Biomedical subjects

B Mannervik

Publications and source records attributed to B Mannervik.

At least 19 recordsLinked to original sources

PubMed1996

Molecular cloning, heterologous expression, and characterization of human glyoxalase II.

A clone encoding glyoxalase II has been isolated from a human adult liver cDNA library. The sequence of 1011 base pairs consists of a full-length coding region of 780 base pairs, corresponding to a protein with a calculated molecular mass of 28,861 daltons. Identities (50-60%) were found to partial 5' and 3' cDNA sequences from Arabidopsis thaliana as well as within a limited region of glutathione transferase I cDNA from corn. A vector was constructed for heterologous expression of glyoxalase II in Escherichia coli. For optimal yield of enzyme, silent random mutations were introduced in the 5' coding region of the cDNA. A yield of 25 mg of glyoxalase II per liter of culture medium was obtained after affinity purification with immobilized glutathione. The recombinant enzyme had full catalytic activity and kinetic parameters indistinguishable from those of the native enzyme purified from human erythrocytes.

Adult↗

PubMed1995

The high activity of rat glutathione transferase 8-8 with alkene substrates is dependent on a glycine residue in the active site.

Rat glutathione transferase (GST) 8-8 displays high catalytic activity with alpha, beta-unsaturated carbonyl compounds, including lipid peroxidation products such as 4-hydroxyalkenals. The catalytic efficiency of the related class Alpha GST 1-1 is substantially lower with the same substrates. Chimeric enzymes were prepared by replacing N-terminal subunit 8 segments of different lengths (6, 25, or 100 residues) with corresponding sequences from subunit 1 using recombinant DNA techniques. The chimeric subunit r1(25)r8, containing 25 amino acid residues from subunit 1, had the same low activity with alkenal substrates as that displayed by subunit 1. Mutation of Ala-12 into Gly in r1(25)r8 gave rise to the high alkenal activity characteristic of subunit 8, showing the importance of amino acid residue 12 for the activity. However, other structural determinants are also essential, as demonstrated by the corresponding Ala-12-->Gly mutation in subunit 1, which did not afford high alkenal activity. The results show that a single point mutation in a GST subunit may give rise to a 100-fold increase in catalytic efficiency with certain substrates. Introduction of such mutations may have contributed to the biological evolution of GST isoenzymes with altered substrate specificities and may also find use in the engineering of GSTs for novel functions.

Alkenes↗

PubMed1995

Isothiocyanates as substrates for human glutathione transferases: structure-activity studies.

The catalytic properties of four human glutathione transferases (GSTs), A1-1, M1-1, M4-4 and P1-1, were examined with 14 isothiocyanate (R-NCS) substrates. The compounds include aliphatic and aromatic homologues, some of which are natural constituents of human food, namely sulphoraphane [1-isothiocyanato-4-(methylsulphinyl)butane], erucin [1-isothiocyanato-4-(methylthio)butane], erysolin [1-isothiocyanato-4-(methylsulphonyl)butane], benzyl-NCS, phenethyl-NCS and allyl-NCS. All isothiocyanates investigated were substrates for the four GSTs. The enzymes promote addition of the thiol group of GSH to the electrophilic central carbon of the isothiocyanate group to form dithiocarbamates [R-NH-C(=S)-SG] which have high UV absorption at 274 nm. Molar absorption coefficients and non-enzymic rate constants as well as standardized enzyme assay conditions for all compounds were established. Of the four isoenzymes investigated, GSTs M1-1 and P1-1 were generally the most efficient catalysts, whereas GST M4-4 was the least efficient. Isothiocyanates are among the GST substrates that are most rapidly conjugated. On the basis of rate-enhancement data and binding energies, the isothiocyanates were compared with 4-hydroxyalkenals, another class of natural GST substrates previously subjected to systematic kinetic analysis. The incremental transition-state stabilization attributable to an increased number of methylene groups in homologous alkyl isothiocyanates is similar to that previously noted for homologous 4-hydroxyalkenals.

