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.     

Expression of Yb1 glutathione S-transferase using a baculovirus expression system

A full-length cDNA clone was isolated for rat liver Yb1 glutathione S-transferase (EC 2.5.1.18). The coding sequence of Yb1 cDNA was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The enzymatically active recombinant Yb1 glutathione S-transferase protein has a native molecular weight of 42,000 daltons (by molecular sieve chromatography), a subunit molecular weight of 26,500 daltons (by SDS-polyacrylamide gel electrophoresis), a pI of 8.4 and an extinction coefficient E1%280 of 5.6 +/- 0.4.     

中华稻蝗谷胱甘肽S-转移酶的分离纯化

采用硫酸铵沉淀法和GSH-agarose亲和层析法,对中华稻蝗Oxya chinensis(Thunberg)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:经硫酸铵沉淀,饱和度在60%-80%下沉淀中GSTs比活力较高,饱和度90%时比活力达到最高...     

Spectroscopic and kinetic evidence for the thiolate anion of glutathione at the active site of glutathione S-transferase

Ultraviolet difference spectroscopy of the binary complex of isozyme 4-4 of rat liver glutathione S-transferase with glutathione (GSH) and the enzyme alone or as the binary complex with the oxygen analogue, gamma-L-glutamyl-L-serylglycine (GOH), at neutral pH reveals an absorption band at 239 nm (epsilon = 5200 M-1 cm-1) that is assigned to the thiolate anion (GS-) of the bound tripeptide. Titration of this difference absorption band over the pH range 5-8 indicates that the thiol of enzyme-bound GSH has a pKa = 6.6, which is about 2.4 pK units less than that in aqueous solution and consistent with the kinetically determined pKa previously reported [Chen et al. (1988) Biochemistry 27, 647]. The observed shift in the pKa between enzyme-bound and free GSH suggests that about 3.3 kcal/mol of the intrinsic binding energy of the peptide is utilized to lower the pKa into the physiological pH range. Apparent dissociation constants for both GSH and GOH are comparable and vary by a factor of less than 2 over the same pH range. Site occupancy data and spectral band intensity reveal large extinction coefficients at 239 nm (epsilon = 5200 M-1 cm-1) and 250 nm (epsilon = 1100 M-1 cm-1) that are consistent with the existence of either a glutathione thiolate (E.GS-) or ion-paired thiolate (EH+.GS-) in the active site. The observation that GS- is likely the predominant tripeptide species bound at the active site suggested that the carboxylate analogue of GSH, gamma-L-glutamyl-(D,L-2-aminomalonyl)glycine, should bind more tightly than GSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Communication between the two active sites of glutathione S-transferase A1-1, probed using wild-type-mutant heterodimers

To study the communication between the two active sites of dimeric glutathione S-transferase A1-1, we used heterodimers containing one wild-type (WT) active site and one active site with a single mutation at either Tyr9, Arg15, or Arg131. Tyr9 and Arg15 are part of the active site of the same subunit, while Arg131 contributes to the active site of the opposite subunit. The V(max) values of Tyr9 and Arg15 mutant enzymes were less than 2% that of WT, indicating their importance in catalysis. In contrast, V(max) values of Arg131 mutant enzymes were about 50-90% of that of WT enzyme while K(m)(GSH) values were approximately 3-8 times that of WT, suggesting that Arg131 plays a role in glutathione binding. The mutant enzyme (with a His(6) tag) and the WT enzyme (without a His(6) tag) were used to construct heterodimers (WT-Y9F, WT-Y9T, WT-R15Q, WT-R131M, WT-R131Q, and WT-R131E) by incubation of a mixture of wild-type and mutant enzyme at pH 7.5 in buffer containing 1,6-hexanediol, followed by dialysis against buffer lacking the organic solvent. The resultant heterodimers were separated from the wild-type and mutant homodimers using chromatography on nickel-nitrilotriacetic acid agarose. The V(max) values of all heterodimers were lower than expected for independent active sites. Our experiments demonstrate that mutation of an amino acid residue in one active site affects the activity in the other active site. Modeling studies show that key amino acid residues and water molecules connect the two active sites. This connectivity is responsible for the cross-talk between the active sites.     

