Purification and properties of glutathione S-transferases from larvae of Wiseana cervinata.

The glutathione S-transferases from the porina moth, Wiseanna cervinata, were purified by affinity chromatography, cation-exchange chromatography and preparative isoelectrofocusing. The major transferase (IV) was purified to homogeneity by a factor of 530-fold with a yield of 83%. Other transferases present were purified to a smaller degree (approx. 50-fold) to a stage of near-homogeneity. The transferases examined all had Mr values about 45 000-50 000. They appeared to be homodimers of either of two types of subunit, of Mr 22 800 and 24 600. Enzymes consisting of the different types of subunit were not immunologically cross-reactive. The major enzyme fractions separated by cation-exchange chromatography were both active with 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, ethacrynic acid and iodomethane, but were inactive with 4-nitropyridine N-oxide, 1,2-epoxy-3-(p-nitrophenoxy)propane, bromosulphophthalein and p-nitrobenzyl chloride. The kinetics of the enzyme-catalysed reaction with enzyme IV were non-Michaelean with respect to both substrates. Both products were inhibitory. The results appear to be compatible with a random steady-state mechanism. It is concluded that these enzymes are very similar, in their physical and chemical constitution, in their catalytic properties and in their relationships with each other, to those enzymes that have been isolated from vertebrate organisms.     

A ligand function of glutathione S-transferase

Glutathione S-transferases (GSTs) are ubiquitous enzymes and abundant in plants. They are intimately involved in plant metabolism and stress defense related to reactive oxygen species. Our project assigned particular reactions including novel ones to certain GST-isoforms. Transformed E. coli was used to express recombinant GST-isoforms from maize. An N-terminal His tag allowed their purification by affinity chromatography. Three GST-monomers had a molecular weight of 26, 27, 29 kDa, and aggregated to dimers when assayed for their enzymic properties. Four dimeric isoforms were used to study how they interact with tetrapyrroles (of the chlorophyll biosynthesis pathway). It was found that protoporphyrin IX (Proto IX), Mg-protoporphyrin and other tetrapyrroles are bound non-covalently ("liganded") to GSTs but not conjugated with reduced glutathione. This binding is non-covalent, and results in inhibition of conjugation activity, the degree depends on type of the porphyrin and GST-isoform. I50-values between 1-10 microM were measured for Proto IX, the inhibition by mesoporphyrin and Mg-protoporphyrin was 2- to 5-fold less. The ligand binding is noncompetitive for the substrate 1-chloro-2,4-dinitrobenzene and competitive for glutathione. The dimer GST 26/26 prevents the (non-enzymic) autoxidation of protoporphyrinogen to Proto IX, which produces phytotoxic reactive oxygen species in the light. GST 27/27 protects hemin against degradation. Protoporphyrinogen is formed in the plastid and then exported into the cytosol. Apparently binding by a suitable GST-isoform ensures that the highly autoxidizable protoporphyrinogen can safely reach the mitochondrium where it is processed to cytochrome.     

Coisolation of glutathione peroxidase, catalase and superoxide dismutase from human erythrocytes

Glutathione peroxidase (GSH-Px; glutathione: hydrogen peroxide oxidoreductase; EC 1.11.1.9), catalase (H2O2: H2O2 oxidoreductase; EC 1.11.1.6) and superoxide dismutase (superoxide: superoxide oxidoreductase; EC 1.15.1.1) were coisolated from human erythrocyte lysate by chromatography on DEAE-cellulose. Glutathione peroxidase was separated from superoxide dismutase and catalase by thiol-disulfide exchange chromatography and then purified to approximately 90% homogeneity by gel permeation chromatography and dye-ligand affinity chromatography. Catalase and superoxide dismutase were separated from each other and purified further by gel permeation chromatography. Catalase was then purified to approximately 90% homogeneity by ammonium sulfate precipitation and superoxide dismutase was purified to apparent homogeneity by hydrophobic interaction chromatography. The results for glutathione peroxidase represent an improvement of approximately 10-fold in yield and 3-fold in specific activity compared with the established method for the purification of this enzyme. The yields for superoxide dismutase and catalase were high (45 mg and 232 mg, respectively, from 820 ml of washed packed cells), and the specific activities of both enzymes were comparable to values found in the literature.     

