than the susceptible strain. However, the enzyme from both the strains had similar K_m values for GSH and DNCB, respectively. The crude enzyme had an optimum pH of 8.3 and its activity was affected by buffer molarity. The enzyme was completely inactivated on dialysis and the stability of the enzyme in the crude supernatant could be maintained in the presence of 1 mM concentrations of either GSH, 2-mercaptoethanol or cysteine. Metal ions had no effect on the stability of the enzyme. Data from Arrhenius plots, column chromatographic techniques and isoelectric focusing suggested the presence of a single form of the enzyme. The enzyme had an isoelectric point of 9.26 and a molecular weight of 36,400.     

Purification and properties of dehydroascorbate reductase from spinach leaves

Dehydroascorbate reductase was detected in the leaves of several plants and has been partially purified from spinach leaves. The enzyme has a MW of ca 25 000, a pH optimum of 7.5, a K_m for glutathione (GSH) of 4.43 ± 0.4 mM and a K_m for dehydroascorbate of 0.34 ± 0.05 mM. High concentrations of dehydroascorbate inhibit the enzyme. Cysteine cannot replace GSH as a donor. The purified dehydroascorbate reductase is extremely unstable and also inhibited by compounds which react with thiol groups. Dehydroascorbate does not protect the enzyme against such inhibition. GSH reduces dehydroascorbate non-enzymically at alkaline pH values.     

The purification and properties of a thiaminase I enzyme from nardoo (Marsilea drummondii)

Fronds of the fern nardoo (Marsilea drummondii) contain a thiaminase I enzyme at very high levels of activity. Highest levels of enzyme activity were found in vigorously growing plant material. The thiaminase I has been purified to a final sp act value of 2.07 μkat/mg protein at 30° and pH 6.5. It was shown to have similar properties to thiaminase I enzymes purified from bracken fern, rock fern and freshwater mussels. These enzymes have MW values in the range 93 000–115 000, energies of activation of 14 000 cal mol, pH optima of 8–9 and are quite stable in the pH range 3 to 12 and to extended incubation at 55°. The temperature for 50 % denaturation is 60–65°. p-CMB, mersalyl acid and HgCl₂ (10^t-6 M) are potent inhibitors, but monoiodacetic acid (10^?4 M) has no effect. A wide range of heterocyclic bases, sulphydryl compounds, and amines, including the non-aromatic amines 6-aminohexanoic acid and ethanolamine, act as co-substrates in the thiaminase I reaction; however, their effectiveness is dependent on both their degrees of basicity and to some extent, their stereochemistry. When the co-substrate activity of a range of substituted anilines were compared, no correlation was found between the degree to which the base activates the reaction and the pK_b (or Hammett's sigma constant) of the base.     

Purification and properties of alcohol oxidase from Tanacetum vulgare

Alcohol oxidase (alcohol: O₂ oxidoreductase) from leaves of Tanacetum vulgare has been purified 5150-fold to homogeneity on disc electrophoresis and gel electrofocussing. The enzyme which is probably flavoprotein, has molecular weight 180 000 daltons and is comprised of two sub-units of 94 000 and 75 000 daltons. It is active over a broad range (pH 5–9) and best accepts primary aliphatic alcohols with 6 to 10 carbons, especially those with a 2-ene group. K_m values for hex-trans-2-ene-1-ol, geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol) and n-octanol were 0.19, 1.56 and 0.49 mM respectively. The significance of the enzyme in the formation of leaf aldehyde (hex-trans-2-ene-1-al) and in terpene metabolism is discussed.     

Properties of potato α-glucosidase

Potato tuber α-glucosidase has an isoelectric point of 4.7 and an apparent MW of 120 000. The enzyme has a neutral pH optimum (pH 6.5–7.0) and a K_m of 0.21 mM for p-nitrophenol-α-D-glucoside at pH 6.8 and 30°. Maltose and higher maltosaccharides are also substrates. The enzyme exhibits transglucosidase activity.     