A general method for the purification of restriction enzymes.

An abbreviated procedure has been developed for the purification of restriction endonucleases. This procedure uses chromatography on phosphocellulose and hydroxylapatite and results in enzymes of sufficient purity to permit their use in the sequencing, molecular cloning, and physical mapping of DNA.     

Recombinant eggshell ovocalyxin-32: expression, purification and biological activity of the glutathione S-transferase fusion protein

The avian eggshell is a highly ordered biomineral composed mainly of calcium carbonate associated with an organic matrix composed of proteins, glycoproteins and proteoglycans. This structure provides the developing embryo with protection from physical damage and microbial invasion. Ovocalyxin-32 (OCX-32) is a 32 kDa eggshell-specific matrix protein which has been cloned and demonstrates 30% identity with the mammalian carboxypeptidase inhibitor, latexin. In order to further study its function, recombinant OCX-32 protein was expressed in E. coli. The protein was extracted from inclusion bodies and purified by sequential DEAE Sepharose and Ni2+ metal ion affinity chromatographies as a 58 kDa GST-fusion protein. The refolded GST-OCX-32 significantly inhibited bovine carboxypeptidase and also inhibited the growth of Bacillus subtilis. The results suggest that OCX-32 may show similar activity to the fusion protein and reinforce the antimicrobial properties of the eggshell by providing protection to the developing avian embryo. OCX-32 is the first example of an eggshell specific protein to be successfully cloned and expressed in a prokaryotic system. The association of an antimicrobial protease inhibitor with the outer eggshell and cuticle of the table egg may enhance the food safety of this product.     

A rapid, novel high performance liquid chromatography method for the purification of glutathione S-transferase: an application to the human placental enzyme

A simple High Performance Liquid Chromatography procedure is detailed for the purification of Glutathione S-transferase. The human placental transferase was used to assess its potential. Unlike conventional methods of purification, the procedure is rapid and resolution of the various forms is achieved in less than 20 min. Since recovery is essentially complete, it is possible to isolate different minor forms. Three forms, one major and two minor, were separated. The major form represented about 97% of the total recovered activity and exhibited a specific activity of 254.94 mumoles/min/mg protein with a purification of 1342-fold. Electrophoresis of the major form revealed the presence of a single band, suggesting homogeneity.     

Secretion and affinity purification of glutathione S-transferase fusion proteins from yeast

Summary Sequences encoding GST-fusion proteins were cloned into the Saccharomyces cerevisiae secretion vector, pYEX-S1, to direct secretion into the culture medium. GST and metallothionein fused to GST were secreted successfully and the fusion proteins purified. With several other GST-fusion proteins however, the proteins were retained inside the cell, indicating limitations to the types of proteins that can be secreted from yeast.     

Expression and purification of biologically active IGF-binding proteins using the LCR/Mel expression system

The anabolic effects and bioavailability of insulin-like growth factors I and II (IGF-I, IGF-II) are regulated in part by a family of IGF-binding proteins (IGFBPs). There are six known members of the IGFBP family, which share distinct structural characteristics and functional activities. To study the binding properties of these proteins, we have expressed recombinant IGFBP-3 and IGFBP-4 using the LCR/Mel expression system. Using this system, we found that recombinant IGFBP-3 was secreted by Mel cells and had a glycosylation pattern similar to that of native IGFBP-3. Recombinant IGFBP-4 secreted from Mel cells had a molecular size identical to that of non-glycosylated native IGFBP-4. The binding kinetics of recombinant IGFBPs was measured using a solid-phase ligand-binding assay, an in vitro solution-binding assay, and a cellular proliferation assay. IGF-I bound with high affinity to recombinant IGFBP-3 and IGFBP-4 with K(D)s of <0.25 nmol. As reported for native IGFBPs, IGF-II bound with affinity higher than IGF-I to recombinant IGFBP-3 and IGFBP-4 (K(D) of <0.05 nmol). Recombinant IGFBP-3 and IGFBP-4 were found to inhibit the IGF-induced proliferation of an NIH3T3 cell line engineered to overexpress the IGF-I receptor. We have compared the binding kinetics of Mel cell-expressed IGFBPs with that of recombinant protein expressed in Escherichia coli and found them to be equivalent. Here, we show that the LCR/Mel expression system represents an effective route for expression of biologically active IGFBPs.     

