Purification of NADPH-dependent dehydroascorbate reductase from rat liver and its identification with 3α-hydroxysteroid dehydrogenase

Rat liver cytosol has been found to reduce dehydroascorbic acid (DHAA) to ascorbic acid in the presence of NADPH. The enzyme responsible for such activity has been purified by ammonium sulphate fractionation, DEAE-Sepharose, Sephadex G-100 SF and Reactive Red column chromatography, with an overall recovery of 27%. SDS/PAGE of the purified enzyme showed one single protein band with an M(r) of 37,500. A similar value (36,800) was found by gel filtration on a Sephadex G-100 SF column. The results indicate that the enzyme is a homogeneous monomer. The Km for DHAA was 4.6 mM and the Vmax. was 1.55 units/mg of protein; for NADPH Km and Vmax. were 4.3 microM and 1.10 units/mg of protein respectively. The optimum pH was around 6.2. Several typical substrates and inhibitors of the aldo-keto reductase superfamily have been tested. The strong inhibition of DHAA reductase effected by steroidal and non-steroidal anti-inflammatory drugs, together with the ability to reduce 5 alpha-androstane-3,17-dione strongly, suggest the possibility that DHAA reductase corresponds to 3 alpha-hydroxysteroid dehydrogenase. Microsequence analysis performed on the electro-transferred enzyme band shows that the N-terminus is blocked. Internal primary structure data were obtained from CNBr-derived fragments and definitely proved the identity of NADPH-dependent DHAA reductase with 3 alpha-hydroxysteroid dehydrogenase.

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Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver

Biochemical Journal ◽

10.1042/bj3010471 ◽

1994 ◽

Vol 301 (2) ◽

pp. 471-476 ◽

Cited By ~ 87

Author(s):

E Maellaro ◽

B Del Bello ◽

L Sugherini ◽

A Santucci ◽

M Comporti ◽

...

Keyword(s):

Rat Liver ◽

Protein Identification ◽

Reductase Activity ◽

Sequence Similarity ◽

Gel Filtration ◽

Dehydroascorbic Acid ◽

Protein Band ◽

Dehydroascorbate Reductase ◽

Apparent Homogeneity ◽

Sds Page

GSH-dependent enzymic reduction of dehydroascorbic acid to ascorbic acid has been studied in rat liver cytosol. After gel filtration of cytosol on Sephadex G-100 SF, dehydroascorbate reductase activity was recovered in two distinct peaks, one corresponding to glutaredoxin (an enzyme already known for its dehydroascorbate reductase activity) and another, much larger one, corresponding to a novel enzyme different from glutaredoxin. The latter was purified to apparent homogeneity. The purification process involved (NH4)2SO4 fractionation, followed by DEAE-Sepharose, Sephadex G-100 SF and Reactive Red chromatography. SDS/PAGE of the purified enzyme in either the presence or absence of 2-mercaptoethanol demonstrated a single protein band of M(r) 31,000. The M(r) determined by both Sephadex G-100 SF chromatography and h.p.l.c. was found to be approx. 48,000. H.p.l.c. of the denatured enzyme gave an M(r) value identical with that obtained by SDS/PAGE (31,000). The apparent Km for dehydroascorbate was 245 microM and the Vmax. was 1.9 mumol/min per mg of protein; for GSH they were 2.8 mM and 4.5 mumol/min per mg of protein respectively. The optimal pH range was 7.5-8.0. Microsequence analysis of the electro-transferred enzyme band showed that the N-terminus is blocked. Data on internal primary structure were obtained from CNBr-and N-chlorosuccinimide-derived fragments. No significative sequence similarity was found to any of the protein sequences contained in the Protein Identification Resource database.

