Kinetic properties of purified sheep lung microsomal NADH-cytochrome b5 reductase.

1. Lung NADH-cytochrome b5 reductase was saturated with its artificial substrate, potassium ferricyanide at approximately 0.1 mM ferricyanide concentration, and the activity of the lung enzyme was inhibited by the higher concentrations of potassium ferricyanide. Ferricyanide at 0.5 and 1.0 mM inhibited the activity of the enzyme by about 20 and 61% respectively. The apparent Km value was calculated as 13.7 microM potassium ferricyanide and 4.3 microM NADH. 2. The Michaelis constants for cytochrome b5 and NADH were determined to be 1.67 and 7.7 microM from the Lineweaver-Burk plots. These results demonstrate that affinity of the lung reductase for its natural substrate is almost 10 times higher than that for potassium ferricyanide. 3. Addition of non-ionic detergent stimulated the rate of reductase-catalyzed reduction of lung cytochrome b5 up to 8.2-fold. 4. Kinetic studies performed with lung reductase by varying NADH and cytochrome b5 concentrations at different fixed concentrations at cytochrome b5 or NADH showed a series of parallel lines indicating a "ping-pong" type of kinetic mechanism for interaction of NADH and cytochrome b5 with lung cytochrome b5 reductase.     

Sheep brain glutathione reductase: purification and general properties

Sheep brain glutathione reductase was purified about 11,000-fold with an overall yield of 40%. The method included ammonium sulphate fractionation, heat denaturation, 2',5'-ADP Sepharose 4B and Sephadex G-200 chromatography steps. Specific activity at the final step was 193 IU/mg. The Mr of the enzyme was found to be 116,000 by gel filtration chromatography. On SDS-PAGE, two identical subunits of Mr 64,000 were obtained. From the spectral data, about 2 mol FAD per mol of enzyme were calculated.     

Kinetic mechanism of sheep liver NADPH-dependent aldehyde reductase.

The kinetic mechanism of the major sheep liver aldehyde reductase (ALR1) was studied with three aldehyde substrates: p-nitrobenzaldehyde, pyridine-3-aldehyde and D-glucuronate. In each case the enzyme mechanism was sequential and product-inhibition studies were consistent with an ordered Bi Bi mechanism, with the coenzymes binding to the free enzyme. Binding studies were used to investigate the interactions of substrates, products and inhibitors with the free enzyme. These provided evidence for the binding of D-glucuronate, L-gulonate and valproate, as well as NADP+ and NADPH. The enzyme was inhibited by high concentrations of D-glucuronate in a non-competitive manner, indicating that this substrate was able to bind to the free enzyme and to the E X NADP+ complex at elevated concentrations. Although the enzyme was inhibited by high pyridine-3-aldehyde concentrations, there was no evidence for the binding of this substrate to the free enzyme. Sheep liver ALR1 was inhibited by the ionized forms of alrestatin, sorbinil, valproate, 2-ethylhexanoate and phenobarbitone, indicating the presence of an anion-binding site similar to that described for the pig liver enzyme, which interacts with inhibitors and substrates containing a carboxy group. Sorbinil, valproate and 2-ethylhexanoate inhibited the enzyme uncompetitively at low concentrations and non-competitively at high concentrations, whereas phenobarbitone and alrestatin were non-competitive and uncompetitive inhibitors respectively. The significance of these results with respect to inhibitor and substrate binding is discussed.     

Interaction of a glutathione S-conjugate with glutathione reductase. Kinetic and X-ray crystallographic studies

S-Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73-76] and by inhibition of the enzyme GSSG reductase (EC 1.6.4.2). The interaction of GSSG reductase with a well-studied conjugate, namely S-(2,4-dinitrophenyl)-glutathione and its electrophilic precursor 1-chloro-2,4-dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 microM and 22 microM respectively. After prolonged incubation, 1-chloro-2,4-dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ. As shown by X-ray crystallography the major binding site of S-(2,4-dinitrophenyl)-glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.     

