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Biomedical subjects

B Mannervik

Publications and source records attributed to B Mannervik.

At least 217 records · Page 12Linked to original sources
PubMed1981

Glutathione S-transferase (transferase pi) from human placenta is identical or closely related to glutathione S-transferase (transferase rho) from erythrocytes.

Glutathione S-transferase (RX: glutathione R-transferase, EC 2.5.1.18) from human placenta has been purified to homogeneity. This enzyme, transferase pi, is an acidic protein (isoelectric point at pH 4.8) composed of two subunits. The molecular weights for the dimer and monomer were determined by independent methods as 47,000 and 23,400, respectively. These properties are not significantly different from those of glutathione S-transferase rho from human erythrocytes. Antibodies to transferase pi reacted with the enzyme from erythrocytes but not with the basic transferases alpha - epsilon and the neutral transferase mu isolated from human liver. Antibodies to the latter enzymes did not react with the transferase from placenta. Further similarities between transferases pi and rho appear in amino acid compositions, kinetic constants and substrate specificities. Both the placental and the erythrocyte enzyme have considerably higher activity with ethacrynic acid than any other of the human glutathione S-transferases. The glutathione S-transferase could be distinguished from two additional acidic glutathione-dependent enzymes, glyoxalase I and selenium-dependent glutathione peroxidase. It is concluded that transferase pi from placenta is identical with or very closely related to transferase rho from erythrocytes.

Amino Acids↗
PubMed1981

Effect of inducers of drug-metabolizing enzymes on glutathione reductase and glutathione peroxidase in rat liver.

Cytosolic glutathione reductase activity of rat liver was shown to increase to about 250% of control values after treatment of the animals by intraperitoneal injections of trans-stilbene oxide. The time course and dose-response relationship of the induction brought about by trans-stilbene oxide were determined. The increase of activity was accompanied by a similar increase of protein precipitable by antibodies to rat liver glutathione reductase. These results strongly indicate that increase of glutathione reductase activity in response to treatment with this inducer is the result of true induction. Phenobarbital and 3-methylcholanthrene increased the activities per mg protein in the cytosol fraction by 80 and 24%, respectively. Glutathione reductase was purified to homogeneity from rats treated with trans-stilbene oxide. The molecular and kinetic properties investigated were not significantly different from those of the enzyme from control animals. Selenium-dependent glutathione peroxidase was not induced in the hepatic cytosol of animals treated with trans-stilbene oxide.

Animals↗
PubMed1981

Purification and immunological studies of glutathione reductase from rat liver. Evidence for an antigenic determinant at the nucleotide-binding domain of the enzyme.

Glutathione reductase has been purified to homogeneity by a method which is an improvement of an earlier procedure (Carlberg, I. and Mannervik, B. (1975) J. Biol. Chem. 250, 5475-5480). The new steps in the purification scheme include affinity chromatography on 2',5' ADP-Sepharose 4B. Antibodies to glutathione reductase from rat liver were raised in rabbits and used for analysis of the enzyme by quantitative 'rocket' immunoelectrophoresis. Glutathione reductase from human erythrocytes, porcine erythrocytes, and calf-liver gave precipitin lines showing partial identity with the rat liver enzyme in Ouchterlony double diffusion experiments. Enzyme from spinach, yeast (Saccharomyces cerevisiae), and the photosynthetic bacterium Rhodospirillum rubrum did not give precipitates with the antibodies to the enzyme from rat liver. Titration of glutathione reductase from the different sources with antibodies confirmed the cross-reactivity of the mammalian enzymes; the human enzyme giving the strongest heterologous reaction. No reaction was observed with the enzyme from spinach, yeast, and Rhodospirillum rubrum. NADPH, NADP+, and 2',5' ADP were found to inhibit the interaction between antibodies and glutathione reductase from rat liver and human erythrocytes. NADH, glutathione, or glutathione disulfide did not protect the enzyme from reacting with the antibodies. It is concluded that glutathione reductase has an antigenic binding site for the antibodies at the pyridine nucleotide-binding site of the enzyme molecule.

Animals↗
PubMed1981

Probing the active site of glyoxalase I from human erythrocytes by use of the strong reversible inhibitor S-p-bromobenzylglutathione and metal substitutions.

Glyoxalase I from human erythrocytes was studied by use of the strong reversible competitive inhibitor S-p-bromobenzylglutathione. Replacements of cobalt, manganese and magnesium for the essential zinc in the enzyme were made by a new procedure involving 10% methanol as a stabilizer of the enzyme. The K(m) value for the adduct of methylglyoxal and glutathione was essentially unchanged by the metal substitutions, whereas the inhibition constant for S-p-bromobenzylglutathione increased from 0.08mum for the Zn-containing enzyme to 1.3, 1.7 and 2.4mum for Co-, Mn- and Mg-glyoxalase I respectively. Binding of the inhibitor to the enzyme caused quenching of the tryptophan fluorescence of the protein, from which the binding parameters could be determined by the use of non-linear regression analysis. The highest dissociation constant was obtained for apoenzyme (6.9mum). The identity of the corresponding kinetic and binding parameters of the native enzyme and the Zn(2+)-re-activated apoenzyme and the clear differences from the parameters of the other metal-substituted enzyme forms give strong support to the previous identification of zinc as the natural metal cofactor of glyoxalase I. Binding to apoenzyme was also shown by the use of S-p-bromobenzylglutathione as a ligand in affinity chromatography and as a protector in chemical modification experiments. The tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl bromide caused up to 85% inactivation of the enzyme. After blocking of the thiol groups (about 8 per enzyme molecule) 6.1 2-hydroxy-5-nitrobenzyl groups were incorporated. Inclusion of S-p-bromobenzylglutathione with the modifying reagent preserved the catalytic activity of the enzyme completely and decreased the number of modified residues to 4.4 per enzyme molecule. The findings indicate the presence of one tryptophan residue in the active centre of each of the two subunits of the enzyme. Thiol groups appear not to be essential for catalytic activity. The presence of at least two categories of tryptophan residues in the protein was also shown by quenching of the fluorescence by KI.