Purification and characterization of three distinct glutathione transferases from mouse liver

Three distinct glutathione transferases in the liver cytosol fraction of male NMRI mice have been purified by affinity chromatography and fast protein liquid chromatofocusing. These enzymes account for approximately 95% of the activity detectable with 1-chloro-2,4-dinitrobenzene as electrophilic substrate. Differences between the three forms are manifested in isoelectric points, apparent subunit molecular mass values, amino acid compositions, N-terminal structures, substrate specificities, and sensitivities to inhibitors, as well as in reactions with specific antibodies raised against glutathione transferases from rat and human tissues. The results indicate strongly that the three mouse enzymes are products of different genes. A comparison of the mouse glutathione transferases with rat and human enzymes revealed similarities between the transferases from different species. Mouse glutathione transferases have been named on the basis of their respective subunit compositions.     

Purification and characterization of a glutathione S-transferase from the fungus Cunninghamella elegans

Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S-transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120000 x g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of M(r) 27000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian alpha-, mu-, and pi-class GSTs, although it showed a small degree of cross-reactivity with a theta-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs.     

Newly identified bile acid binders in rat liver cytosol. Purification and comparison with glutathione S-transferases

Gel filtration of male rat liver cytosol preincubated with radiolabeled lithocholic, chenodeoxycholic, and glycochenodeoxycholic acids, and taurocholic acid revealed two major peaks of radioactivity, one co-eluting with the glutathione S-transferases and the other with a separate fraction, respectively. Chromatofocusing of the pooled fractions containing the new bile acid binding activity resulted in a separation of bile acid binding from the previously described organic anion binding activity in this fraction. Two binding peaks for lithocholic acid (pI 5.6, Binder I, and pI 5.5, Binder II) were identified on chromatofocusing and were further purified to apparent homogeneity by hydroxyapatite chromatography. The two Binders were monomers having identical molecular weight (33,000) and similar amino acid compositions. Bile acid binding to purified Binders I and II and glutathione S-transferases A, B, and C was studied by inhibition of the fluorescence of bound 1-anilino-8-naphthalenesulfonate (ANS). Confirmatory experiments using equilibrium dialysis produced comparable results. Glutathione S-transferase B had greater affinity for bile acids than transferases A or C. Binder II, which had greater affinity than Binder I for most bile acids, had greater affinity for chenodeoxycholic acid than transferase B but comparable or lower affinities for the other bile acids. All bile acids studied diminished ANS fluorescence with Binder II. Taurocholic and cholic acids increased ANS fluorescence with Binder I without affecting KANS, whereas lithocholic and chenodeoxycholic acids diminished ANS fluorescence with Binder I. In summary, we have identified and isolated two proteins (Binders I and II) which, along with glutathione S-transferase B, are the major hepatic cytosol bile acid binding proteins; these proteins have overlapping but distinct specificities for various bile acids.     

Characterization of two forms of asparaginase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae X2180-1A synthesizes two forms of asparaginase: L-asparaginase I, an internal constitutive enzyme, and asparaginase II, an external enzyme which is secreted in response to nitrogen starvation. The two enzymes are biochemically and genetically distinct. The structural gene for asparaginase I (asp 1) is closely linked to the trp 4 gene on chromosome IV. The gene controlling the synthesis of asparaginase II is not linked to either the trp 4 or asp 1 genes. The rate of biosynthesis of asparaginase II is unaltered in yeast strains carrying the structural gene mutation for asparaginase I. Asparaginase II has been purified approximately 300-fold from crude extracts of Saccharomyces by heat and pH treatment, ethanol fractionation, ammonium sulfate fractionation followed by Sephadex G-25 chromatography, and DEAE-cellulose chromatography. Multiple activity peaks were obtained which, upon gas chromatographic analysis, exhibit varying mannose to protein ratios. Asparaginase I has been purified approximately 100-fold from crude extracts of Saccharomyces by protamine sulfate treatment, ammonium sulfate fractionation, gel permeation chromatography, and DEAE-cellulose chromatography. No carbohydrate component was observed upon gas chromatographic analysis. Comparative kinetic and analytic studies show the two enzymes have little in common except their ability to hydrolyze L-asparagine to L-aspartic acid and ammonia.     