Kinetic and structural properties of biocatalytically active sheep lung microsomal NADPH-cytochrome c reductase

NADPH-cytochrome c reductase was purified to electrophoretic homogeneity from detergent solubilized sheep lung microsomes. The specific activity of the purified enzyme ranged from 56 to 67 mumol cytochrome c reduced/min/mg protein and the yield was 48-52% of the initial activity in lung microsomes. The reductase had Mr of 78,000 and contained 1 mol each of FAD and FMN. Km values obtained in 0.3 M phosphate buffer, pH 7.8 at 37 degrees C for NADPH and cytochrome c were 11.1 +/- 0.70 microM and 20.0 +/- 2.15 microM. Lung reductase was inhibited by its substrate, cytochrome c when its concentration was above 160 microM. The lung reductase exhibited a ping-pong type kinetic mechanism for NADPH mediated cytochrome c reduction. Purified lung reductase was biocatalytically active in supporting benzo(a)pyrene hydroxylation reaction when coupled with lung cytochrome P-450 and lipid.     

Rabbit liver glutathione reductase. Purification and properties

Hepatic glutathione reductase can be obtained in relatively good amounts from rabbit by a procedure which is fairly simple and sufficiently rapid. The purified flavoprotein shows absorbance ratios at 274, 379, and 463 nm of 8.2:0.92:1.0, respectively; the FAD fluorescence is nearly completely quenched by the protein. Gradient ultracentrifugation and sodium dodecyl sulfate gel electrophoresis indicate that the enzyme is a dimer, consisting of subunits of about 56,000 molecular weight; flavin content suggests one FAD per chain. Gel filtration under a variety of conditions, on the other hand, yields a molecular weight in the range 56,000–67,000. It is proposed that rabbit liver glutathione reductase can be active also as monomer. Kinetic parameters of the enzyme have been determined under optimal conditions. The rabbit liver glutathione reductase is, at physiological pH, absolutely specific for NADPH.     

Developmental study of rat brain glutathione peroxidase and glutathione reductase

Glutathione peroxidase and glutathione reductase activities were measured in whole rat brains at selected ages from birth to adulthood. On a wet weight basis glutathione peroxidase activity increased 70% during development and glutathione reductase activity increased 160%. On a protein basis glutathione peroxidase declined slightly in activity during the first two weeks of life and then maintained the 14-day activity into adulthood while glutathione reductase showed a 30% increase in activity. While less than the developmental changes in many enzymes involved in aerobic glycolysis or catecholamine metabolism, these increases do suggest a role in CNS metabolism.     

Distribution of glutathione peroxidases glutathione reductase in rat brain mitochondria

The distribution of glutathione reductase (GR), glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in isolated rat brain mitochondria was investigated. using a fractionation procedure for the separation of inner and outer membranes, contact sites between the two membranes and a soluble fraction mainly originating from the mitochondrial matrix. The data indicate that GR and GPx are concentrated in the soluble fraction, with a minor portion of the two enzymes being associated with the contact sites. PHGPx is localized largely in the inner membrane. The possible functional significance of these findings is discussed.     

Superoxide dismutase, glutathione peroxidase, and glutathione reductase in sheep organs

The enzyme activities of the superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione reductase (GR) and thiobarbituric acid reactive substances (TBARS) content were measured in tissue extracts of the liver, kidney and lung of sheep in a nonpolluted control area (C), a polluted area pasture (PP) and those from polluted areas but fed in the laboratory with an experimental emission supplement diet (EEF). Compared with the control SOD, activity was significantly increased (1.75 times) only in the liver of the PP group. In the EEF group there was a tendency toward lower activities in all organs. The Cu,Zn-SOD isoenzymes pattern analyzed by isoelectrofocusing was different in the organs of the animals exposed to pollutants when compared with those of the controls. In the liver, two new isoenzymes with pI 5.30 and 5.70 were found in the PP group and an additional isoenzyme with pI 5.10 in the EEF group. The kidney isoenzymes with pl 5.30 and 5.40 were inhibited in the EEF group. In the lung, two new isoenzymes appeared with pl 5.30 and 5.40 in the PP group and two new isoenzymes with pI 6.10 and 6.50 in the EEF group. GSHPx activity was inhibited in the liver and kidney of the sheep exposed to pollutants. GR activity was significantly changed only in the liver. The activity in the PP group was 2.30 and 2.10 times higher than in the C and EEF groups, respectively. TBARS content was increased in the liver and kidney of the EEF group compared with the control.     

Human erythrocyte glutathione reductase. I. Purification and properties.