A mammalian expression system for rapid production and purification of active MAP kinase phosphatases

Expression of enzymatically active mammalian proteins in Escherichia coli can proven to be a challenging task due to poor solubility, improper folding, and lack of adequate posttranslational modification. Expression of mammalian proteins using baculovirus or yeast systems is time-consuming and may also be subject to inadequate modification. In order to overcome these technical difficulties, we have developed a mammalian expression system for the convenient subcloning of cDNA fragments, high-level expression, and one-step purification of enzymatically active proteins. The mammalian expression vector pEBG that expresses glutathione S-transferase fusion proteins was modified to create an SrfI restriction site in the multiple cloning site. The protein coding sequences of MAP kinase phosphatase-1 (MKP-1), MAP kinase phosphatase-2 (MKP-2), and the tumor suppressor PTEN were PCR-amplified using Pfu DNA polymerase and cloned into the SrfI site through SrfI digestion-coupled ligation. The resulting plasmids were transiently transfected into 293T cells using FuGENE 6 transfection reagent. Forty eight hours after transfection, cells were harvested and bioactive recombinant proteins were purified by glutathione-Sepharose beads. Protein yield, which ranged from 200 to 700 microg, was more than adequate for biochemical studies. The usefulness of this versatile system for studying protein function and its potential application for proteomics research are discussed.     

A Mu-class glutathione S-transferase from gills of the marine shrimp Litopenaeus vannamei: purification and characterization

Glutathione S-transferases (GSTs) are a family of detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thereby increasing the solubility of GSH and aiding its excretion from the cell. In this study, a glutatione S-transferase from the gills of the marine shrimp Litopenaeus vannamei was purified by affinity chromatography using a glutathione-agarose affinity column. GST was purified to homogeneity as judged by reducing SDS-PAGE and zymograms. This enzyme is a homodimer composed of approximately 25-kDa subunits and identified as a Mu-class GST based on its activity against 1-chloro-2,4-dinitrobenzene (CDNB) and internal peptide sequence. The specific activity of purified GST was 440.12 micromol/(min mg), and the K(m) values for CDNB and GSH are very similar (390 and 335 microM, respectively). The intersecting pattern of the initial velocities of this enzyme in the Lineweaver-Burke plot is consistent with a sequential steady-state kinetic mechanism. The high specific activity of shrimp GST may be related to a highly effective detoxification mechanism necessary in gills since they are exposed to the external and frequently contaminated environment.     

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.     

Evidence for the involvement of tryptophan 38 in the active site of glutathione S-transferase P.

Glutathione S-transferase P (GST-P) exists as a homodimeric form and has two tryptophan residues, Trp28 and Trp38, in each subunit. In order to elucidate the role of the two tryptophan residues in catalytic function, we examined intrinsic fluorescence of tryptophan residues and effect of chemical modification by N-bromosuccinimide (NBS). The quenching of intrinsic fluorescence was observed by the addition of S-hexylglutathione, a substrate analogue, and the enzymatic activity was totally lost when single tryptophan residue was oxidized by NBS. To identify which tryptophan residue is involved in the catalytic function, each tryptophan was changed to histidine by site-directed mutagenesis. Trp28His GST-P mutant enzyme showed a comparable enzymatic activity with that of the wild type one. Trp38His mutant neither was bound to S-hexylglutathione-linked Sepharose nor exhibited any GST activity. These findings indicate that Trp38 is important for the catalytic function and substrate binding of GST-P.     