Absorption↗

PubMed1995

Cloning, sequencing, and regulation of the glutathione reductase gene from the cyanobacterium Anabaena PCC 7120.

Glutathione reductase (GR) was purified from the cyanobacterium Anabaena PCC 7120. A 3-kilobase genomic DNA fragment containing the coding sequence for the GR gene (gor) was identified and cloned by polymerase chain reaction based on sequences of selected peptides isolated from proteolyzed GR. The coding sequence encompassing 458 amino acid residues, as well as 360 base pairs of the 5'-flanking region and 430 base pairs of the 3'-flanking region, were determined. Genomic Southern analysis indicates that gor is a single-copy gene. A gor antisense RNA probe hybridized with a 1.4-kilobase transcript, suggesting that the gene is not part of an operon including additional genes. The deduced GR amino acid sequence shows 41 to 48% identity with those of human, Escherichia coli, Pseudomonas aeruginosa, pea, and Arabidopsis thaliana GR. The coding sequence of GR was overexpressed in a GR-deficient E. coli strain, SG5, and the recombinant protein was purified. Anabaena GR is NADPH-linked, but a Lys residue replaces an Arg residue involved in NADPH binding in GR from other species. In addition, Anabaena GR carries the GXGXXG "fingerprint" motif which otherwise characterizes NAD(H)-dependent enzymes. These differences may contribute to the lack of affinity for 2',5'-ADP-Sepharose 4B of Anabaena GR. Three E. coli-type promoter sequences and a BifA/NtcA binding motif were found upstream of the open reading frame. The middle and the proximal promoters were shown to be active. However, the use of the middle promoter was dependent on the nitrogen source in the culture medium. Both GR activity and GR protein concentration increased in ammonium grown cultures in which both the middle and proximal promoters were used for transcriptional initiation. The BifA/NtcA-binding site overlaps the middle promoter sequence and may thus be involved in regulation of differential transcription.

Amino Acid Sequence↗

PubMed1995

Glutathione transferases with novel active sites isolated by phage display from a library of random mutants.

Human glutathione transferase A1-1 can be expressed as a fusion protein with coat protein III of filamentous phage f1 in a form that allows selection among variant mutant forms based on specific adsorption to immobilized active-site ligands. A library of mutant enzymes differing in the active-site region was generated by random mutagenesis of ten amino acid residues involved in the binding of electrophilic substrates. Novel glutathione transferases with altered specificity for active-site ligands were isolated by adsorption of the fusion protein on the surface of phage to analogs of an electrophilic substrate. Thus, phage display of glutathione transferase affords a system for engineering novel binding specificities onto the pre-existing protein framework of the enzyme.

Amino Acid Sequence↗

PubMed1995

Functional significance of arginine 15 in the active site of human class alpha glutathione transferase A1-1.

Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase A1-1 have shown that N epsilon of this amino acid residue is adjacent to the sulfur atom of a glutathione derivative bound to the active site, suggesting the presence of a hydrogen bond. The phenolic hydroxyl group of Tyr9 also forms a hydrogen bond to the sulfur atom of glutathione, and removal of this hydroxyl group causes partial inactivation of the enzyme. The present study demonstrates by use of site-directed mutagenesis the functional significance of Arg15 for catalysis. Mutation of Arg15 into Leu reduced the catalytic activity by 25-fold, whereas substitution by Lys caused only a threefold decrease, indicating the significance of a positively charged residue at position 15. Mutation of Arg15 into Ala or His caused a substantial reduction of the specific activity (200 or 400-fold, respectively), one order of magnitude more pronounced than the effect of the Tyr9-->Phe mutation. Double mutations involving residues 9 and 15 demonstrated that the effects of mutations at the two positions were additive except for the substitution of His for Arg15, which appeared to cause secondary structural effects. The pKa value of the phenolic hydroxyl of Tyr9 was determined by UV absorption difference spectroscopy and was found to be 8.1 in the wild-type enzyme. The corresponding pKa values of mutants R15K, R15H and R15L were 8.5, 8.7 and 8.8, respectively, demonstrating the contribution of the guanidinium group of Arg15 to the electrostatic field in the active site. Addition of glutathione caused an increased pKa value of Tyr9; this effect was not obtained with S-methylglutathione. These results show that Tyr9 is protonated when glutathione is bound to the enzyme at physiological pH values. The involvement of an Arg residue in the binding and activation of glutathione is a feature that distinguishes class Alpha glutathione transferases from members in other glutathione transferase classes.