Effects of some metal ions on rainbow trout erythrocytes glutathione S-transferase enzyme: an in vitro study

Glutathione S-transferase enzyme (GST) (EC 2.5.1.18) was purified from rainbow trout erythrocytes, and some characteristics of the enzyme and effects of some metal ions on enzyme activity were investigated. For this purpose, erythrocyte glutathione S-transferase enzyme which has 16.54 EU/mg protein specific activities was purified 11,026-fold by glutathione-agarose affinity chromatography with a yield of 59%. Temperature was kept under control (+4°C) during purification. Enzyme purification was checked by performing SDS-PAGE. Optimal pH, stable pH, optimal temperature, and K_M and V_max values for GSH and 1-chloro-2, 4-dinitrobenzene (CDNB) were also determined for the enzyme. In addition, IC₅₀ values, K_i constants and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as Ag⁺; Cd²⁺, Cr²⁺ and Mg²⁺.     

Glutathione transferases in primary rat hepatomas: the isolation of a form with GSH peroxidase activity

A previously uncharacterized glutathione (GSH) transferase which is not apparent in normal liver, accounts for at least 25% of the soluble GSH transferase content of primary hepatomas induced by feeding N,N-dimethyl-4-aminoazobenzene. This enzyme is readily isolated, has an isoelectric point of 6.8, is composed of two identical subunits of apparent M_r 26 000 and has GSH transferase activity towards a number of substrates including benzo(a)pyrene-7,8-diol-9,10-oxide. It is unusual in that it has GSH peroxidase activity towards fatty acid hydroperoxides but not towards the model substrates, cumene hydroperoxide and t-butyl hydroperoxide. It has been shown by tryptic peptide analysis to be distinct from GSH transferases composed of subunits 1, 2, 3,4 or 6 and has been designated GSH transferase 7-7.     

Purification and properties of chalcone isomerase from cell suspension cultures of Phaseolus vulgaris

Chalcone isomerase (EC 5.5.1.6) from cell suspension cultures of Phaseolus vulgaris has been purified about 400-fold. The molecular weight, as estimated by gel-filtration and SDS-polyacrylamide gel electrophoresis, is approx. 28 000. No isoenzymic forms are observed. The enzyme, which appears to require no cofactors, catalyses the isomerisation of both 6′-hydroxy and 6′-deoxy chalcones to the corresponding flavones. Likewise, a range of both 5-hydroxy and 5-deoxy flavonoids and isoflavonoids act as competitive inhibitors. The most potent inhibitors include the naturally occurring antimicrobial comcpounds kievitone (K_i 9.2 μM) and coumestrol (K_i 2.5 μM). The kinetics of the isomerisation of 2′,4,4′-trihydroxychalcone to the flavanone liquiritigenin have been investigated at a range of pH values. The pH optimum was around 8.0 and K_m changed with pH in a manner consistent with control by groups which ionise with pK_a values of 7.05 and 8.7 respectively. At pH 8.0, the energy of activation was 17.56 kJ/mol in the range 25–40°C. The role of the enzyme in the induced accumulation of flavonoid/isoflavonoid derivatives inthe Frech bean in discussed.     

Effect of cadmium on antioxidative enzymes,glutathione content,and glutathionylation in tall fescue

The aim of this work was to assess the effect of different Cd2+concentrations on some antioxidant enzymes in Festuca arundinacea. Increased activities of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione S-transferase, and glutathione reductase were ascertained in response to low Cd²⁺ concentrations (0–20 μM), whereas the enzyme activities were less increased or decreased at a higher Cd²⁺ dosage (50 μM) and a longer exposure. The content of reduced glutathione (GSH) decreased significantly with increasing Cd²⁺ concentrations, whereas the content of oxidized glutathione (GSSG) increased proportionally to the amount of Cd²⁺ applied. Further experiments, performed by incubating the enzyme extracts with oxidized glutathione, evidenced that the addition of GSSG to the incubation mixtures caused significant decreases of some enzymatic activities. Finally, the effect of glutathione S-transferase, FaGST I, extracted from fescue seedlings and purified till homogeneity, on these enzyme activities was investigated. It was found that FaGST I enhanced the decreased enzymatic activities caused by GSSG.     

