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Biomedical subjects

B Mannervik

Publications and source records attributed to B Mannervik.

At least 199 records · Page 11Linked to original sources
PubMed1983

Relative contributions of thioltransferase-and thioredoxin-dependent systems in reduction of low-molecular-mass and protein disulphides.

Two enzyme systems capable of reducing disulphide bonds both in low-Mr compounds and in polypeptides and proteins exist. One consists of thioltransferase in combination with reduced glutathione and glutathione reductase, and the second consists of thioredoxin in combination with thioredoxin reductase. Their relative effectiveness in catalysing disulphide reduction of various substrates in rat liver cytosol was evaluated in the present study. The thioltransferase-dependent system was found to be more efficient in reducing small molecules. Insulin was most effectively reduced by the thioredoxin system. Bovine trypsin was a better substrate for thioltransferase, and partially proteolysed bovine serum albumin was equally good for the two systems. Thus, in the case of protein disulphide bonds, the nature of the particular substrate used determines which of the two reducing systems is the more important.

Animals↗
PubMed1983

A set of inhibitors for discrimination between the basic isozymes of glutathione transferase in rat liver.

A set of inhibitors including hematin, bromosulfophthalein, and triethyltin bromide was used for discrimination and identification of the major basic isozymes of glutathione transferase in rat liver cytosol. Six enzymes are formed as binary combinations of 4 protein subunits: A, B, C, and L. Discrimination between the transferases can be based on the differences of the subunits in susceptibility to the inhibitors. The identification of transferase subunits is further supported by the combined use of specific substrates and inhibitors.

Animals↗
PubMed1983

Reversal of the reaction catalyzed by glyoxalase I. Calculation of the equilibrium constant for the enzymatic reaction.

Glyoxalase I catalyzes the formation of S-D-lactoyl-glutathione via the hemimercaptal adduct of methylglyoxal and glutathione. This enzymatic reaction, which has been considered virtually irreversible, was found to be reversible under such conditions that glutathione liberated from the thiolester was trapped. The reverse reaction could be monitored spectrophotometrically by use of 5,5'-dithiobis-(2-nitrobenzoate). In addition to 5,5'-dithiobis-(2-nitrobenzoate), 2,2'-dithiobispyridine and cystamine were used to promote the reverse reaction. S-D-Lactoylglutathione did not hydrolyze in the presence of glyoxalase I under the conditions investigated, as shown by its stability in the absence of thioltrapping agents. Proof of the reversal of the reaction was obtained by demonstrating the formation of stoichiometric amounts of methylglyoxal and glutathione from S-D-lactoylglutathione. Catalysis of the reverse reaction was dependent upon the presence of a bivalent metal ion in the active site of the enzyme. Apoenzyme, obtained by removal of the essential Zn2+ from the active site, did not catalyze the reverse reaction, but catalytic activity was restored by addition of Zn2+, Mg2+, Mn2+, or Co2+. The reverse reaction was also catalyzed by glyoxalase I from yeast. Linear competitive inhibition (Ki = 0.64 mM) was obtained with 5,5'-dithiobis-(2-nitrobenzoate), which necessitated correction of the apparent kinetic parameters of the reverse reaction. The corrected values for the reverse reaction catalyzed by glyoxalase I from human erythrocytes with S-D-lactoylglutathione as substrate were kcat = 3.6 s-1 and Km = 1.9 mM. Combination of these values with the corresponding parameters for the forward reaction allowed calculation, through the Haldane relation, of the equilibrium constant, Keq = 1.1 X 10(4), for the isomerization between the hemimercaptal of methylglyoxal and glutathione and S-D-lactoylglutathione. The strong reversible competitive inhibitor of the forward reaction, S-p-bromobenzylglutathione, also inhibited the reverse reaction competitively (Ki = 0.38 microM).

Disulfides↗
PubMed1983

13C NMR studies of the product complex of glyoxalase I.