Binding Sites↗
PubMed1980

Product inhibition studies of human liver formaldehyde dehydrogenase.

The steady-state kinetic mechanism of human liver formaldehyde dehydrogenase (formaldehyde:NAD+ oxidoreductase (glutathione-formylating), EC 1.2.1.1) was investigated by product inhibition of the forward and the reverse reactions catalyzed by the enzyme. The results are compatible with a mechanism which contains the random addition to the enzyme of NAD+ and S-hydroxymethylglutathione (the adduct of glutathione and formaldehyde), or NADH and S-formylglutathione, and free glutathione as the allosteric activator of the enzyme (Uotila, L. and Mannervik, B. (1979) Biochem. J. 177, 869-878).

Aldehyde Oxidoreductases↗
PubMed1980

Induction of glutathione S-transferases A, B and C in rat liver cytosol by trans-stilbene oxide.

The cytoplasmic glutathione S-transferase activity of rat liver has been shown to increase to 300--400% of control values after treatment of the animals with trans-stilbene oxide and this phenomenon has been further characterized in the present study. Quantitative immunological determinations showed that the content of glutathione S-transferases A, B and C together constituted 4.5% of the soluble proteins in the hepatic cytoplasm of untreated rats. The content rose to 12.9 and 17.4% after treatment with trans-stilbene oxide or a combination of trans-stilbene oxide, 3-methylcholanthrene and phenobarbital, respectively. It was demonstrated that the cytosolic fraction from induced liver contains 4.2 times as much antigen which can be precipitated with antiglutathione S-transferase B antiserum as does control cytosol. Antiglutathione S-transferase C, which intereacts with transferases A and C, precipitates 3.3 times as much protein from the induced cytosol compared with control. Crossed immunoelectrophoresis and purification demonstrated that both A and C are increased in amount after treatment with trans-stilbene oxide. Thus, cytosolic glutathione S-transferases A, B, and C in liver are all induced by treatment of rats with trans-stilbene oxide. Immunological crossreaction, similar behavior during chromatography on CM-cellulose and hydroxyapatite and similar specific activities suggest that the control and induced enzymes are essentially identical, trans-Stilbene oxide was found to serve as a relatively poor second substrate for glutathione S-transferases A, B and C and can thus be said to cause substrate induction of these enzymes.

Animals↗
PubMed1980

Role of cytoplasmic thioltransferase in cellular regulation by thiol-disulphide interchange.

Cytoplasmic thioltransferase purified from rat liver [Axelsson, Eriksson & Mannervik (1978) Biochemistry 17, 2978--2984] catalyses the formation and decomposition of mixed disulphides of proteins and glutathione. The enzyme was found to catalyse the reversible thiol-disulphide interchange between glutathione disulphide and a crude thiol-containing protein fraction from rat liver. This finding indicates a role of the thioltransferase in the regulation of the 'glutathione status' of the cell. Specifically, it was found that thioltransferase catalyses the reactivation of pyruvate kinase from rat liver that had previously been inactivated by glutathione disulphide. It is suggested that thioltransferase may have a general role in regulatory processes involving thiol-disulphide interchange.

Animals↗
PubMed1980

General specificity of cytoplasmic thioltransferase (thiol:disulfide oxidoreductase) from rat liver for thiol and disulfide substrates.

The substrate specificity of thioltransferase (thiol:disulfide oxidoreductase) purified from rat liver cytosol (Axelsson, K., Eriksson, S. and Mannervik, B. (1978) Biochemistry 17, 2978-2984) has been investigated. Various low-molecular-weight thiols were found to be substrates in the scission of sulfur-sulfur bonds in cystamine and glutathione disulfide as well as in mixed disulfides of proteins and glutathione. Protein sulfhydryl groups could also serve as thiol substrates in enzyme-catalyzed thiol-disulfide interchange. Thiol-transferase also catalyzed the reduction of sulfur-sulfur bonds in disulfide-containing proteins by glutathione provided that the proteins had been denatured by proteolysis or urea treatment. It is proposed that thioltransferase may have a role in the intracellular protein degradation by cleavage of disulfide bonds. The finding that thioltransferase catalyzes the reversible formation of mixed disulfides of glutathione and the bulk of proteins in rat liver cytosol is relevant for the catalysis of changes in the 'glutathione status' in the cell. In conclusion, the results of the present investigation show that cytoplasmic thiol-transferase has a very broad substrate specificity, which implies that the enzyme may participate in a multitude of thiol-disulfide interchange reactions.

Animals↗