Arginine↗

PubMed1995

Reversible conjugation of isothiocyanates with glutathione catalyzed by human glutathione transferases.

Rates were determined for the nonezymatic (second order rate constants) and enzyme-catalyzed conjugations with glutathione of four isothiocyanates that are present in edible plants (allyl-, benzyl-, phenethyl-isothiocyanates, and sulforaphane). Of four cloned human glutathione transferases studied, GSTP1-1 and GSTM1-1 were the most efficient catalysts. GSTA1-1 was less efficient, and GSTM2-2 was the least efficient. Conjugation of benzyl-NCS is the most rapid and that of sulforaphane [CH3S(O)(CH2)4-NCS] is the slowest. The large enzymatic rate enhancements and the abundance of the enzymes suggest that the glutathione transferases play important roles in the metabolic disposition of isothiocyanates in humans. Enzymatic cleavage of the GSH conjugates of isothiocyanates (dithiocarbamates) is catalyzed by glutathione transferases. The importance of these reverse reactions is probably limited because they are slow and inhibited by high intracellular concentrations of glutathione.

Glutathione↗

PubMed1995

High-level bacterial expression of human glutathione transferase P1-1 encoded by semisynthetic DNA.

A cDNA clone, lambda GTHP1del, encoding glutathione transferase (GST) P1-1, was isolated from a human K562 erythroleukemia cell line cDNA library. The coding sequence was lacking the codons for the N-terminal 34 amino acids. A DNA segment was designed in order to obtain the missing portion and a structure representing the entire protein. The synthetic DNA sequence was constructed to achieve efficient base pairing with Escherichia coli 16S ribosomal RNA, avoidance of internal secondary structure, and optimal codon usage for high-level protein expression in accord with the known preferences in E. coli. The truncated GST P1-1 cDNA sequence and the synthetic segment were ligated into a plasmid to give an inducible expression system. Among the resulting clones a limited number was selected by immunodetection for highest yield of GST P1-1. Maximal expression was obtained from a spontaneously mutated sequence with altered as well as deleted bases as compared to the original construct. This clone, pKXHP1, allowed heterologous expression in E. coli in yields of > 200 mg enzyme per liter culture medium. The physicochemical and catalytic properties of the recombinant protein were indistinguishable from those of the enzyme purified from human placenta.

Base Sequence↗

PubMed1995

The expression of glutathione transferase isoenzymes in human malignant lymphoma biopsies.

Glutathione transferase (GST) activity as well as the expression of different classes of GST isoenzymes were studied in 14 lymphoma biopsies. The GST activity measured with 1-chloro-2,4-dinitrobenzene as a substrate, varied almost 9-fold. The expression of GSTs classes Pi, Alpha and Mu was studied by immunoblotting using antibodies against human GSTs. All lymphoma samples displayed high levels of class Pi GST. Class Alpha and Mu GSTs expression varied from not detectable to high. The observations were confirmed by quantitation of the three classes of GST with an ELISA technique. Nine of the patients were treated with bifunctional alkylating agents. A correlation between a clinical complete response to chemotherapy and low expression of GST Alpha was noted (p < 0.02).

Alkylating Agents↗

PubMed1995

Glutathione transferase P1-1 expression in human melanoma metastases: correlation to N-RAS mutations and expression.