Isolation and some properties of extracellular d-glucosyltransferases and d-fructosyltransferases from Streptococcus mutans serotypes c, e, and f

Extracellular d-glucosyltransferases (GTase) and d-fructosyltransferases (FTase) were isolated from Streptococcus mutans IB (serotype c), B14 (e), and OMZ175 (f) by chromatofocusing, followed by hydroxyapatite column chromatography. The GTases isolated from serotypes c, e, and f are basic proteins (pI 7.4). The serotype c and e enzymes have two protein components having M_r 173 000 and 158 000 and the enzyme of the serotype f one component having M_r 156 000. The GTases of all the serotypes showed a K_m value for sucrose of 10–14mm and an optimum pH 5.5–6.0 for enzyme activity, and their activities were enhanced by the presence of primer Dextran T10. The α-d-glucans synthesized by the purified GTases are water soluble and primarily consist of (1→6)-α-d-glucosidic linkage (41–66 mol/100 mol) and α-d-(1→3,6)-branch linkage (6–20 mol/100 mol), but significant proportions of α-d-(1→3), α-d-(1→4), and α-d-(1→3,4) linkages (11, 6, and 14 mol/100 mol, respectively) were detected in the serotype c α-d-glucan. The isolated FTases of the serotypes c, e, and f are acidic enzymes (pI 4.6) and consist of two components having M_r 84 000 and 76 000 for the serotype c enzyme, and 106 000 and 84 000 for the serotypes e and f enzymes, respectively. The K_m value for sucrose was 6, 10, and 17mm for the serotypes c, e, and f enzymes, respectively, and the optimum pH of enzymic activity 5.5–6.0. Reactivity with Concanavalin A, susceptibility to acid hydrolysis, and paper chromatography of the hydrolyzates suggested that the water-soluble β-d-fructans synthesized by the purified FTases were of the inulin-type and had chemical structures somewhat different among the serotypes.     

Some properties of glutamine synthetase and glutamate synthase from Derxia gummosa

The incorporation of ¹⁵N into washed cells of Derxia gummosa from labelled-(NH₄)₂SO₄ and -KNO₃ respectively was inhibited by both L-methionine-DL-sulphoximine and azaserine. Glutamine synthetase purified to homogeneity from this bacterium had a molecular weight of 708 000 and was composed of 12 similar subunits each of 59 000. The enzyme assayed by γ-glutamyltransferase method had K_m values for L-glutamine and hydroxylamine of 12.5 and 1.2 mM, respectively. Optimal pH values for adenylylated and deadenylylated forms were pH 7.0 and pH 8.0, respectively. The adenylylated enzyme was deadenylylated by treatment with snake venom phosphodiesterase. The inhibitions by both glutamate and ammonia were competitive. The activity was markedly inhibited by L-methionine-DL-sulphoximine, alanine, glycine and serine and to a lesser extent by aspartate, phenylalanine and lysine. Various tri-, di- and mono-phosphate nucleotides, organic acids (pyruvate, oxalate and oxaloacetate) were also inhibitory. Glutamate synthase purified 167-fold had specific requirements for NADH, L-glutamine and 2-ketoglutarate. The K_m values for NADH, glutamine and 2-ketoglutarate were 9.6, 270 and 24 μM respectively. Optimal pH range was 7.2–8.2. The enzyme was inhibited by azaserine, methionine, aspartate, AMP, ADP and ATP.     