Characterization of Cytosolic Glutathione S-Transferase in Spruce Needles

Active glutathione S-transferase (GST) has been purified from needles of Norway spruce (Picea abies L. Karst.). Two isoforms of the enzyme which exhibit different physico-chemical and catalytic properties were separated by (NH₄)₂SO₄ fractionation, affinity chromatography on epoxy-activated 4% cross-linked beaded agarose, using glutathione as the ligand, ion-exchange chromatography, and isoelectric focusing. The isozymes have pI values of 5.5 (GST I) and 4.3 (GST II). Both GST isozymes are homodimeric proteins with subunit sizes of 26 kD (GST I), and 23 kD (GST II). The kinetic properties of the enzymes are described and compared with other plants GSTs. Only GST II is able to conjugate the pesticides fluorodifen and alachlor.     

In vitro effects of some antibiotics on glutathione reductase from sheep liver

The effects of gentamicin sulphate, thiamphenicol, ofloxacin, levofloxacin, cefepime, and cefazolin were investigated on the in vitro enzyme activity of glutathione reductase. The enzyme was purified 1,850-fold with a yield 18.76% from sheep liver using ammonium sulphate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). The enzyme activity was measured spectrophotometrically at 340 nm, according to the method of Carlberg and Mannervik. From these six antibiotics, Ofloxacin, levofloxacin, cefepime, and cefazolin inhibited the activity of the purified enzyme; gentamicin sulphate and thiamphenicol showed little effect on the enzyme activity. The I50 values for these four antibiotics were 0.150 mM, 0.154 mM, 3.395 mM, and 18.629 mM, respectively. The Ki constants were 0.047 +/- 0.034 mM, 0.066 +/- 0.038 mM, 4.885 +/- 3.624 mM, and 6.511 +/- 1.894 mM, respectively and they were competitive inhibitors.     

东亚飞蝗谷胱甘肽S-转移酶分离纯化

通过硫酸铵沉淀技术和GSH-agarose亲和层析对东亚飞蝗Locusta migratoria manilensis(Meyen)5龄若虫谷胱甘肽S-转移酶(glutathione S-transferases,GSTs)进行了分离纯化。结果表明GSTs活性在硫酸铵各沉淀段均有分布,但在55%～100%沉淀段活性较高,在硫酸铵饱和度为85%时比活力最高,达到420.33μmol/min/mg protein,纯化倍数为18.86。根据硫酸铵粗沉淀谷胱甘肽S-转移酶结果,选择硫酸铵浓度为60%～90%沉淀段进行GSH-agarose亲和层析,纯化后比活力最高达到1365.29μmol/min/mg protein,纯化倍数达到61.25。经SDS-PAGE鉴定,得到的GST为1条带,亚基的分子量约为24kDa。     

Purification and subunit-structural and immunological characterization of five glutathione S-transferases in human liver, and the acidic form as a hepatic tumor marker