Expression of the detoxication enzyme glutathione transferase P1-1 (GST P1-1) at elevated levels has been noted in many types of human tumors, including melanomas. The products of the human H-RAS, K-RAS and N-RAS genes play a key role in intracellular signal transduction leading to transcriptional activation of AP-1 (Fos/Jun) responsive genes. The oncogenic mutated forms of the ras proteins are constitutively active and interfere with normal signal transduction. Mutated RAS genes as well as increased expression of wild-type ras proteins are common features in human tumors including melanoma. We have characterized 30 melanoma metastases from 23 melanoma patients with reference to N-RAS expression and mutation as well as to GST P1 expression (immunohistochemistry and genetic analysis). Twenty-three of 30 samples (70%) had high N-Ras p21 and/or N-RAS codon 61 mutations and 18 of these 23 samples also had high GST P1-1 immunoreactivity. Seven of 30 (23%) samples had low N-Ras p21 immunoreactivity and no detectable N-RAS codon 61 mutations. Six of these 7 samples (86%) also had low GST P1-1 immunoreactivity. The results indicate a statistically significant correlation (Spearman correlation coefficient, r = 0.56, p = 0.001, 2-tailed test) and provide, for the first time, indirect evidence for a possible coregulation of N-RAS and GST P1 in human malignant melanoma which should be further evaluated.

Biomarkers, Tumor↗

PubMed1994

Contribution of amino acid residue 208 in the hydrophobic binding site to the catalytic mechanism of human glutathione transferase A1-1.

Glutathione transferases (GSTs) catalyze the nucleophilic attack of the thiolate of glutathione on a variety of noxious, often hydrophobic, electrophiles. The interactions responsible for the binding of glutathione have been deduced in great detail from the 3-dimensional structures that have been solved for three different GSTs, each a member of a distinct structural class. However, the interactions of the electrophilic substrates with these enzymes are still largely unexplored. The contribution of the active-site Met208 to aromatic and benzylic chloride substitution reactions catalyzed by human class Alpha GST A1-1 has been evaluated by comparison of wild-type enzyme with variants mutated in position 208. The results show that the amino acid residue at position 208 primarily affects the aromatic substitution reaction, tested with 1-chloro-2,4-dinitrobenzene as substrate, possibly by interacting with the delocalized negative charge of the substituted ring structure in the transition state.

Base Sequence↗

PubMed1994

Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1.

The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher levels of radioisotope incorporation for the Mu than the Alpha families. Taking rat GST 3-3, 1.18 (+/- 0.05) mol of radiolabel from S-(2-nitro-4-azidophenyl)glutathione was incorporated per mol of dimeric enzyme, which could be blocked by the presence of the strong competitive inhibitor, S-tritylglutathione (Ki = 1.4 x 10(-7) M). Radiolabelling of the protein paralleled the loss of enzyme activity. Photoaffinity labelling by tritiated S-(2-nitro-4-azidophenyl)glutathione on a preparative scale (in the presence and absence of S-tritylglutathione) followed by tryptic digestion and purification of the labelled peptides indicated that GST 3-3 was specifically photolabelled; the labelled peptides were sequenced. Similarly, preparative photoaffinity labelling by S-(2-nitro-4-azidophenyl)glutathione of the rat liver 1-1 isoenzyme, the human GST A1-1 and the human-rat chimaeric GST, H1R1/1, was carried out with subsequent sequencing of radiolabelled h.p.l.c.-purified tryptic peptides. The results were interpreted by means of molecular-graphics analysis to locate photoaffinity-labelled peptides using the X-ray-crystallographic co-ordinates of rat GST 3-3 and human GST A1-1. The molecular-graphical analysis indicated that the labelled peptides are located within the immediate vicinity of the region occupied by S-substituted glutathione derivatives bound in the active-site cavity of the GSTs investigated.

Affinity Labels↗

PubMed1994

Similar toxic effect of 1,3-bis(2-chloroethyl)-1-nitrosourea on lymphocytes from human subjects differing in the expression of glutathione transferase M1-1.

Sixteen healthy donors were investigated for the presence or absence of glutathione transferase (GST) M1-1 in lymphocytes by immunodetection with polyclonal antibodies against human GST M1-1. Nine out of 16 individuals (56%) were categorized as GST M1-1 positive. Phytohaemagglutinin stimulated lymphocytes from GST M1-1 positive and negative donors were treated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and compared regarding inhibition of [3H]thymidine incorporation as a measure of cytotoxicity. No significant differences in the effect of BCNU were observed between the two groups, indicating that GST M1-1 is not an important resistance factor for BCNU.

