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B Mannervik

Publications and source records attributed to B Mannervik.

At least 145 records · Page 8Linked to original sources
PubMed1989

Chromosomal localization of human glutathione transferase genes of classes alpha, mu and pi.

The numerous human glutathione transferases may be divided into three classes, mu, alpha and pi. Using a panel of human-rodent somatic cell hybrids and DNA probes specific for each of the three classes, we have mapped a class mu gene to chromosome 3, a class alpha gene to chromosome 6 and a class pi gene to chromosome 11. The two latter assignments confirm earlier reports, whereas the assignment of the class mu gene represents a new addition to the human gene map.

Animals↗
PubMed1989

Glutathione transferase catalyzed conjugation of benzo[a]pyrene 7,8-diol 9,10-epoxide with glutathione in human skin.

Glutathione transferase (GST) activity towards racemic as well as the resolved enantiomers of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene (anti-BPDE) and 1-chloro-2,4-dinitrobenzene (CDNB) was measured in post-microsomal supernatants (PMS) obtained from eight human skin samples. All preparations showed significant activity towards anti-BPDE and an almost exclusive preference for the more tumourigenic (+)-enantiomer. The specific activity towards (+)-anti-BPDE varied about five-fold between different PMS (range 147-781 pmol/min per mg protein) whereas the variation in specific activities towards CDNB was about two-fold (range 30-71 nmol/min per mg protein). The activities obtained with PMS at saturating concentrations of racemic anti-BPDE were about half of the activity towards the (+)-enantiomer indicating that (-)-anti-BPDE competitively inhibits conjugation of the (+)-form. No correlation was evident between the activities towards (+)-anti-BPDE and CDNB implying that different classes of GST isoenzymes participated in the two different reactions. Immunoblot analysis revealed the presence of Class Alpha and Pi isoenzymes whereas Class Mu isoenzymes seemed to be absent in the human skin samples analyzed. Quantitatively, the Class Pi isoenzyme(s) predominated in all skin samples and the amount of enzyme was about 1-3 micrograms GST Pi/mg PMS protein. The almost exclusive conjugation of (+)-anti-BPDE by PMS and previous results with GST Pi enzymes from human placenta suggested that this type of enzymes catalysed the conjugation reaction. The five-fold variation in specific activity towards (+)-anti-BPDE observed among the different PMS may be explained by individual differences in GST Pi content or by the presence of endogenous modifiers of GST activity towards the diol-epoxide.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1988

Isoenzymes of glutathione transferase in rat small intestine.

The major glutathione transferases in the rat small-intestine cytosol were isolated and characterized. The enzymes active with 1-chloro-2,4-dinitrobenzene as second substrate were almost quantitatively recovered after affinity chromatography on immobilized S-hexylglutathione. The different basic forms of glutathione transferase, which account for 90% of the activity, were resolved by chromatofocusing. Fractions containing enzymes with lower isoelectric points were not further resolved. The isolated fractions were characterized by their elution position in chromatofocusing, apparent subunit Mr, reactions with specific antibodies, substrate specificities and inhibition characteristics. The major basic forms identified were glutathione transferases 1-1, 4-4 and 7-7. In addition, evidence for the presence of a variant form of subunit 1, as well as trace amounts of subunits 2 and 3, was obtained. A significant amount of transferase 8-8 in the fraction of acidic enzyme forms was demonstrated by immunoblot and Ouchterlony double-diffusion analysis. In the comparison of the occurrence of the different forms of glutathione transferase in liver, lung, kidney and small intestine, it was found that the small intestine is the richest source of glutathione transferase 7-7.

Animals↗
PubMed1988

Relaxed thiol substrate specificity of glutathione transferase effected by a non-substrate glutathione derivative.

Rat glutathione transferase 4-4 catalyzed the conjugation of 2-mercaptoethanol with 1-chloro-2,4-dinitrobenzene in the presence of S-methyl-glutathione. The reaction was linearly dependent on enzyme concentration and saturation was seen with respect to both 2-mercaptoethanol and S-methyl-glutathione concentration. High concentrations of S-methyl-glutathione were inhibitory. The results suggest that the natural substrate glutathione has two distinct functions in the normal catalytic reaction, (i) induction of a catalytically competent conformation of the enzyme and (ii) provision of the substrate sulfhydryl group in the reaction catalyzed.

Animals↗
PubMed1988

Paradoxical inhibition of rat glutathione transferase 4-4 by indomethacin explained by substrate-inhibitor-enzyme complexes in a random-order sequential mechanism.