Five glutathione S-transferase (GST, EC 2.5.1.18) forms were purified from human liver by S-hexylglutathione affinity chromatography followed by chromatofocusing, and their subunit structures and immunological relationships to rat liver glutathione S-transferase forms were investigated. They were tentatively named GSTs I, II, III, IV and V in order of decreasing apparent isoelectric points (pI) on chromatofocusing. Their subunit molecular weights assessed on SDS-polyacrylamide gel electrophoresis were 27 (Mr X 10(-3)), 27, 27.7,27 and 26, respectively, (26, 26, 27, 26, and 24.5 on the assumption of rat GST subunit Ya, Yb and Yc as 25, 26.5 and 28, respectively), indicating that all forms are composed of two subunits identical in size. However, it was suggested by gel-isoelectric focusing in the presence of urea that GSTs I and IV are different homodimers, consisting of Y1 and Y4 subunits, respectively, which are of identical Mr but different pI, while GST II is a heterodimer composed of Y1 and Y4 subunits. This was confirmed by subunit recombination after guanidine hydrochloride treatment. GST III seemed to be identical with GST-mu with regard to Mr and pI. GST V was immunologically identical with the placental GST-pi. On double immunodiffusion or Western blotting using specific antibodies to rat glutathione S-transferases, GST I, II and IV were related to rat GST 1-1 (ligandin), GST III(mu) to rat GST 4-4 (D), and GST V (pi) to rat GST 7-7 (P), respectively. GST V (pi) was increased in hepatic tumors.     

A rapid purification procedure for camel kidney glutathione S-transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37,000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 mumol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100. The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29,000 D and 26,000 D to give a native molecular weight of 55,000 D. The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. 1-chloro-2,4-dinitrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse. Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Characterisation of glutathione transferases and glutathione peroxidases in pea (Pisum sativum)

Glutathione peroxidases (GPOXs) and glutathione transferases, also termed glutathione S-transferases (GST, EC 2.5.1.18), with activities toward a range of xenobiotic substrates including herbicides, have been characterized in etiolated pea (Pisum sativum L. cv. Feltham's First) seedlings. Crude extracts showed high activity toward a range of GST substrates including 1-chloro-2,4-dinitrobenzene (GSTC activity) and the herbicide fluorodifen (GSTF) but low activities toward chloroacetanilides and atrazine. Treatment of the pea seedlings with the herbicide safener dichlormid selectively increased the activity of GSTC and the GST which detoxified atrazine. This induction was restricted to the roots and was not observed with any of the other GST or GPOX activities. In contrast, treatment with CuCl₂ increased GPOX activity in the root but had no effect on any GST activity, while treatment of epicotyls with elicitors of the phytoalexin response increased GST activity toward ethacrynic acid, but had no effect on other GST or GPOX activities. The major enzymes with GSTC, GSTF and GPOX activities were purified from pea epicotyls 3609-fold, 1431-fold and 1554-fold, respectively. During purification by hydrophobic interaction chromatography and affinity chromatography using S-hexyl-glutathione as ligand all three activities co-eluted but could be partially resolved by anion exchange chromatography and gel filtration chromatography. Both GSTC and GPOX had a molecular mass of 48 kDa and their activities were associated with a similar 27.5-kDa subunit but distinct 29-kDa subunits. GSTF could be resolved into two isoenzymes with molecular masses of 49.5 and 54 kDa. GSTF activity was associated with a unique 30-kDa subunit in addition to 27.5- and 29-kDa peptides, suggesting that the two isoenzymes were composed of differing subunits. These results demonstrate that peas contain multiple GST isoenzymes some of which have GPOX activity and that the various activities are differentially responsive to biotic and abiotic stress.     

Peroxidase-mediated conjugation of glutathione to unsaturated phenylpropanoids. Evidence against glutathione S-transferase involvement

A number of plant species are thought to possess a glutathione S-transferase enzyme (GST: EC 2.5.1.18) that will conjugate glutathione (GSH) to trans -cinnamic acid (CA) and para -coumaric acid (4-CA). However, we present evidence that this activity is mediated by peroxidase enzymes and not GSTs. The N-terminal amino acid sequence of the GSH-conjugating enzyme purified from etiolated corn shoots exhibited a strong degree of homology to cytosolic ascorbate peroxidase enzymes (APX: EC 1.11.1.11) from a number of plant species. The GSH-conjugating and APX activities of corn could not be separated during chromatography on hydrophobic-interaction. anion-exchange, and gel filtration columns. Spectral analysis of the enzyme revealed that the protein had a Soret band at 405 nm. When the enzyme was reduced with dithionite, the peak was shifted to 423 nm with an additional peak at 554 nm. The spectrum of the dithionite-reduced enzyme in the presence of 0.1 m M KCN exhibited peaks at 430, 534 and 563 nm. These spectra are consistent with the presence of a heme moiety. The GSH-conjugating and APX activities of the enzyme were both inhibited by KCN. NaN₃, p -chloromercuribenzoate ( p CMB), and iodoacetate. The APX specific activity of the enzyme was 1.5-fold greater than the GSH-conjugating specific activity with 4-CA. In addition to the corn enzyme, a pea recombinant APX (rAPX) and horseradish peroxidase (HRP; EC 1.11.1.7) were also able to conjugate GSH to CA and 4-CA. The peroxidase enzymes may generate thiyl free radicals of GSH that react with the alkyl double bond of CA and 4-CA resulting in the formation of a GSH conjugate.     