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Purification of p-hydroxyphenylpyruvate hydroxylase from rat liver—requirements for cofactors

Canadian Journal of Biochemistry ◽

10.1139/o76-061 ◽

1976 ◽

Vol 54 (5) ◽

pp. 423-431 ◽

Cited By ~ 5

Author(s):

Kun-Tsan Lin ◽

John C. Crawhall

Keyword(s):

Molecular Weight ◽

Enzyme Activity ◽

Rat Liver ◽

Gel Filtration ◽

Protein Band ◽

Final Concentration ◽

Assay Method ◽

Enzyme Extract ◽

Crude Enzyme Extract ◽

Single Protein

Theenzyme p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27)from rat liver was studied with the assay method which measures the release of 14CO2 from p-hydroxyphenyl [carboxyl-,14C]pyruvate. Extensive dialysis of the crude enzyme extract against Tris buffer or purification involving ammonium sulfate, gel filtration, and ion exchange results in loss of enzyme activity that can be reactivated by Fe2+, dichlorophenolindophenol, and various other agents. The effect of these activators depends critically on their final concentration in the assay media.A 70-fold purification of the enzyme fraction yielded a preparation which behaved as a single protein band in Sephadex G-150. It had an isoelectric point at 5.85 and molecular weight of 63 000. The enzyme obtained appears to be different in some respects from those described by other workers from the liver of dog, human, chicken, and frog.

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Purification and properties of a 3α-hydroxysteroid dehydrogenase of rat liver cytosol and its inhibition by anti-inflammatory drugs

Biochemical Journal ◽

10.1042/bj2220601 ◽

1984 ◽

Vol 222 (3) ◽

pp. 601-611 ◽

Cited By ~ 143

Author(s):

T M Penning ◽

I Mukharji ◽

S Barrows ◽

P Talalay

Keyword(s):

Rat Liver ◽

Aromatic Aldehydes ◽

Hydroxysteroid Dehydrogenase ◽

Liver Cytosol ◽

Purification And Properties ◽

Competitive Inhibitors ◽

Dihydrodiol Dehydrogenase ◽

Inflammatory Drugs ◽

Anti Inflammatory ◽

Rat Liver Cytosol

An NAD(P)-dependent 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) was purified to homogeneity from rat liver cytosol, where it is responsible for most if not all of the capacity for the oxidation of androsterone, 1-acenaphthenol and benzenedihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene). The dehydrogenase has many properties (substrate specificity, pI, Mr, amino acid composition) in common with the dihydrodiol dehydrogenase (EC 1.3.1.20) purified from the same source [Vogel, Bentley, Platt & Oesch (1980) J. Biol. Chem. 255, 9621-9625]. Since 3 alpha-hydroxysteroids are by far the most efficient substrates, the enzyme is more appropriately designated a 3 alpha-hydroxysteroid dehydrogenase. It also promotes the NAD(P)H-dependent reductions of quinones (e.g. 9,10-phenanthrenequinone, 1,4-benzoquinone), aromatic aldehydes (4-nitrobenzaldehyde) and aromatic ketones (4-nitroacetophenone). The dehydrogenase is not inhibited by dicoumarol, disulfiram, hexobarbital or pyrazole. The mechanism of the powerful inhibition of this enzyme by both non-steroidal and steroidal anti-inflammatory drugs [Penning & Talalay (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4504-4508] was examined with several substrates. Most non-steroidal anti-inflammatory drugs are competitive inhibitors (e.g. Ki for indomethacin, 0.20 microM for 9,10-phenanthrenequinone reduction at pH 6.0, and 0.835 microM for androsterone oxidation at pH 7.0), except for salicylates, which act non-competitively (e.g. Ki for aspirin, 650 microM for androsterone oxidation). The inhibitory potency of these agents falls sharply as the pH is increased from 6 to 9. Most anti-inflammatory steroids are likewise competitive inhibitors, except for the most potent (betamethasone and dexamethasone), which act non-competitively. The enzyme is inhibited competitively by arachidonic acid and various prostaglandins.

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Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

Biochemical Journal ◽

10.1042/bj20041053 ◽

2005 ◽

Vol 387 (1) ◽

pp. 271-280 ◽

Cited By ~ 58

Author(s):

Seonghun KIM ◽

Sun Bok LEE

Keyword(s):

Molecular Mass ◽

Gel Filtration ◽

Maximal Activity ◽

Genome Database ◽

Sds Page ◽

Key Enzyme ◽

Bivalent Metal Ions ◽

Ammonium Sulphate Fractionation ◽

Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

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The kinetic mechanism catalysed by homogeneous rat liver 3α-hydroxysteroid dehydrogenase. Evidence for binary and ternary dead-end complexes containing non-steroidal anti-inflammatory drugs

Biochemical Journal ◽

10.1042/bj2780835 ◽

1991 ◽

Vol 278 (3) ◽

pp. 835-841 ◽

Cited By ~ 53

Author(s):