The paramagnetic effects of Mn2+ . glyoxalase I on the 13C relaxation rates of the reaction product, S-(D-lactoyl)glutathione, separately enriched in the lactoyl carbonyl (C-1) and hydroxymethylene (C-2) carbons, have been measured at 62.8 MHz. The 1/fT1p values of C-1 (1100 +/- 120 s-1) and C-2 (712 +/- 290 s-1) and the previously determined tau c (0.74 ns) yield Mn2+ to carbon distances of 5.7 +/- 0.3 and 6.1 +/- 0.5 A, respectively. These distances, together with previously determined Mn2+-proton distances (Sellin, S., Rosevear, P.R., Mannervik, B., and Mildvan, A.S. (1982) J. Biol. Chem. 257, 10023-10029) constrain the thioester carbonyl group of the product to point toward the metal, with the oxygen positioned to accept a hydrogen bond from a water ligand, in a kinetically competent, second sphere complex. Model-building studies indicate that any averaging of multiple second sphere complexes would require as a major contributor at least one conformation with the lactoyl carbonyl oxygen within hydrogen-bonding distance of an intervening water ligand. Such a structure would facilitate polarization of the carbonyl group in the reverse glyoxalase reaction.

Erythrocytes↗
PubMed1983

Octahedral metal coordination in the active site of glyoxalase I as evidenced by the properties of Co(II)-glyoxalase I.

Co(II)-glyoxalase I has been prepared by reactivation of apoenzyme from human erythrocytes with Co2+. The visible absorption spectrum showed maxima at 493 and 515 nm and shoulders at 465 and 615 nm. The absorption coefficients at 493 and 515 nm were 35 and 33 M-1 cm-1/cobalt ion, respectively; i.e. 70 and 66 M-1 cm-1 for the dimeric metalloprotein. The product of the enzymatic reaction, S-D-lactoylglutathione, although binding to Co(II)-glyoxalase I, had no demonstrable effect on the visible absorption spectrum, indicating binding outside the first coordination sphere of the metal. The EPR spectrum at 3.9 K was characterized by g1 approximately 6.6, g2 approximately 3.0, and g3 approximately 2.5, and eight hyperfine lines with A1 = 0.025 cm-1. Binding of the strong competitive inhibitor S-p-bromobenzylglutathione to Co(II)-glyoxalase I gave three g values: 6.3, 3.4, and 2.5, indicating a conformational change affecting the environment of the metal ion. Both optical and EPR spectra strongly suggest a high spin Co2+ with octahedral coordination in the active site of the enzyme. The similarities in kinetic properties between native Zn(II)-glyoxalase I and enzyme substituted with Mg2+, Mn2+, or Co2+ is consistent with the view that these enzyme forms have the same metal coordination in the protein.

Binding Sites↗
PubMed1983

An essential role of cytosolic thioltransferase in protection of pyruvate kinase from rabbit liver against oxidative inactivation.

Pyruvate kinase from rabbit liver is inactivated spontaneously in the presence of air. Glutathione in physiological concentrations gives partial protection against inactivation. Full protection is obtained with glutathione and purified cytosolic thioltransferase supplemented with a glutathione-regenerating system. It is suggested that thioltransferase plus glutathione serve a general function in protecting protein thiol groups against oxidation.

Animals↗
PubMed1983

Enzymes involved in glutathione metabolism in rat liver and blood after carbon tetrachloride intoxication.

Some glutathione-metabolizing enzymes in rat liver cytosol, blood plasma and urine of rats administered carbon tetrachloride (CCl4) were investigated. After CCl4 at different doses (0.3, 1.2, 2.5 ml/kg at 24, 12 and 3 or 6 h, respectively) liver glutathione reductase (GR), glyoxalase I (GLY-I) and glutathione S-transferases (GST) significantly decreased, while thioltransferase and glutathione peroxidase (GPX) did not change their activity. At the times under investigation the glutathione level significantly increased in the liver and the above-mentioned enzymes in plasma, thus evidencing a liver injury. The GR, GPX and GST assayed in urine within 72 h after CCl4 administration showed a maximum of activity at 35-48 h.

Animals↗
PubMed1982

Fluorescence and nuclear relaxation enhancement studies of the binding of glutathione derivatives to manganese-reconstituted glyoxalase I from human erythrocytes. A model for the catalytic mechanism of the enzyme involving a hydrated metal ion.