The purification of the hepatic glutathione S-transferases of rainbow trout by glutathione affinity chromatography alters their isoelectric behaviour.

1. The basic glutathione S-transferases from rainbow-trout liver were more stable than the acidic ones. 2. The apparent pI values of these enzymes were lowered when they were eluted from a glutathione affinity column by reduced glutathione at pH 8.85. 3. The pI effect was not a function of the high pH alone, was diminished under conditions less favourable to glutathione oxidation, and did not occur when S-hexylglutathione affinity chromatography was used instead.     

Purification and properties of a glutathione peroxidase from Southern bluefin tuna (Thunnus maccoyii) liver

A glutathione peroxidase (GPX) protein was purified approximately 1000-fold from Southern bluefin tuna (Thunnus maccoyii) liver to a final specific activity of 256 micromol NADPH oxidised min(-1) mg(-1) protein. Gel filtration chromatography and denaturing protein gel electrophoresis of the purified preparation indicated that the protein has a native molecular mass of 85 kDa and is most likely a homotetramer with subunits of approximately 24 kDa. The Km values of the purified enzyme for hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and glutathione were 12, 90, 90 and 5900 microM, respectively. The Km values for cumene hydroperoxide and t-butyl hydroperoxide were approximately 8-fold greater than the Km value for hydrogen peroxide. Thus, the SBT liver GPX has a considerably greater affinity for hydrogen peroxide than for the other two substrates. The pH optimum of the purified enzyme was pH 8.0. Immunoblotting experiments with polyclonal antibodies, raised against a recombinant human GPX, provided further evidence that the purified SBT enzyme is a genuine GPX.     

Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [gamma-32P]ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit. A low-Km (0.1 microM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (Ki = 5 microM) and stimulated 2-3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (approximately equal to 100 microM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi. Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate (Km greater than 50 microM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.     

Host-Pathogen Interactions: XVI. PURIFICATION AND CHARACTERIZATION OF A beta-GLUCOSYL HYDROLASE/TRANSFERASE PRESENT IN THE WALLS OF SOYBEAN CELLS