Under standard assay conditions, with 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrate, rat glutathione transferase 4-4 is strongly inhibited (I50 = 1 microM) by indomethacin. No other glutathione transferase investigated is significantly inhibited by micromolar concentrations of indomethacin. Paradoxically, the strong inhibition of glutathione transferase 4-4 was dependent on high (millimolar) concentrations of CDNB; at low concentrations of this substrate or with other substrates the effect of indomethacin on the enzyme was similar to the moderate inhibition noted for other glutathione transferases. In general, the inhibition of glutathione transferases can be explained by a random-order sequential mechanism, in which indomethacin acts as a competitive inhibitor with respect to the electrophilic substrate. In the specific case of glutathione transferase 4-4 with CDNB as substrate, indomethacin binds to enzyme-CDNB and enzyme-CDNB-GSH complexes with an even greater affinity than to the corresponding complexes lacking CDNB. Under presumed physiological conditions with low concentrations of electrophilic substrates, indomethacin is not specific for glutathione transferase 4-4 and may inhibit all forms of glutathione transferase.

Animals↗
PubMed1988

Leukotriene C synthase in mouse mastocytoma cells. An enzyme distinct from cytosolic and microsomal glutathione transferases.

Leukotriene C4 synthesis was studied in preparations from mouse mastocytoma cells. Enzymic conjugation of leukotriene A4 with glutathione was catalysed by both the cytosol and the microsomal fraction. The specific activity of the microsomal fraction (7.8 nmol/min per mg of protein) was 17 times that of the cytosol fraction. The cytosol fraction of the mastocytoma cells contained two glutathione transferases, which were purified to homogeneity and characterized. A microsomal glutathione transferase was purified from mouse liver; this enzyme was shown by immunoblot analysis to be present in the mastocytoma microsomal fraction at a concentration one-tenth or less of that in the liver microsomal fraction. Both the cytosolic and the microsomal glutathione transferases in the mastocytoma cells were identified with enzymes previously characterized, by determining specific activities with various substrates, sensitivities to inhibitors, reactions with antibodies, and physical properties. The purified microsomal glutathione transferase from liver was inactive with leukotriene A4 or its methyl ester as substrate. The cytosolic enzymes displayed activity with leukotriene A4, but their specific activities and intracellular concentrations were too low to account for the leukotriene C4 formation in the mastocytoma cells. The microsomal fraction of the cells contained an enzyme distinguishable by various criteria from the previously studied glutathione transferases. This membrane-bound enzyme, leukotriene C synthase (leukotriene A4:glutathione S-leukotrienyltransferase), appears to carry the main responsibility for the biosynthesis of leukotriene C4.

Animals↗
PubMed1988

Activation and inhibition of microsomal glutathione transferase from mouse liver.

Mouse liver microsomal glutathione transferase was purified in an N-ethylmaleimide-activated as well as an unactivated form. The enzyme had a molecular mass of 17 kDa and a pI of 8.8. It showed cross-reactivity with antibodies raised against rat liver microsomal glutathione transferase, but not with any of the available antisera raised against cytosolic glutathione transferases. The fully N-ethylmaleimide-activated enzyme could be further activated 1.5-fold by inclusion of 1 microM-bromosulphophthalein in the assay system. The latter effect was reversible, which was not the case for the N-ethylmaleimide activation. At 20 microM-bromosulphophthalein the activated microsomal glutathione transferase was strongly inhibited, while the unactivated form was activated 2.5-fold. Inhibitors of the microsomal glutathione transferase from mouse liver showed either about the same I50 values for the activated and the unactivated form of the enzyme, or significantly lower I50 values for the activated form compared with the unactivated form. The low I50 values and the steep slope of the activity-versus-inhibitor-concentration curves for the latter group of inhibitors tested on the activated enzyme indicate a co-operative effect involving conversion of activated enzyme into the unactivated form, as well as conventional inhibition of the enzyme.

Animals↗
PubMed1988

Conjugation of styrene oxide by the basic and acidic forms of glutathione transferase in the human fetal liver.