Immobilization of Bacillus subtilis α-amylase on zirconia-coated alkylamine glass with glutaraldehyde

Bacillus subtilis α-amylase (EC 3.2.1.1) has been immobilized on zirconia-coated alkylamine glass by using the process of glutaraldehyde coupling. The immobilized enzyme preparation exhibited 52% of the initial enzyme activity and a conjugation yield of 28 mg/g support. The K_m value of the immobilized α-amylase was decreased by immobilization while V_max was unaltered. E_a of the enzyme was decreased upon conjugation. The soluble enzyme was optimally active at pH 5.6 while the immobilized enzyme exhibited optimal activity in the pH range 5.4–6.2. The alkylamine-immobilized enzyme has also been characterized through its isoelectric point. The industrial importance of this work is discussed.     

Cadmium(II) complex formation with glutathione

Complex formation between heavy metal ions and glutathione (GSH) is considered as the initial step in many detoxification processes in living organisms. In this study the structure and coordination between the cadmium(II) ion and GSH were investigated in aqueous solutions (pH 7.5 and 11.0) and in the solid state, using a combination of spectroscopic techniques. The similarity of the Cd K-edge and L₃-edge X-ray absorption spectra of the solid compound [Cd(GS)(GSH)]ClO₄·3H₂O, precipitating at pH 3.0, with the previously studied cysteine compound {Cd(HCys)₂·H₂O}₂·H₃O⁺·ClO₄ ^? corresponds to Cd(S–GS)₃O (dominating) and Cd(S–GS)₄ four-coordination within oligomeric complexes with mean bond distances of 2.51 ± 0.02 Å for Cd–S and 2.24 ± 0.04 Å for Cd–O. For cadmium(II) solutions (C _Cd(II) ~ 0.05 M) at pH 7.5 with moderate excess of GSH (C _GSH/C _Cd(II) = 3.0–5.0), a mix of Cd(S–GS)₃O (dominating) and Cd(S–GS)₄ species is consistent with the broad ¹¹³Cd NMR resonances in the range 632–658 ppm. In alkaline solutions (pH 11.0 and C _GSH/C _Cd(II) = 2.0 or 3.0), two distinct peaks at 322 and 674 ppm are obtained. The first peak indicates six-coordinated mononuclear and dinuclear complexes with CdS₂N₂(N/O)₂ and CdSN₃O₂ coordination in fast exchange, whereas the second corresponds to Cd(S–GS)₄ sites. At high ligand excess the tetrathiolate complex, Cd(S–GS)₄, characterized by a sharp δ(¹¹³Cd) NMR signal at 677 ppm, predominates. The average Cd–S distance, obtained from the X-ray absorption spectra, varied within a narrow range, 2.49–2.53 Å, for all solutions (pH 7.5 and 11.0) regardless of the coordination geometry.     

Isolation, Purification, and Some Properties of Penicillium chrysogenum Tannase

Tannase isolated from Penicillium chrysogenum was purified 24-fold with 18.5% recovery after ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. Optimum enzyme activity was recorded at pH 5.0 to 6.0 and at 30 to 40°C. The enzyme was stable up to 30°C and within the pH range of 4.0 to 6.5. The K_m value was found to be 0.48 × 10⁻⁴ M when tannic acid was used as the substrate. Metal salts at 20 mM inhibited the enzyme to different levels.     

Fructose-1,6-diphosphatase from spinach leaf chloroplasts: Purification and heterogeneity

The enzyme fructose- 1,6-diphosphatase (FDPase), involved in the reductive cycle of the pentose phosphate pathway, has been purified from spinach leaves by heating (30 min at 60°), “salting out” with ammonium sulphate (between 30–70% of saturation), filtration through Sephadex G-100 and G-200, fractionation on DEAE-52 cellulose and preparative electrophoresis on polyacrylamide gel. Filtration through DEAE-cellulose led to the isolation of two active fractions (fractions I and II) with very close MWs and isoelectric points. By electrophoresis on acrylamide gel, both fractions gave two active fractions (fractions I_a-I_b and II_a-II_b). The fractions with low electrophoretic migration rate—I_b and II_b—are stable in acid and neutral pH, have a MW between 90 000 and 110 000 and constitute the native form of the photosynthetic enzyme. The fractions of faster migration rate—I_a and II_a-originate from the corresponding fractions I_b and II_b under alkaline conditions, show half the MW of the respective fractions, and behave as subunits of the original dimer form. Measured by electrofocusing, the four active fractions have isoclectric points in the range 4·10–4.30.     