1. Glutathione reductase (NAD(P)H:oxidized-glutathione oxidoreductase, EC. 1.6.4.2) from human erythrocytes was purified 49 000-fold with an overall yield of 15% and a 280/460 nm absorbance ratio of 6.03. The procedure used was the method of Worthington and Rosemeyer modified by addition of heating and recrystallization. 2. It was concluded from the results of purification, electrofocusing and inhibition studies that glutathione reductase is a single enzyme which used both NADPH and NADH as hydrogen donors. 3. Apoenzyme cross-reacts with the antibody to the holoenzyme but has a slightly reduced affinity to the antibody. Apoenzyme can be removed from the hemolysate by heating and centrifugation without loss of holoenzyme. 4. Indirect immunological assay of the specific activity of the erythrocyte glutathione reductase is possible in the enzyme saturated with FAD.     

Purification and properties of the glutathione reductase of Chromatium vinosum.

The Chromatium vinosum glutathione reductase [NAD(P)H: glutathione disulfide oxidoreductase, EC 1.6.4.2] was purified to apparent homogeneity. The enzyme was found to require reduced nicotinamide adenine dinucleotide (NADH) as a reductant and to be specific for oxidized glutathione (GSSG). The polypeptide molecular weight in sodium dodecyl sulfate was found to be 52,000. Incubation of enzyme with NADH in the absence of GSSG resulted in a significant loss in activity. The enzyme was stimulated by phosphate and sulfate ion, but was inhibited by chloride ion, heavy metals, and sulfhydryl reagents. Adenylate nucleotides were inhibitory, and the data suggested that they were acting as competitive inhibitors of flavin adenine dinucleotide (FAD). The Km values of 7 X 10-3 for GSSG and 6 X 10-5 M for NADH were the highest reported of any previously investigated glutathione reductase. The order of addition of components markedly affected the response of the enzyme to FAD. A requirement for FAD (Km 5.2 X 10-7 M) was seen if the enzyme was incubated with NADH prior to GSSG addition, whereas no FAD was required if the order was reversed.     

Kinetic properties and regulation of biliverdin reductase

Transverse tubules (t-tubules) were prepared from muscle by dissociation of intact triads during centrifugation in ion-free sucrose gradients. They were further purified by the removal of contaminating sarcoplasmic reticulum after loading with calcium phosphate. Purification was accompanied by enrichment in markers specific for t-tubules, e.g., nitrendipine binding sites. According to gel electrophoresis the purified t-tubules contained three major protein bands of 104, 70, and 30 kDa. When solubilized with detergents there was a two- to threefold increase in Mg2+-ATPase activity, and a corresponding increase in the 30-kDa protein band. The 104-kDa protein was shown to be a (Na+ + K+)-ATPase because of its phosphorylation by [gamma-32P]ATP in the presence of sodium ions. The orientation of the t-tubule membrane was predominantly inside-out.     

Chloroplast glutathione reductase: Purification and properties

Glutathione reductase was partially purified from isolated pea chloroplasts ( Pisum sativum L. cv. Progress #9). A 1600-fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)⁻¹ min⁻¹. The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The K_m for oxidized glutathione was 11 μ M and the K_m for NADPH was 1.7 μ M . Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 m M . The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H₂O₂ by reducing dehydroascorbate for ascorbate-linked reduction of H₂O₂. Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity.     

Kinetic properties of aldehyde dehydrogenase from sheep liver mitochondria.

The kinetics of the NAD+-dependent oxidation of aldehydes, catalysed by aldehyde dehydrogenase purified from sheep liver mitochondria, were studied in detail. Lag phases were observed in the assays, the length of which were dependent on the enzyme concentration. The measured rates after the lag phase was over were directly proportional to the enzyme concentration. If enzyme was preincubated with NAD+, the lag phase was eliminated. Double-reciprocal plots with aldehyde as the variable substrate were non-linear, showing marked substrate activation. With NAD+ as the variable substrate, double-reciprocal plots were linear, and apparently parallel. Double-reciprocal plots with enzyme modified with disulfiram (tetraethylthiuram disulphide) or iodoacetamide, such that at pH 8.0 the activity was decreased to 50% of the control value, showed no substrate activation, and the plots were linear. At pH 7.0, the kinetic parameters Vmax. and Km NAD+- for the oxidation of acetaldehyde and butyraldehyde by the native enzyme are almost identical. Formaldehyde and propionaldehyde show the same apparent maximum rate. Aldehyde dehydrogenase is able to catalyse the hydrolysis of p-nitrophenyl esters. This esterase activity was stimulated by both NAD+ and NADH, the maximum rate for the NAD+ stimulated esterase reaction being roughly equal to the maximum rate for the oxidation of aldehydes. The mechanistic implications of the above behaviour are discussed.     

Kinetic studies with the low-Km aldehyde reductase from ox brain.

