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Biomedical subjects

B Mannervik

Publications and source records attributed to B Mannervik.

At least 163 records · Page 9Linked to original sources
PubMed1986

Rat glutathione transferase 8-8, an enzyme efficiently detoxifying 4-hydroxyalk-2-enals.

Rat glutathione transferase 8-8 is one of the less abundant cytosolic glutathione transferases, accounting for approx. 1% of the total activity with 1-chloro-2,4-dinitrobenzene in liver. The enzyme is eluted at pH 6.3 upon chromatofocusing and has so far been identified in liver, kidney, lung and testis. Characteristic properties include high relative activity with ethacrynic acid (70% of the specific activity with 1-chloro-2,4-dinitrobenzene) and an apparent subunit Mr of 24 500. The most significant property noted is the high catalytic activity in the conjugation of 4-hydroxyalk-2-enals, major products of lipid peroxidation. The catalytic efficiency with these substrates exceeds corresponding values for all known substrates tested with any glutathione transferase, which suggests that transferase 8-8 may have evolved to detoxify 4-hydroxyalk-2-enals.

Animals↗
PubMed1986

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.

Amino Acids↗
PubMed1986

Two distinct forms of glutathione transferase from human foetal liver. Purification and comparison with isoenzymes isolated from adult liver and placenta.

Isoelectric focusing of a cytosol fraction from human foetal liver revealed the existence of an acidic and a basic isoenzyme of GSH transferase. The acidic and basic forms of GSH transferase were purified in good yield by use of ion-exchange chromatography on DEAE-cellulose followed by affinity chromatography on S-hexyl-GSH coupled to epoxy-activated Sepharose 6B. The content of the acidic and the basic isoenzymes of GSH transferase together was calculated to constitute 1-2% of the soluble proteins in the hepatic cytoplasm. Physical, catalytic and immunological analyses of the acidic and the basic isoenzymes from foetal liver demonstrated unambiguously that the two forms are different structures with distinct properties. On the other hand, the results show clearly extensive similarities between the foetal acidic transferase and transferase pi from human placenta as well as between the foetal basic form and the basic isoenzymes isolated from adult liver. An exception is that both foetal enzymes seem to be considerably more efficient in catalysing the conjugation of GSH with styrene 7,8-epoxide than the corresponding adult forms of GSH transferase.

Female↗
PubMed1986

Error structure as a function of substrate and inhibitor concentration in enzyme kinetic experiments.

Optimal design of experiments as well as proper analysis of data are dependent on knowledge of the experimental error. A detailed analysis of the error structure of kinetic data obtained with acetylcholinesterase showed conclusively that the classical assumptions of constant absolute or constant relative error are inadequate for the dependent variable (velocity). The best mathematical models for the experimental error involved the substrate and inhibitor concentrations and reflected the rate law for the initial velocity. Data obtained with other enzymes displayed similar relationships between experimental error and the independent variables. The new empirical error functions were shown superior to previously used models when utilized in weighted non-linear-regression analysis of kinetic data. The results suggest that, in the spectrophotometric assays used in the present study, the observed experimental variance is primarily due to errors in determination of the concentrations of substrate and inhibitor and not to error in measuring the velocity.

Acetylcholine↗
PubMed1986

Cytosolic rat liver glutathione transferase 4-4. Primary structure of the protein reveals extensive differences between homologous glutathione transferases of classes alpha and mu.

The primary structure of the class Mu glutathione transferase 4-4 from rat liver was determined. The structural data characterize a class Mu protein within an enzyme family for which three classes have been distinguished (Alpha, Mu, Pi). The structure was determined by analysis of peptides obtained after treatment with trypsin. Glu-specific protease and CNBr. The protein is composed of two identical subunits, each with 217 amino acid residues. No evidence for microheterogeneity or for the presence of modified residues was encountered. The primary structure was found to be strictly homologous with corresponding parts in known regions of other class Mu enzymes of rat, mouse, human and bovine origin. Relationships to the cytosolic enzyme of other classes (Pi and Alpha) are considerably more distant. A comparison with the entire chain of the class Alpha subunit 1 from rat liver was carried out by three methods, alignment of amino acid sequences, correlation of hydrophilicity plots and predictions of secondary structures. All methods reveal weak similarities but also large differences. The overall positional identity is only 26%. Combined, the results establish the first complete class Mu structure, show distant inter-class relationships, and relate subunit 4 (class Mu) and subunit 1 (class Alpha) in a family of enzymes rather than in a group of isoenzymes.