Carmustine↗

PubMed1994

Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases.

Radiation and chemical reactions that give rise to free radicals cause the formation of highly cytotoxic base propenals, degradation products of DNA. Human glutathione transferases (GSTs; RX:glutathione R-transferase, EC 2.5.1.18) of classes Alpha, Mu, and Pi were shown to promote the conjugation of glutathione with base propenals and related alkenes. GST P1-1 was particularly active in catalyzing the reactions with the propenal derivatives, and adenine propenal was the substrate giving the highest activity. The catalytic efficiency of GST P1-1 with adenine propenal (kcat/Km = 7.7 x 10(5) M-1.s-1) is the highest so far reported with any substrate for this enzyme. In general, GST A1-1 and GST M1-1, in contrast to GST P1-1, were more active with 4-hydroxyalkenals (products of lipid peroxidation) than with base propenals. The adduct resulting from the Michael addition of glutathione to the alkene function of one of the base propenals (adenine propenal) was identified by mass spectrometry. At the cellular level, GST P1-1 was shown to provide protection against alpha, beta-unsaturated aldehydes. GST P1-1 added to the culture medium of HeLa cells augmented the protective effect of glutathione against the toxicity of adenine propenal and thymine propenal. No protective effect of the enzyme was observed in the presence of the competitive inhibitor S-hexylglutathione. GST P1-1 introduced into Hep G2 cells by electroporation was similarly found to increase their resistance to acrolein. The results show that glutathione transferases may play an important role in cellular detoxication of electrophilic alpha, beta-unsaturated carbonyl compounds produced by radical reactions, lipid peroxidation, ionizing radiation, and drug metabolism.

Acrolein↗

PubMed1994

Isoenzyme-specific quantitative immunoassays for cytosolic glutathione transferases and measurement of the enzymes in blood plasma from cancer patients and in tumor cell lines.

Enzyme-linked immunoassays (ELISAs) based on the double-antibody sandwich technique have been developed for the quantitative analysis of the major human cytosolic class Pi, Mu and Alpha glutathione transferases (GSTs). The procedures were optimized with respect to antibody concentration for coating of plates as well as other parameters in order to achieve high sensitivity and accuracy. No cross-reactivity was detected between members of the three different classes of GSTs or among the Mu class GSTs M2-2, M3-3 and M4-4 with the ELISA for GST M1-1. The ELISAs have been applied to establish the cytosolic GST profiles of 10 cell lines and to monitor the plasma GST levels in cancer patients. The results revealed that the class Pi GST was the dominant isoenzyme in six (LS 174T, HCT-8, Hu 549 Pat, K-562, U-937 and Hu 549) out of nine tumor cell lines and immortalized hepatocytes (Chang Liver). The isoenzymes A1-1 and M1-1 were determined to be the major GST components in Hep G2 and HeLa cells, respectively. In a clinical study, the majority of the patients with urinary bladder cancer were found to have increased plasma levels of both GST A1-1 and GST P1-1 (10/15), while patients with renal cancer frequently showed increases only in GST P1-1 (5/8). The results demonstrate that the ELISAs are suitable for analyzing GST phenotypes in both normal and tumor cells and in monitoring plasma levels of GSTs in cancer patients.

Cross Reactions↗

PubMed1994

Co-variation of glutathione transferase expression and cytostatic drug resistance in HeLa cells: establishment of class Mu glutathione transferase M3-3 as the dominating isoenzyme.