L J Askonas ◽

J W Ricigliano ◽

T M Penning

Keyword(s):

Rat Liver ◽

Initial Velocity ◽

Equilibrium Dialysis ◽

Hydroxysteroid Dehydrogenase ◽

Pyridine Nucleotide ◽

Free Enzyme ◽

Binary Complex ◽

Dead End ◽

Inflammatory Drugs ◽

Inhibition Studies

Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) (EC 1.1.1.50) is an NAD(P)(+)-dependent oxidoreductase that is potently inhibited at its active site by non-steroidal anti-inflammatory drugs (NSAIDs). Initial-velocity and product-inhibition studies performed in either direction at pH 7.0 are consistent with a sequential ordered Bi Bi mechanism in which pyridine nucleotide binds first and leaves last. This mechanism is supported by fluorescence titrations of the E-NADH complex, and by the failure to detect the binding of either [3H]androsterone or [3H]androstanedione to free enzyme by equilibrium dialysis. Dead-end inhibition studies with NSAIDs also support this mechanism. Initial-velocity studies with indomethacin show that this drug is an uncompetitive inhibitor against NAD+, but a potent competitive inhibitor against androsterone, indicating the ordered formation of an E.NAD+.indomethacin complex. Calculation of the individual rate constants reveals that the binding and release of pyridine nucleotide is rate-limiting and that isomerization of the central complex is favoured in the forward direction. Equilibrium dialysis experiments with [14C]indomethacin reveal the presence of two abortive NSAID complexes, a high-affinity ternary complex corresponding to E.NAD+.indomethacin (Kd = 1-2 microM for indomethacin) and a low-affinity binary complex corresponding to E.indomethacin (Kd = 22 microM for indomethacin). Since indomethacin has a low affinity for free enzyme, the formation of this abortive binary complex does not complicate kinetic measurements which are made in the presence of NAD+, but may contribute to the inhibition of the enzyme by NSAIDs. Using either pro-R-[4-3H]NADH or pro-S-[4-3H]NADH as cofactor, radiolabelled androsterone was formed only when the pro-R-[4-3H]NADH was used, confirming that purified 3 alpha-HSD is a Class A dehydrogenase.

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Rat liver cytosol contains NADPH- and GSH-dependent factors able to restore ornithine decarboxylase inactivated by removal of thiol reducing agents

Biochemical Journal ◽

10.1042/bj2500053 ◽

1988 ◽

Vol 250 (1) ◽

pp. 53-58 ◽

Cited By ~ 8

Author(s):

F Flamigni ◽

C Guarnieri ◽

C M Caldarera

Keyword(s):

Ornithine Decarboxylase ◽

Rat Liver ◽

Gel Filtration ◽

Enzymic Activity ◽

Effective Concentration ◽

The Other ◽

Limited Effect ◽

Liver Cytosol ◽

Rat Liver Cytosol ◽

Dependent Mechanism

Removal of dithiothreitol (DTT) from partially purified ornithine decarboxylase (ODC) led to an almost complete inhibition of enzymic activity. The inactivation was reversed by addition of millimolar concentrations of DTT, whereas natural reductants such as NADPH or NADH were ineffective, and GSH had only a limited effect. Addition of rat liver cytosol to the incubation mixture resulted in a noticeable re-activation of ODC; however, dialysed cytosol had little effect unless NADPH or GSH was present. Fractionation of rat liver cytosol by gel filtration on Sephadex G-75 yielded two fractions involved in the NADPH- and GSH-dependent re-activation of ODC: one designated ‘A’, eluted near the void volume (Mr greater than or equal to 60,000), and the other designated ‘B’, eluted later (Mr approx. 12,000). The NADPH-dependent mechanism required both fractions A and B for maximal ODC re-activation; the most effective concentration of NADPH was 0.15 mM, although a significant effect was observed at a concentration more than 10-fold lower. The GSH-dependent mechanism involved the mediation of Fraction B only, and operated at millimolar concentrations of GSH. These results suggest the existence of reducing systems in the cytosol, which may play a role in maintaining, and potentially in regulating, ODC activity by modulation of its thiol status.