Amino Acid Sequence↗
PubMed1986

Reduction of 2,4,6-trinitrobenzenesulfonate by glutathione reductase and the effect of NADP+ on the electron transfer.

Glutathione reductase has been found to catalyze an NAD(P)H-dependent electron transfer to 2,4,6-trinitrobenzenesulfonate (TNBS). In the presence of oxygen TNBS is not consumed in the reaction, but is rapidly reoxidized with concomitant production of hydrogen peroxide. Cytochrome c can replace oxygen as the final electron acceptor, indicating that a one-electron transfer takes place. The rate is slightly higher in the absence than in the presence of oxygen, ruling out superoxide anion as an obligatory intermediate in cytochrome c reduction. In the absence of oxygen (or cytochrome c), TNBS limits the reaction and accepts a total of four electrons. The TNBS-dependent NADPH (or NADH) oxidation is markedly stimulated by NADP+, and to a smaller extent also by NAD+. The TNBS-dependent reactions are inhibited by excess of NADPH but not by NADH. The kinetics of these reactions are consistent with a branching reaction mechanism in which a pathway including a ternary complex between the two-electron reduced enzyme and NADP+ has the highest turnover. NADPH-dependent reductions of ferricyanide or 2,6-dichloroindophenol catalyzed by glutathione reductase are also markedly influenced by NADP+. Evidently NADP+ facilitates a shift of the catalyzed reaction from the normal two-electron reduction of glutathione disulfide to a more unspecific one-electron reduction of other acceptors. Spectral as well as kinetic data suggest that the rate of radical formation limits the reactions with the artificial electron acceptors and that NADP+ promotes this rate-limiting step.

Cytochrome c Group↗
PubMed1986

Simple inhibition studies for distinction between homodimeric and heterodimeric isoenzymes of glutathione transferase.

Simple inhibition studies in which fractional velocity is measured as a function of inhibitor concentration were used to distinguish heterodimeric from homodimeric isoenzymes of glutathione transferase. Homodimeric isoenzymes from rat, mouse, and human tissues were shown to give graphs of fractional velocity versus the logarithm of inhibitor concentration that were characterized by a sigmoid curve shape and a maximal slope of -0.58 at 50% inhibition, characteristic for linear inhibition of monomeric or non-cooperative oligomeric enzymes. In contrast, inhibition curves for heterodimeric isoenzymes from rat liver displayed significant deviations from these characteristics. The basis for the identification of heterodimers was the finding that the kinetic properties of subunits were additive such that the inhibition curve of a heterodimeric isoenzyme could be predicted from those of the corresponding homodimers. The method should be valuable in the differentiation between the multiple forms of glutathione transferase in tissues not previously characterized. It is suggested that the method should be applicable for discrimination also in other isoenzyme families consisting of oligomeric structures of identical and nonidentical subunits.

Animals↗
PubMed1986

Glutathione transferases in rat lung: the presence of transferase 7-7, highly efficient in the conjugation of glutathione with the carcinogenic (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The enzyme-catalysed conjugation of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE] with glutathione (GSH) by cytosolic GSH transferases isolated primarily from rat lung has been studied. GSH transferase 4-4 was active in the GSH conjugation of anti-BPDE, whereas transferases 2-2 and 3-3 showed little activity. GSH transferase 1-1 did not contribute to the activity since significant amounts were not detected in the rat lung. Activity was also obtained with several acidic pulmonary GSH transferases and with a newly described form, transferase 7-7, also isolated from rat kidney and from hyperplastic liver nodules. The catalytic efficiency (kcat/Km) of transferase 7-7 was seven times that of transferase 4-4, the most active rat transferase previously identified. When the GSH concentration was varied at constant (+/-)-anti-BPDE concentration in the presence of transferases 4-4, 7-7 or the major acidic transferase, non-linear Lineweaver-Burk plots were obtained. Resolution of the GSH conjugates of the two enantiomers of (+/-)-anti-BPDE by h.p.l.c. showed that all isoenzymes with notable activity were selective (greater than or equal to 97%) for the (+)-enantiomer of anti-BPDE, which is generally considered to be the most carcinogenic form of BPDE. The possibility that one enantiomer inhibits the conjugation of the other enantiomer with GSH cannot be excluded and may quantitatively affect the results obtained.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1986

Differences in stereoselectivity and catalytic efficiency of three human glutathione transferases in the conjugation of glutathione with 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene.