Two forms of glutathione transferase were isolated by means of isoelectric focusing of human fetal liver cytosol preparations. The enzyme activity was measured with 1-chloro-2,4-dinitrobenzene as the electrophilic substrate. One peak focused at pH 9-10 (basic form) and the other at pH 4-5 (acidic form). The basic and the acidic forms are representatives of glutathione transferase classes alpha and tau, respectively. These classes constitute two of the three classes defined for cytosolic forms of the enzyme in several mammalian species [Mannervik et al., Proc. natn. Acad. Sci. USA 82: 7202-7206, 1985]. Only the basic fraction isolated from human fetal liver catalyzed the conjugation of styrene oxide with glutathione at a significant rate. The kinetics of this form were studied keeping the concentration of styrene oxide constant (6 mM) and varying the glutathione concentration from 0.05 to 25 mM. The enzyme activity displayed non-Michaelis-Menten kinetics. The basic and acidic forms of glutathione transferase from a fetal liver were purified to homogeneity. Both purified forms catalyzed the conjugation of glutathione with styrene oxide. The kinetics were studied at varying glutathione concentrations and for both forms, it was found to be of a non-Michaelis-Menten type. The results are consistent with previous findings in the cytosolic fraction [Pacifici et al., Biochem. Pharmac. 30: 3367-3371, 1981] and show that the non-Michaelian kinetics observed with glutathione in human fetal liver cytosol are reflections of the intrinsic properties of the basic as well as the acid form of this enzyme and not primarily depending on the simultaneous catalytic action of the two forms.

Cytosol↗
PubMed1988

Glutathione transferases--structure and catalytic activity.

The glutathione transferases are recognized as important catalysts in the biotransformation of xenobiotics, including drugs as well as environmental pollutants. Multiple forms exist, and numerous transferases from mammalian tissues, insects, and plants have been isolated and characterized. Enzymatic properties, reactions with antibodies, and structural characteristics have been used for classification of the glutathione transferases. The cytosolic mammalian enzymes could be grouped into three distinct classes--Alpha, Mu, and Pi; the microsomal glutathione transferase differs greatly from all the cytosolic enzymes. Members of each enzyme class have been identified in human, rat, and mouse tissues. Comparison of known primary structures of representatives of each class suggests a divergent evolution of the enzyme proteins from a common precursor. Products of oxidative metabolism such as organic hydroperoxides, epoxides, quinones, and activated alkenes are possible "natural" substrates for the glutathione transferases. Particularly noteworthy are 4-hydroxyalkenals, which are among the best substrates found. Homologous series of substrates give information about the properties of the corresponding binding site. The catalytic mechanism and the active-site topology have been probed also by use of chiral substrates. Steady-state kinetics have provided evidence for a "sequential" mechanism.

Catalysis↗
PubMed1988

Structural classes of glutathione transferase: distinctions between isoenzymes and enzymes.

The amino acid sequences of the five cytosolic rat glutathione transferases 1-1, 2-2, 3-3, 4-4, and 7-7 of three different classes have been compared. Alignments demonstrate 68%-78% positional identity between isoenzymes within the same class, and 29%-32% between the enzymes of different classes. Of the 209-221 residues in the structures, those strictly conserved are limited to 24, over half of which are charged residues and Leu, while few are Gly and Pro that in related proteins otherwise are often maintained because of space restrictions and conserved conformations. In spite of the limited sequence homologies, hydropathy profiles and predictions of secondary structures emphasize the relationship between the three enzyme classes. The predictions indicate alternating alpha-helices and beta-strands, a chain fold typical for alpha/beta protein structures. Glutathione transferases within a class are highly similar and may be regarded as true isoenzymes, while transferases of different classes appear to occupy an intermediate stage between isoenzymes and discrete enzymes.

Amino Acid Sequence↗
PubMed1987

Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases.

The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism.

Aldehydes↗
PubMed1987

Electron paramagnetic resonance study of the active site of copper-substituted human glyoxalase I.

Zn2+ in native glyoxalase I from human erythrocytes can be replaced by Cu2+, giving an inactive enzyme. Cu2+ was demonstrated to compete with the activating metals Zn2+ and Mn2+, indicating a common binding site on the enzyme for these metal ions. The electron paramagnetic resonance (EPR) spectra of 63Cu(II) glyoxalase I at 77 K and of its complexes with glutathione and some glutathione derivatives are characteristic of Cu2+ in an elongated octahedral coordination (g parallel = 2.34, g perpendicular = 2.09, and A parallel = 14.2 mT). The low-field bands of the free enzyme are asymmetric and become symmetrical upon addition of glutathione or S-(p-bromobenzyl)glutathione but not S-(D-lactoyl)glutathione. The results indicate the existence of two conformations of Cu(II) glyoxalase I, in agreement with the effects caused by these compounds on the protein fluorescence. The copper hyperfine line at low field in the EPR spectrum of the S-(p-bromobenzyl)glutathione complex of 63Cu(II) glyoxalase I shows a triplet structure, indicative of coupling to one nitrogen ligand in the equatorial plane. Similar results were obtained with the glutathione complex. By addition of the spectrum of the S-(p-bromobenzyl)glutathione complex and a spectrum corresponding to two nitrogen ligands with two different coupling constants, a good fit was obtained for the low-field region of the asymmetric spectrum of free 63Cu(II) glyoxalase I. The first two spectra are assumed to correspond to two separate conformational states of the enzyme. The results demonstrate that at least one nitrogen ligand is involved in the binding of Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1987