Qualitative and quantitative analyses of glutathione, glutathione transferases (GSTs) and other glutathione-linked enzymes in HeLa cells have been made in order to study their significance in cellular resistance to electrophilic cytotoxic agents. The cytosolic concentrations of three GSTs, GST M1-1 (53 +/- 9 ng/mg of cytosolic protein), GST P1-1 (11 +/- 3 ng/mg) and GST A1-1 (1.1 +/- 0.4 ng/mg) were quantified by isoenzyme-specific enzyme-linked immunoassays. Electrophoretic analysis and immunoblotting demonstrated another component, GST M3-3, which was identified by amino acid sequence analysis. GST M3-3 was quantified (1550 +/- 250 ng/mg) by slot-blot immunoanalysis and was the most abundant GST in HeLa cells. An additional cytosolic 13 kDa protein with high affinity for immobilized glutathione or S-hexyglutathione was found to be identical with a macrophage migration-inhibitory factor, previously identified as a lymphokine. Cells grown in roller bottles (HR) rather than in ordinary culture flasks contain a significantly lower concentration of all the GSTs and were found to be more sensitive to the cytostatic agents doxorubicin (2.3-fold), cisplatin (1.7-fold) and melphalan (1.4-fold). The cytosolic concentrations of glutathione reductase and glyoxalase I were also lower in HR cells, whereas the total glutathione concentration was unchanged and the glutathione peroxidase activity was increased. The results indicate that GSTs contribute to the cellular resistance phenotype.

Amino Acid Sequence↗

PubMed1994

A comparison of the enzymatic and physicochemical properties of human glutathione transferase M4-4 and three other human Mu class enzymes.

The multigene family of cytosolic glutathione S-transferases (GSTs) consists of four classes (Alpha, Mu, Pi, and Theta), all involved in the detoxication of reactive electrophiles. The human Mu class GSTs consist of at least four expressed isozyme subunits, GST M1, GST M2, GST M3, and GST M4, which have 70-90% amino acid sequence identity. The gene and cDNA sequences for GST M4 have been determined recently (K. E. Comstock, K. J. Johnson, D. Rifenbery, and W. D. Henner, J. Biol. Chem. (1993) 268, 16958-16965). Cloning of GST M4 cDNA into an Escherichia coli expression system permitted the production of the corresponding protein. The enzyme was purified and shown to have a relatively low specific activity with the standard GST substrate 1-chloro-2,4-dinitrobenzene (1.4 +/- 0.2 mumol min-1 mg-1 protein), but an activity equivalent to other Mu class enzymes with other tested substrates. The protein forms functional dimers composed of subunits with a M(r) of approximately 26,400. A detailed comparison of the activity with various substrates and inhibitors was performed between GST M4-4 and other human Mu class GSTs, GST M1a-1a, GST M2-2, and GST M3-3, produced in bacterial expression systems. Despite the high level of amino acid sequence identity, the enzymatic properties of these enzymes were quite different. Comparisons with the crystallographic structure of a homologous rat GST, GST 3-3, indicate that a number of the nonconserved amino acid residues can be assigned to the putative active site of GST M4-4. This suggests that diversification in the evolution of these genes has occurred primarily in the substrate binding regions to cope with an increasing variety of foreign compounds.

Amino Acid Sequence↗

PubMed1994

Cell cycle dependent sensitivity of human melanoma cells to melphalan is correlated with the activity and cellular concentration of glutathione transferases.

Glutathione transferases (GSTs) are enzymes involved in the resistance of tumor cells to bifunctional alkylating cytostatic drugs. We investigated the melphalan sensitivity together with activity and cellular concentration of GST isoenzymes of human melanoma cell line RPMI 8322 in different phases of the cell cycle. By centrifugal elutriation three cell fractions containing different proportions of cells in the G1 phase were isolated. Melphalan sensitivity was estimated by the colony formation assay. The cell fraction with the largest proportion of G1 cells was more sensitive to the drug than the fractions enriched in S and G2 cells. The GST activity of the cell fractions was measured with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate and the concentrations of GST P1-1, GST M1-1 and GST A1-1 were quantitated by use of isoenzyme-specific ELISA. The results show that there were less GST activity and lower GST P1-1 and A1-1 concentrations in the G1 cell enriched fraction, demonstrating a cell cycle dependence of GST expression. Thus, the cell fraction most sensitive to melphalan had the highest proportion of G1 cells and displayed the lowest GST activity, suggesting that the cell cycle dependent sensitivity to melphalan may at least partially depend on the expression of GSTs.

Cell Cycle↗