The kinetics of the enzyme-catalyzed conjugation of glutathione with (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha -oxy-7,8,9,10 -tetrahydrobenzo(a)pyrene [(+/-)-anti-BPDE] have been studied with the following human cytosolic glutathione transferases: the basic (alpha-epsilon) and near-neutral (mu) isoenzymes from liver, and the acidic (pi) isoenzyme from placenta. When the BPDE concentration was varied (using 5 mM glutathione) the apparent Vmax values for transferases alpha-epsilon, mu, and pi were 38, 570, and 825 nmol X mg-1 X min-1, respectively, with corresponding apparent Km values of 88, 27, and 54 microM. The apparent Km values for glutathione [using 80 microM (+/-)-anti-BPDE] were 0.4, 0.7, and 0.1 mM for transferase alpha-epsilon, mu, and pi, respectively. The glutathione conjugates formed with the two enantiomers of (+/-)-anti-BPDE were resolved by high performance liquid chromatography. The percentages of conjugates derived from the highly carcinogenic (+)-enantiomer were 59, 60, and greater than or equal to 90% for transferases alpha-epsilon, mu, and pi, respectively. The separate enantiomers of anti-BPDE were assayed in experiments with transferases mu and pi. Both enantiomers were substrates for transferase mu, but only the (+)-enantiomer gave measurable activity with transferase pi. A 3-fold increase in Vmax and Km values for transferase pi was obtained with (+)-anti-BPDE as compared with the racemic substrate and could be quantitatively accounted for by the finding that (-)-anti-BPDE serves as a competitive inhibitor for transferase pi.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1985

Kinetic independence of the subunits of cytosolic glutathione transferase from the rat.

The steady-state kinetics of the dimeric glutathione transferases deviate from Michaelis-Menten kinetics, but have hyperbolic binding isotherms for substrates and products of the enzymic reaction. The possibility of subunit interactions during catalysis as an explanation for the rate behaviour was investigated by use of rat isoenzymes composed of subunits 1, 2, 3 and 4, which have distinct substrate specificities. The kinetic parameter kcat./Km was determined with 1-chloro-2,4-dinitrobenzene, 4-hydroxyalk-2-enals, ethacrynic acid and trans-4-phenylbut-3-en-2-one as electrophilic substrates for six isoenzymes: rat glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4. It was found that the kcat./Km values for the heterodimeric transferases 1-2 and 3-4 could be predicted from the kcat./Km values of the corresponding homodimers. Likewise, the initial velocities determined with transferases 3-3, 3-4 and 4-4 at different degrees of saturation with glutathione and 1-chloro-2,4-dinitrobenzene demonstrated that the kinetic properties of the subunits are additive. These results show that the subunits of glutathione transferase are kinetically independent.

Animals↗
PubMed1985

Isoenzymes of glutathione transferase in rat kidney cytosol.

Glutathione transferases from rat kidney cytosol were purified about 40-fold by chromatography on S-hexylglutathione linked to epoxy-activated Sepharose 6B. Further purification by fast protein liquid chromatography with chromatofocusing in the pH interval 10.6-7.6 resolved five major peaks of activity with 1-chloro-2,4-dinitrobenzene as the second substrate. Four of the peaks were identified with rat liver transferases 1-1, 1-2, 2-2 and 4-4 respectively. The criteria used for identification included physical properties, reactions with specific antibodies, substrate specificities and sensitivities to several inhibitors. The fourth major peak is a 'new' form of transferase, which has not been found in rat liver. This isoenzyme, glutathione transferase 7-7, has a lower apparent subunit Mr than any of the transferases isolated from rat liver cytosol, and does not react with antibodies raised against the liver enzymes. Glutathione transferases 3-3 and 3-4, which are abundant in liver, were only present in very small amounts. In a separate chromatofocusing separation in a lower pH interval, an additional peak was eluted at pH 6.3. This isoenzyme is characterized by its high activity with ethacrynic acid.

Animals↗
PubMed1985

Selective expression of glutathione transferase isoenzymes in chemically induced preneoplastic rat hepatocyte nodules.