The fact that fungal glucans will stimulate soybeans to accumulate phytoalexins prompted an investigation of soybean cell beta-1,3-glucanases and beta-glucosidases, as well as the ability of these enzymes to hydrolyze the fungal glucans. Several beta-1,3-glucanases and beta-glucosidases can be solubilized from the walls of suspension-cultured soybean cells by treatment with 1.0 molar sodium acetate buffer. An enzyme, which has been termed beta-glucosylase I, is the dominant beta-1,3-glucanase in the cell wall extracts. Utilizing CM-Sephadex chromatography, hydroxylapatite chromatography, and affinity chromatography, beta-glucosylase I has been purified 71-fold, with 39% recovery, from the mixture of cell wall enzymes. The affinity chromatography column material was prepared by covalently attaching p-aminophenyl-1-beta-d-glucopyranoside, an analog of a beta-glucosylase I substrate, to Sepharose. beta-Glucosylase I, purified by this procedure, yields a single band on isoelectric focusing gels (pH 8.9). However, the purified beta-glucosylase I yields a darkly-staining protein band at an apparent molecular weight of 69,000 and several lightly-staining protein bands in sodium dodecyl sulfate polyacrylamide gels. Additional purification procedures fail to remove these lightly-staining protein bands.beta-Glucosylase I will hydrolyze the beta-glucan substrates, laminarin (3-linked) and lichenan (3- and 4-linked), and therefore, possesses beta-glucanase activity. Studies of the progressive hydrolysis of laminarin by beta-glucosylase I demonstrate that the enzyme hydrolyzes polysaccharide substrates in an exo manner. beta-Glucosylase I will also hydrolyze a variety of low molecular weight beta-glucosides including various beta-linked diglucosides. Thus, beta-glucosylase I also possesses beta-glucosidase activity.Several lines of evidence are presented that the beta-glucanase and the beta-glucosidase activities exhibited by purified beta-glucosylase I preparations are catalyzed by the same enzyme. This evidence includes inhibition studies which indicate that the beta-glucanase and the beta-glucosidase activities of beta-glucosylase I are catalyzed at the same active site. beta-Glucosylase I will also catalyze glucosyl transfer. This catalytic activity is responsible for the observed ability of the enzyme to synthesize di- and trisaccharides from laminarin. The disaccharides formed by beta-glucosylase I-catalyzed transglucosylation are the beta-anomers of the 6-, 4-, 3-, and 2-linked diglucosides in the relative proportions of 10:1:1:1. The ability of beta-glucosylase I to catalyze glucosyl transfer indicates that beta-glucosylase I is biochemically more similar to previously studied beta-glucosidases than to beta-glucanases. This conclusion is supported by the observation that beta-glucosylase I is strongly inhibited by 1,5-d-gluconolactone, an inhibitor of beta-glucosidases but not of beta-glucanases.     

In vitro effects of some drugs on human erythrocyte glutathione reductase

The effects of ketotifen, meloxicam, phenyramidol-HCl and gadopentetic acid on the enzyme activity of GR were studied using human erythrocyte glutathione reductase (GR) enzymes in vitro. The enzyme was purified 209-fold from human erythrocytes in a yield of 19% with 0.31?U/mg. The purification procedure involved the preparation of haemolysate, ammonium sulphate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies. In the in vitro studies, IC(50) values and K(i) constants were 0.012?mM and 0.0008?±?0.00021?mM for ketotifen; 0.029?mM and 0.0061?±?0.00127?mM for meloxicam; 0.99?mM and 0.4340?±?0.0890?mM for phenyramidol-HCl; 138?mM and 28.84?±?4.69?mM for gadopentetic acid, respectively, showing the inhibition effects on the purified enzyme. Phenyramidol-HCl showed competitive inhibition, whereas the others showed non-competitive inhibition.     

Purification of rat kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2',5'-ADP Sepharose 4B affinity in a single chromatography step

The enzymes of glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and glutathione reductase (GR) were purified from rat kidney in one chromatographic step consisting of the use of the 2',5'-ADP Sepharose 4B by using different elution buffers. This purification procedure was accomplished with the preparation of the homogenate and affinity chromatography on 2',5'-ADP Sepharose 4B. The purity and subunit molecular weights of the enzymes were checked on SDS-PAGE and purified enzymes showed a single band on the gel. The native molecular weights of the enzymes were found with Sephadex G-150 gel filtration chromatography. Using this procedure, G6PG, having the specific activity of 32 EU/mg protein, was purified 531-fold with a yield of 88%; 6PGD, having the specific activity of 25 EU/mg protein, was purified 494-fold with a yield of 73%; and GR, having the specific activity of 33 EU/mg protein, was purified 477-fold with a yield of 76%. Their native molecular masses were estimated to be 144 kDa for G6PD, 110 kDa for 6PGD, and 121 kDa for GR and the subunit molecular weights were found to be 68, 56, and 61 kDa, respectively. A new modified method to purify G6PD, 6PGD, and GR, namely one chromatographic step using the 2',5'-ADP Sepharose 4B, is described for the first time in this study. This procedure has several advantages for purification of enzymes, such as, rapid purification, produces high yield, and uses less chemical materials.