Dissecting the Catalytic Mechanism of Trypanosoma brucei Trypanothione Synthetase by Kinetic Analysis and Computational Modeling

In pathogenic trypanosomes, trypanothione synthetase (TryS) catalyzes the synthesis of both glutathionylspermidine (Gsp) and trypanothione (bis(glutathionyl)spermidine (T(SH)₂)). Here we present a thorough kinetic analysis of Trypanosoma brucei TryS in a newly developed phosphate buffer system at pH 7.0 and 37 °C, mimicking the physiological environment of the enzyme in the cytosol of bloodstream parasites. Under these conditions, TryS displays K_m values for GSH, ATP, spermidine, and Gsp of 34, 18, 687, and 32 μm, respectively, as well as K_i values for GSH and T(SH)₂ of 1 mm and 360 μm, respectively. As Gsp hydrolysis has a K_m value of 5.6 mm, the in vivo amidase activity is probably negligible. To obtain deeper insight in the molecular mechanism of TryS, we have formulated alternative kinetic models, with elementary reaction steps represented by linear kinetic equations. The model parameters were fitted to the extensive matrix of steady-state data obtained for different substrate/product combinations under the in vivo-like conditions. The best model describes the full kinetic profile and is able to predict time course data that were not used for fitting. This system''s biology approach to enzyme kinetics led us to conclude that (i) TryS follows a ter-reactant mechanism, (ii) the intermediate Gsp dissociates from the enzyme between the two catalytic steps, and (iii) T(SH)₂ inhibits the enzyme by remaining bound at its product site and, as does the inhibitory GSH, by binding to the activated enzyme complex. The newly detected concerted substrate and product inhibition suggests that TryS activity is tightly regulated.     

Characterization of an alkaline elastase from alkalophilic Bacillus Ya-B

The alkaline elastase produced by alkalophilic Bacillus Ya-B was a new type of proteinase which had a very high optimum pH and high elastolytic activity. It also had a high hydrolyzing activity against keratin and collagen. The molecular weight was determined to be 23 700 and 25 000 by ultracentrifugation analysis and SDS-polycrylamide gel electrophoresis, respectively. The isoelectric point was 10.6. The optimum reaction temperature was 60°C. Like many alkaline proteinases, this enzyme required Ca²⁺ for stability. The optimum reaction pH was 11.75 toward casein and elastin-orcein. The K_cat/K_m values of the enzyme to synthetic substrates were constant from pH 8.5 up to 12.75. The enzyme was stable in the pH range 5.0–10.0. The enzyme was inhibited by alkaline proteinase inhibitors Streptomyces subtilisin inhibitor and microbial alkaline proteinase inhibitor, but not by elastatinal or the metalloproteinase inhibitor metalloproteinase inhibitor. Sodium chloride inhibited the elastolytic activity but not the caseinolytic activity at a concentration below 0.2 M. The inhibitory effect of sodium chloride to elastolytic activity was much more prominent at pH 9.0 than at pH 11.5. More than 50% of the enzyme bound onto elastin in the pH range below the isoelectric point of this enzyme. The amino-terminal sequence of the enzyme was determined, and compared with those of subtilisin BPN′ and subtilisin Carlsberg. Extensive sequence homology was noted among these three enzymes.     