Isoenzymes of glutathione transferase were shown to occur at selectively altered levels in rat hepatocyte nodules produced by 2-acetylaminofluorene treatment. Changes were measured by different substrates, antibodies raised against purified glutathione transferases, and by purification of the major isoenzymes. Isoenzymes composed of subunits 1, 2 and 3, expressed in normal liver tissue, all occurred at increased concentrations in nodules, whereas the level of transferase 4-4 was decreased. The most conspicuous change was the appearance of glutathione transferase 7-7 (or transferase P), the concentration of which in negligible in normal liver.

2-Acetylaminofluorene↗
PubMed1985

Purification of major basic glutathione transferase isoenzymes from rat liver by use of affinity chromatography and fast protein liquid chromatofocusing.

Seven major isoenzymes of glutathione transferase with isoelectric points ranging from pH 6.9 to 10 were isolated from rat liver cytosol. The purification procedure included affinity chromatography on immobilized S-hexylglutathione followed by high-performance liquid chromatofocusing. Characteristics, such as physical properties, reactions with antibodies, specific activities with various substrates, kinetic constants, and sensitivities to a set of inhibitors, are given for discrimination and identification of the different isoenzymes. The multiple forms of the enzyme correspond to glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4, and 4-4 in the recently introduced nomenclature [W.B. Jakoby et al. (1984) Biochem. Pharmacol. 33, 2539-2540]. A seventh form appears to be a heterodimeric protein composed of subunit 3 and an as yet unidentified subunit.

Animals↗
PubMed1985

Leukotriene C4 formation catalyzed by three distinct forms of human cytosolic glutathione transferase.

The ability of three distinct types of human cytosolic glutathione transferase to catalyze the formation of leukotriene C4 from glutathione and leukotriene A4 has been demonstrated. The near-neutral transferase (mu) was the most efficient enzyme with Vmax= 180 nmol X min-1 X mg-1 and Km= 160 microM. The Vmax and Km values for the basic (alpha-epsilon) and the acidic (pi) transferases were 66 and 24 nmol X min-1 X mg-1 and 130 and 190 microM, respectively. The synthetic methyl ester derivative of leukotriene A4 was somewhat more active as a substrate for all the three forms of the enzyme.

Arachidonic Acids↗
PubMed1985

Structural evidence for three different types of glutathione transferase in human tissues.

Cytosolic glutathione transferase was purified from human placenta and human liver. Three different forms of the enzyme were obtained, the acidic (pi), the near-neutral (mu), and the basic (alpha-epsilon) forms; two had free alpha-amino groups (pi, mu) and one had a blocked alpha-amino group (alpha-epsilon). N-terminal sequence analyses and total compositions gave clearly different results for each form, although transferases pi and mu showed 35% sequence homology in the N-terminal regions, with a 1-residue shift in starting position. Consequently, the proteins are concluded to be products of three discrete but related genes.

Amino Acid Sequence↗
PubMed1985

Differences in the occurrence of glutathione transferase isoenzymes in rat lung and liver.

Cytosolic GSH transferases have been purified from rat lung by affinity chromatography followed by chromatofocusing. On the criteria of order of elution, substrate specificity, apparent subunit Mr, sensitivity to inhibitors, and reaction with antibodies, transferase subunits equivalent to subunits 2, 3, and 4, in the binary combinations occurring in liver, were identified. However, subunit 1 (and therefore transferases 1-1 and 1-2) was not detected. The most conspicuous difference is the presence in lung of a new form, eluting at pH 8.7, which is not detected in rat liver. This isoenzyme (transferase "pH 8.7") is characterized by its low apparent subunit Mr and high efficiency in the conjugation of glutathione with anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, considered the ultimate carcinogen of benzo(a)-pyrene.

Animals↗
PubMed1985

Inhibitors for distinction of three types of human glutathione transferase.

A set of inhibitors that are useful for distinction of three types of human cytosolic glutathione transferase is presented. The near-neutral transferase is inhibited most effectively by Cibacron blue (I50 = 0.05 microM), the acidic transferase by Cibacron blue (I50 = 0.5 microM), and the basic transferase by tributyltin acetate (I50 = 0.1 microM). The use of any of these two compounds makes possible differentiation between all three types of human transferase.

Cytosol↗