Initial-rate studies of the low-Km aldehyde reductase-catalysed reduction of pyridine-3-aldehyde by NADPH gave families of parallel double-reciprocal plots, consistent with a double-displacement mechanism being obeyed. Studies on the variation of the initial velocity with the concentration of a mixture of the two substrates were also consistent with a double-displacement mechanism. In contrast, the initial-rate data indicated that a sequential mechanism was followed when NADH was used as the coenzyme. Product-inhibition studies, however, indicated that a compulsory-order mechanism was followed in which NADPH bound before pyridine-3-aldehyde with a ternary complex being formed and the release of pyrid-3-ylcarbinol before NADP+. The apparently parallel double-reciprocal plots obtained in the initial-rate studies with NADPH and pyridine-3-aldehyde were thus attributed to the apparent dissociation constant for the binary complex between the enzyme and coenzyme being finite but very low.     

Kinetic properties of plasmalogenase from bovine brain.

Abstract— Plasmalogenase was assayed by measuring the disappearance of the plasmalogen by two-dimensional thin-layer chromatography. The enzyme was present in a glycerol-bicarbonate extract of an acetone-dried powder from bovine brain. With ethanolamine plasmalogens as the substrate, the K_m was 180 μM. Diacyl glycerophosphorylcholines, diacyl glycerophosphorylethanolamines and choline plasmalogens were competitive inhibitors. With choline plasmalogens as the substrate, the K_m was 208 μM and competitive inhibition was observed with diacyl glycerophosphorylcholines and ethanolamine plasmalogens. The same enzyme may be responsible for the hydrolysis of the alk-1-enyl moiety from both plasmalogens. Plasmalogenase activity was 5.1 μmol/h/g of dog brain, 3.9 μmol/h/g of rat brain and 3.4 μmol/h/g of gerbil brain. A lysophospholipase was also found in the glycerol-bicarbonate extract from the acetone-dried powder. The lysophospholipase was more active in hydrolysing acyl groups from 2-acyl-sn-glycero-3-phosphorylethanolamines than the plasmalogenase was active in hydrolyzing alk-1-enyl groups from 1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines.     

Kinetic and mechanistic properties of biotin sulfoxide reductase

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin. Initial rate studies of the homogeneous recombinant enzyme, expressed in Escherichia coli, have demonstrated that the purified protein utilizes NADPH as a facile electron donor in the absence of any additional auxiliary proteins. We have previously shown [Pollock, V. V., and Barber, M. J. (1997) J. Biol. Chem. 272, 3355-3362] that, at pH 8 and in the presence of saturating concentrations of BSO, the enzyme exhibits, a marked preference for NADPH (k(cat,app) = 500 s(-1), K(m,app) = 269 microM, and k(cat,app)/K(m,app) = 1.86 x 10(6) M(-1) s(-1)) compared to NADH (k(cat,app) = 47 s(-1), K(m,app) = 394 microM, and k(cat,app)/K(m,app) = 1.19 x 10(5) M(-1) s(-1)). Production of biotin using NADPH as the electron donor was confirmed by both the disk biological assay and by reversed-phase HPLC analysis of the reaction products. The purified enzyme also utilized ferricyanide as an artificial electron acceptor, which effectively suppressed biotin sulfoxide reduction and biotin formation. Analysis of the enzyme isolated from tungsten-grown cells yielded decreased reduced methyl viologen:BSO reductase, NADPH:BSO reductase, and NADPH:FR activities, confirming that Mo is required for all activities. Kinetic analyses of substrate inhibition profiles revealed that the enzyme followed a Ping Pong Bi-Bi mechanism with both NADPH and BSO exhibiting double competitive substrate inhibition. Replots of the 1/v-axes intercepts of the parallel asymptotes obtained at several low concentrations of fixed substrate yielded a K(m) for BSO of 714 and 65 microM for NADPH. In contrast, utilizing NADH as an electron donor, the replots yielded a K(m) for BSO of 132 microM and 1.25 mM for NADH. Slope replots of data obtained at high concentrations of BSO yielded a K(i) for BSO of 6.10 mM and 900 microM for NADPH. Kinetic isotope studies utilizing stereospecifically deuterated NADPD indicated that BSO reductase uses specifically the 4R-hydrogen of the nicotinamide ring. Cyanide inhibited NADPH:BSO and NADPH:FR activities in a reversible manner while diethylpyrocarbonate treatment resulted in complete irreversible inactivation of the enzyme concomitant with molybdenum cofactor release, indicating that histidine residues are involved in cofactor-binding.     