Biosynthesis of dimethyl selenide from sodium selenite in rat liver and kidney cell-free systems

A pathway for the synthesis of dimethyl seledine from sodium selenite was studied in rat liver and kidney fractions under anaerobic conditions in the presence of GSH, a NADPH-generating system, and S-adenosylmethionine. Chromatography of liver or kidney soluble fraction on Sephadex G-75 yielded a Fraction C (30 000 molecular weight) which synthesized dimethyl selenide, but at a low rate. Addition of proteins eluting at the void volume (Fraction A) to Fraction C restored full activity. Fractionation of Fraction A on DEAE-cellulose revealed that its ability to stimulate Fraction C was associated with two fractions, one containing glutathione reductase and the other a NADPH-dependent disulfide reductase. It was concluded that Fraction C contains a methyltransferase acting on small amounts of hydrogen selenide produced non-enzymically by the reaction of selenite with GSH, and that stimulation by Fraction A results partly from the NADPH-linked formation of hydrogen selenide catalyzed by glutathione reductase present in Fraction A. Washed liver microsomal fraction incubated with selenite plus 20 mM GSH also synthesized dimethyl selenide, but addition of soluble fraction stimulated activity. A synergistic effect was obtained when liver soluble fraction was added to microsomal fraction in the presence of a physiological level of GSH (2 mM), whereas at 20 mM GSH the effect was merely additive. The microsomal component of the liver system was labile, had maximal activity around pH 7.5, and was exceedingly sensitive to NaAsO₂ (93% inhibition by 10^?6 M arsenite in the presence of a 20 000-fold excess of GSH). The microsomal activity apparently results from a Se-methyltransferase, possibly a dithiol protein, that methylates hydrogen selenide produced enzymically by the soluble fraction or non-enzymically when a sufficiently high concentration of GSH is used.     

Purification of rabbit kidney cytokinase and a comparison of its properties with human urokinase

1. The cytokinase (tissue activator of plasminogen) content of several mammalian tissues was evaluated by a quantitative casein hydrolysis method. 2. An alkaline (pH10·5) extraction of cytokinase from rabbit kidney lysosome–microsome fraction, followed by chromatography on DEAE-cellulose at pH7·6 with stepwise or linear increase in concentration of phosphate buffer, gave an 86-fold purification of the enzyme. The purified material was non-proteolytic against casein and heated fibrin and was freeze-dried without significant loss of activity or solubility. 3. Cytokinase is a protein with E^0·1%_1cm.=0·87 at 280mμ, and does not possess sufficient hexose or sialic acid to be classified as a glycoprotein. It has S_20,w 2·9–3·1s and molecular weight 50000 when measured on a calibrated Sephadex G-100 column. It has an isoelectric point between pH8 and pH9, and is maximally active and stable at pH8·5. It is inactivated by heat at 78°. 4. Cytokinase and human urokinase have the same K_m value and are inhibited in a partially competitive manner by -aminohexanoic acid and aminomethylcyclohexanecarboxylic acid. They are also inhibited by cysteine and arginine, but are unaffected by iodoacetamide and p-chloromercuribenzoate. 5. On the basis of this and other evidence it is suggested that rabbit kidney cytokinase and human urokinase are similar, if not identical, enzymes.     

Isolation and characterization of the polyphenoloxidase from senescent leaves of black poplar

The polyphenoloxidase (PPO) from black poplar senescent leaves has been purified to almost complete homogeneity by a combination of ammonium sulphate precipitation, Sephadex G75 filtration and DEAE-cellulose chromatography. The purified enzyme has a MW of 60 000 and is probably a Cu⁺ enzyme. Peroxidase (PO) activity co-purifies with PPO and has the same MW as it. The two enzymes differ in pH optimum and in response to the effect of ionic strength. Natural phenols are either substrates, inhibitors or activators of black poplar PPO. This enzyme is an o-diphenoloxidase which binds substrates with K_m in the millimolar range. With caffeic and chlorogenic acids inhibition by excess substrate is observed. Benzoic acid phenols and cinnamic acid phenols are either competitive or non-competitive inhibitors of PPO. Hydroquinone is a highly potent non-competitive inhibitor of the enzyme (K_i  90 μM). Ferulic acid is a potent activator of the PPO-catalysed oxidation of catechol (K_a  0.34 mM, ν_sat/ν_o  7.7).