The apoenzyme of glyoxalase I (EC 4.4.1.5) from human erythrocytes was prepared by removal of Zn2+ with ethylenediaminetetraacetic acid (EDTA). Methanol was used as a stabilizing agent. Extended dialysis was required to remove EDTA from the resulting solution of apoenzyme. Reconstitution with Mn2+ was followed by measuring enzyme activity, electron paramagnetic resonance of free Mn2+ ions, and nuclear magnetic resonance of water protons. The holoenzyme contained two Mn2+ per protein dimer and had approximately 50% of the catalytic activity of the native enzyme. The binding of the cosubstrate glutathione (gamma-L-glutamyl-L-cysteinylglycine), the product S-D-lactoyl-glutathione, and the competitive inhibitor S-(p-bromo-benzyl)glutathione was monitored by the quenching of the intrinsic tryptophan fluorescence and by the proton relaxation enhancement of water bound to Mn2+ in the active site of the enzyme. The dissociation constants were 1.1 mM, 0.42 mM, and 0.54 microM for glutathione, S-D-lactoylglutathione, and S-(p-bromobenzyl)glutathione, respectively. The temperature and frequency dependences of the longitudinal and transverse paramagnetic relaxation rates, 1/T1p and 1/T2p, were studied for water. The results were analyzed in terms of correlation and exchange times. In addition proton and deuteron relaxation rates were measured in parallel at two different magnetic fields. Good agreement between the two approaches of analysis was noticed. The data show that two water molecules are bound in the first coordination sphere of Mn2+ in the active site of glyoxalase I. When S-(p-bromobenzyl)glutathione or S-D-lactoylglutathione is bound to the enzyme, only one exchangeable water molecule could be detected, indicating occlusion of the second water molecule. An enediol mechanism involving the metal-bound water is proposed for the catalysis effected by glyoxalase I.

Apoenzymes↗
PubMed1982

Essential arginine residues in the pyridine nucleotide binding sites of glutathione reductase.

Glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2) from human erythrocytes as well as from other sources was inactivated irreversibly by the arginine modifying reagents 2,3-butanedione, 1,2-cyclohexanedione and phenylglyoxal. The inactivation caused by these reagents increased with time and was biphasic in nature. Inactivation by 2,3-butanedione in borate buffer could be partially reversed by dialysis against phosphate buffer or by incubation with hydroxylamine. The dicarbonyl reagents simultaneously inactivated the transhydrogenase activity of glutathione reductase that is expressed by the pyridine nucleotide sites. Amino acid analysis of enzyme modified with 2,3-butanedione showed a decrease by about 5 in the number of arginine residues. Analysis of enzyme inactivated with phenylglyoxal indicated modification of two arginine residues. NADPH, NADP/ and other adenosine 2'-phosphate nucleotides partially protected the enzyme against inactivation. GSSG did not protect the enzyme significantly. Modified glutathione reductase displayed a diminished affinity for the nucleotides as shown by affinity chromatography on 2',5'-ADP-Sepharose and by stopped-flow kinetics of the reduction of the enzyme. Modification did not cause any gross structural changes of enzyme molecule. It is concluded that some of the arginine residues modified are located at the pyridine-nucleotide-binding sites of the enzyme.

Adenine Nucleotides↗
PubMed1982

Benzo(a)pyrene metabolism by rat liver microsomes: effects of adding purified glutathione S-transferases A, B, and C.

We have examined the effects of adding glutathione and isolated cytosolic glutathione S-transferases A, B, and C to rat liver microsomes metabolizing benzo(a)pyrene. Addition of glutathione alone resulted in the conjugation of 15 to 20% of the total metabolites of benzo(a)pyrene, and this conjugation could be inhibited almost entirely by bromosulfophthalein (an inhibitor of glutathione S-transferases), indicating that it is catalyzed by the glutathione S-transferase present in microsomes. Addition of purified cytosolic glutathione S-transferases A, B, and C yielded about 30 to 40% conjugate formation. Analysis of metabolites by high-pressure liquid chromatography demonstrated that the formation of 4,5-diol of benzo(a)pyrene was decreased by at least 80% by conjugation and that the 7,8-diol was also decreased significantly (40 to 60%). In addition, it was found that glutathione S-transferase B is capable of conjugating benzo(a)pyrene 1,6- and 3,6-quinones.

Animals↗