In vitro effects of some antibiotics on glutathione reductase from sheep liver

The effects of gentamicin sulphate, thiamphenicol, ofloxacin, levofloxacin, cefepime, and cefazolin were investigated on the in vitro enzyme activity of glutathione reductase. The enzyme was purified 1,850-fold with a yield 18.76% from sheep liver using ammonium sulphate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). The enzyme activity was measured spectrophotometrically at 340 nm, according to the method of Carlberg and Mannervik. From these six antibiotics, Ofloxacin, levofloxacin, cefepime, and cefazolin inhibited the activity of the purified enzyme; gentamicin sulphate and thiamphenicol showed little effect on the enzyme activity. The I50 values for these four antibiotics were 0.150 mM, 0.154 mM, 3.395 mM, and 18.629 mM, respectively. The Ki constants were 0.047 +/- 0.034 mM, 0.066 +/- 0.038 mM, 4.885 +/- 3.624 mM, and 6.511 +/- 1.894 mM, respectively and they were competitive inhibitors.     

Kinetic properties of spermine synthase from bovine brain.

Phosphofructokinase (EC 2.7.1.11) from a citric acid-producing strain of Aspergillus niger was partially purified by the application of affinity chromatography on Blue Dextran--Sepharose and the use of fructose 6-phosphate and glycerol as stabilizers in the working buffer. The resulting preparation was still impure, but free of enzyme activities interfering with kinetic investigations. Kinetic studies showed that the enzyme exhibits high co-operativity with fructose 6-phosphate, but shows Michaelis--Menten kinetics with ATP, which inhibits at concentrations higher than those for maximal activity. Citrate and phosphoenolpyruvate inhibit the enzyme; citrate increases the substrate (fructose 6-phosphate) concentration for half-maximal velocity, [S]0.5, and the Hill coefficient, h. The inhibition by citrate is counteracted by NH4+, AMP and phosphate. Among univalent cations tested only NH4+ activates by decreasing the [S]0.5 for fructose 6-phosphate and h, but has no effect on Vmax. AMP and ADP activate at low and inhibit at high concentrations of fructose 6-phosphate, thereby decreasing the [S]0.5 for fructose 6-phosphate. Phosphate has no effect in the absence of citrate. The results indicate that phosphofructokinase from A. niger is a distinct species of this enzyme, with some properties similar to those of the yeast enzyme and in some other properties resembling the mammalian enzyme. The results of determinations of activity at substrate and effector concentrations resembling the conditions that occur in vivo support the hypothesis that the apparent insensitivity of the enzyme to citrate during the accumulation of citric acid in the fungus is due to counteraction of citrate inhibition by NH4+.     

Synthesis and properties of glutathione reductase in stressed peas

We have subjected peas (Pisum sativum L.) to four different oxidative stresses: cold conditions (4 °C) in conjunction with light, treatment with paraquat, fumigation with ozone, and illumination of etiolated seedlings (greening). In crude extracts of leaves from stressed plants, an increase (up to twofold) in activity of glutathione reductase (GR) was observed which was consistent with previous reports from several laboratories. In all cases, except for ozone fumigation, the increase in activity was not due to an elevation in the steady-state levels of GR protein. None of the applied stresses had any effect on steady-state levels of GR mRNA. In contrast to the small increase in GR activity, the K _m of GR for glutathione disulphide showed a marked decrease when determined for extracts of stressed leaves, compared with that from unstressed plants. This indicates that GR from stressed plants has an increased affinity for glutathione disulphide. The profile of GR activity bands fractionated on non-denaturing acrylamide gels varied for extracts from differently stressed leaves and when compared with GR from unstressed plants. The changes in GR-band profiles and the alteration in the kinetic properties are best explained as changes in the isoform population of pea GR in response to stress.Abbreviations GR glutathione reductase - GSSG glutathione disulphide - Rubisco Ribulose-1,5-bisphosphate carboxylase-oxygenase - RNase A/T1 ribonucleases A and T1 We are grateful to Prof. Alan Wellburn and Dr. Phil Beckett (Division of Biological Sciences, University of Lancaster, UK) for providing ozone-fumigated material and Dr. Jeremy Harbinson for providing material grown at 4° C. This work was supported by a grant-in-aid to the John Innes Institute from the Agricultural and Food Research Council. E.A.E. and C.E. gratefully acknowledge the support of a John Innes Foundation studentship and a European Molecular Biology Organisation Fellowship respectively.