The trans-stilbene oxide-active glutathione transferase in human mononuclear leucocytes is identical with the hepatic glutathione transferase mu.

A glutathione transferase from human mononuclear leucocytes with high activity towards trans-stilbene oxide (GT-tSBO) was purified. GT-tSBO is expressed in only about 50% of the individuals studied. As judged from activity measurements, immunological studies and the fact that only those individuals who express glutathione transferase mu have high activity towards trans-stilbene oxide, it is concluded that the hepatic transferase mu is identical with the glutathione transferase (GT-tSBO) in mononuclear leucocytes.

Dinitrochlorobenzene↗
PubMed1987

Identification of a novel glutathione transferase in human skin homologous with class alpha glutathione transferase 2-2 in the rat.

Six forms of glutathione transferase with pI values of 4.6, 5.9, 6.8, 7.1, 8.5 and 9.9 have been isolated from the cytosol fraction of normal skin from three human subjects. The three most abundant enzymes were an acidic Class Pi transferase (pI 4.6; apparent subunit Mr 23,000), a basic Class Alpha transferase (pI 8.5; apparent subunit Mr 24,000) and an even more basic glutathione transferase of Class Alpha (pI 9.9; apparent subunit Mr 26,500). The last enzyme, which was previously unknown, accounts for 10-20% of the glutathione transferase in human skin. The novel transferase showed greater similarities with rat glutathione transferase 2-2, another Class Alpha enzyme, than with any other known transferase irrespective of species. The most striking similarities included reactions with antibodies, amino acid compositions and identical N-terminal amino acid sequences (16 residues). The close relationship between the human most basic and the rat glutathione transferase 2-2 supports the classification of the transferases previously proposed and indicates that the similarities between enzymes isolated from different species are more extensive than had been assumed previously.

Amino Acids↗
PubMed1987

Expression of class Pi glutathione transferase in human malignant melanoma cells.

The occurrence of glutathione transferase in human malignant melanoma cell lines and solid tumor material has been analyzed and compared with the enzyme composition in fibroblasts and naevus samples. All cells and tissues investigated contained essentially only the acidic class Pi glutathione transferase as demonstrated by SDS-PAGE and immunoblotting. The enzyme was purified from tumor material and characterized. Its intracellular concentration was significantly higher in all the melanoma cell preparations analyzed than in the non-malignant cells, supporting the view that the class Pi glutathione transferase may contribute to the drug resistance that is characteristic of malignant melanoma.

Breast Neoplasms↗
PubMed1987

Detoxification of styrene oxide by human liver glutathione transferase.

Cytosolic glutathione transferase (GST) was investigated in four human livers. The profile of GST activity was determined by isoelectric focusing using 1-chloro-2,4-dinitrobenzene as the electrophilic substrate. Three livers contained at least one basic and a near-neutral isoenzyme (GST mu). GST mu was not detectable in the fourth liver. The kinetics of GST with styrene oxide as the electrophilic substrate were studied in the cytosolic fraction, with the pooled fractions from isoelectric focusing containing high activity of GST mu transferase, and with GST mu purified to homogeneity. The cytosol obeyed Michaelis-Menten kinetics when styrene oxide was used as the variable substrate. The average (+/- s.e.m.) of the Vmax and Km were 21.9 +/- 7.9 nmol min-1mg-1 and 4.9 +/- 0.4 mM, respectively. At varying concentrations of glutathione, the enzyme did not obey Michaelis-Menten kinetics. Such kinetics were also observed with the fractions from isoelectric focusing and with the homogeneous GST mu fraction. The Eadie-Hofstee plot showed two phases: one with a low and another with a high Km value. The apparent Km values for the cytosol were 0.035 +/- 0.022 and 0.88 +/- 0.36 mM. The kinetic pattern of purified GST mu is consistent with that found in the cytosol.

Adult↗