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Identification of rat liver phosphatidylinositol synthase as a 21 kDa protein

Biochemical Journal ◽

10.1042/bj3040301 ◽

1994 ◽

Vol 304 (1) ◽

pp. 301-305 ◽

Cited By ~ 12

Author(s):

M E Monaco ◽

M Feldman ◽

D L Kleinberg

Keyword(s):

Heat Treatment ◽

Enzyme Activity ◽

Affinity Chromatography ◽

Molecular Mass ◽

Rat Liver ◽

Protein Band ◽

Absolute Requirement ◽

Sds Page ◽

Sepharose 4B ◽

Post Mitochondrial Supernatant

Substantial purification of rat liver phosphatidylinositol (PtdIns) synthase has been achieved by a combination of Hecameg extraction, heat treatment, affinity chromatography and chromatography on PBE-94. The activity chromatographs as a single peak which has an apparent molecular mass between 150 and 200 kDa on Sepharose 4B. When analysed by SDS/PAGE, two major bands are seen. The enzyme activity is correlated with a protein band of 21 kDa. A second band, at 51 kDa, is eluted from a PBE-94 column slightly ahead of the activity. Manganese is an absolute requirement for stabilization of activity in the presence of detergent. The effect of manganese is optimal at 0.5 mM; magnesium at a concentration of 10 mM is only minimally effective. Substrate Kms are 1.3 mM and 9.5 microM for inositol and CDP-diacylglycerol respectively. The activity eluting from the PBE-94 column is purified 5000-fold over the post-mitochondrial supernatant.

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Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver

Biochemical Journal ◽

10.1042/bj3080983 ◽

1995 ◽

Vol 308 (3) ◽

pp. 983-989 ◽

Cited By ~ 31

Author(s):

I N Fleming ◽

S J Yeaman

Keyword(s):

Phosphatidic Acid ◽

Rat Liver ◽

Lysophosphatidic Acid ◽

Gel Filtration ◽

Purification And Characterization ◽

Sds Page ◽

Chromatography Columns ◽

Triton X ◽

Second Peak

N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.

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Purification and properties of a new glutathione-dependent thiol:disulphide oxidoreductase from rat liver

Biochemical Journal ◽

10.1042/bj2070133 ◽

1982 ◽

Vol 207 (1) ◽

pp. 133-138 ◽

Cited By ~ 20

Author(s):

M G Battelli ◽

E Lorenzoni

Keyword(s):

Rat Liver ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Xanthine Dehydrogenase ◽

Enzymic Activity ◽

Oxidized Glutathione ◽

Adult Rats ◽

Purification And Properties ◽

Rat Liver Cytosol

A new GSSG-dependent thiol:disulphide oxidoreductase was extensively purified from rat liver cytosol. The enzymic protein shows molecular weight 40 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and 43 000 as determined by thin-layer gel filtration on Bio-Gel P-100. The pI is 8.1. This enzyme converts rat liver xanthine dehydrogenase into an oxidase, in the presence of oxidized glutathione. Other disulphide compounds are either inactive or far less active than oxidized glutathione in the enzymic oxidation of rat liver xanthine dehydrogenase. The enzyme also catalyses the reduction of the disulphide bond of ricin and acts as a thioltransferase and as a GSH:insulin transhydrogenase. The enzymic activity was measured in various organs of newborn and adult rats.

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A murine hybridoma with large cytoplasmic inclusions of kappa light chains.

Journal of Experimental Medicine ◽

10.1084/jem.165.2.578 ◽

1987 ◽

Vol 165 (2) ◽

pp. 578-583 ◽

Cited By ~ 3

Author(s):

D C Reason

Keyword(s):

Cell Line ◽

Inclusion Bodies ◽

Protein Band ◽

Light Chains ◽

Cytoplasmic Inclusions ◽

Single Protein Band ◽

Sds Page ◽

Single Protein ◽

Kappa Light Chains

A murine hybridoma cell line has been established that consistently forms large cytoplasmic inclusions. These structures bind antibody specific for mouse kappa L chain when stained in situ. SDS-PAGE analysis of isolated inclusion bodies produce a single protein band of approximately 26,000 Mr that reacts with anti-kappa antibody when transferred to nitrocellulose. No carbohydrate was detected in association with the purified protein. These data are consistent with the intracellular retention and deposition of complete kappa L chain protein.

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