Schistosoma mansoni: single-step purification and characterization of glutathione S-transferase isoenzyme 4.

A soluble glutathione S-transferase isoenzyme, designated SmGST-4 was purified to apparent homogeneity in a single step from the cytosol of adult Schistosoma mansoni by selective elution of the enzyme from a glutathione-agarose affinity column using glutathione disulfide. SmGST-4, which comprised about 5% of the bound glutathione S-transferase activity, could be distinguished from the previously characterized glutathione S-transferase isoenzyme family (SmGST-1/2/3), by its unique chromatographic behavior, lower subunit M(r) (26,000), differences in substrate specificity and inhibitor sensitivity, and a lack of reactivity with antiserum to SmGST-3. The purified isoenzyme catalyzed the conjugation of several model xenobiotics including 1-chloro-2,4-dinitrobenzene, ethacrynic acid, and trans-4-phenyl-3-buten-2-one. Like the SmGST-1/2/3 isoenzyme family, SmGST-4 failed to catalyze the conjugation of a model epoxide substrate, 1,2-epoxy-3-(p-nitrophenoxy)propane. Because glutathione S-transferases from other organisms play a role in protecting cells against the toxic products of lipid peroxidation, SmGST-4 and the members of the SmGST-1/2/3 isoenzyme family were tested for their capacity to reduce cumene hydroperoxide and to catalyze the conjugation of 4-hydroxyalk-2-enals. Although all four isoenzymes catalyzed both reactions, the specific activity of SmGST-1, SmGST-2, and SmGST-3 toward cumene hydroperoxide was at least 10-fold greater than that of SmGST-4. In contrast, the latter more effectively conjugated a homologous series of 4-hydroxyalk-2-enal isomers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the involvement of histidine at the active site of glutathione S-transferase psi from human liver

The inhibition of catalytic activity of glutathione S-transferase psi (pI 5.5) of human liver by diethylpyrocarbonate (DEPC) has been studied. It is demonstrated that DEPC causes a concentration dependent inactivation of GST psi with a concomitant modification of 1-1.3 histidyl residues/subunit of the enzyme. This inactivation of GST psi could be reversed by treatment with hydroxylamine. Glutathione afforded complete protection to the enzyme from inactivation by DEPC. It is suggested that a functional histidyl residue is essential for the catalytic activity of the enzyme and that this residue is most likely to be present at or near the glutathione binding site (G-site).     

Substrate specificity of rat liver glutathione S-transferase isoenzymes for a series of glutathione analogues, modified at the gamma-glutamyl moiety.

The substrate specificity of purified rat liver glutathione S-transferases (GSTs) for a series of gamma-glutamyl-modified GSH analogues was investigated. GST isoenzyme 3-3 catalysed the conjugation of 1-chloro-2,4-dinitrobenzene with six out of the nine analogues. alpha-L-Glu-L-Cys-Gly and alpha-D-Glu-L-Cys-Gly showed catalytic efficiencies of 40% and 130% that of GSH respectively. The GSH analogue with an alpha-D-glutamyl moiety appeared to be a highly isoenzyme-3-3-specific co-substrate: kcat./Km with GST isoenzyme 4-4 was only about 5% that with GST isoenzyme 3-3, and no enzymic activity was detectable with GST isoenzymes 1-1 and 2-2. GST isoenzyme 4-4 showed some resemblance to GST 3-3: five out of nine co-substrate analogues were accepted by this second isoenzyme of the Mu multigene family. Isoenzymes 1-1 and 2-2, of the Alpha multigene family, accepted only two alternative co-substrates, which indicates that their GSH-binding site is much more specific.