No strict coupling of vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone)-dependent carboxylation and vitamin K1 epoxidation in detergent-solubilized microsomal fractions from rat liver.

NAD(P)H dehydrogenase ('DT-diaphorase', EC 1.6.99.2) and vitamin K epoxidase were removed by affinity chromatography from detergent-solubilized microsomal fractions. Thereby the microsomal fractions normally carrying out vitamin K1-dependent carboxylation of the microsomal precursor proteins of the prothrombin complex were inactivated. Purified NAD(P)H dehydrogenase added to this system restored carboxylation in the presence of vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone) plus NADH. Vitamin K1 hydroquinone (2-methyl-3-phytyl-1,4-naphthoquinol) had no effect, in contrast with its effect in the intact system, where it can substitute for vitamin K1 plus NADH. The ability of NAD(P)H dehydrogenase to restore carboxylation in a system without vitamin K epoxidase activity shows that there is no obligatory coupling of the vitamin K1-dependent carboxylation with vitamin K1 epoxidation. These results suggest that the form of vitamin K1 that is active in the carboxylation reaction can be produced independently in two reactions: by NAD(P)H dehydrogenase in the reduction of the quinone and by vitamin K epoxidase in the epoxidation of the hydroquinone.     

Vitamin K-dependent carboxylation and vitamin K epoxidation. Evidence that the warfarin-sensitive microsomal NAD(P)H dehydrogenase reduces vitamin K1 in these reactions.

Passage of a Triton X-100-solubilized microsomal systems in vitro that are used to study these reactions is the warfarin-sensitive NAD(P)H dehydrogenase.     

Effect of n-octylamine on the reduction of mammalian hepatic microsomal cytochrome b5

1. In a preceding paper evidence was presented for the endogenous reduction of NAD(P)+ by mammalian hepatic microsomes and the concomitant reduction of cytochrome b5. The experiments reported here demonstrate that low concentrations of n-octylamine, in the presence of limiting quantities of NAD+, cause an increased level of cytochrome b5 reduction by mouse hepatic microsomes and also delays its reoxidation. 2. These effects are both NAD+ and n-octylamine dependent and appear to be due to an activation of the microsomal enzyme causing endogenous reduction of NAD(P)+ and also, in part, to inhibition of the autooxidation of reduced cytochrome b5. 3. Protection from the inhibitory action of sulfhydryl reagents on NADH-cytochrome b5 reductase was also observed in the presence of n-octylamine. 4. The results suggest that the enzyme(s) involved in the endogenous reduction of NAD(P)+ is not the microsomal alcohol dehydrogenase.     

Purification and immunochemical characterization of malate synthase from Euglena gracilis.

A simple three-step method was established for the purification of NAD(P)H dehydrogenase (quinone) ('DT-diaphorase', EC 1.6.99.2) from rat liver by affinity chromatography with a recovery of above 50%. The final enzyme preparation was purified about 750-fold and was electrophoretically homogeneous. Gel filtration showed that the enzyme had a mol.wt. of about 55 000, and one molecule of FAD was found per 55 000 mol.wt. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave a mol.wt. of about 27 000. Two N-terminal amino acids, asparagine/aspartic acid and glutamine/glutamic acid, were found in about equal yield, suggesting the presence of two non-identical polypeptide chains in the enzyme. NAD(P)H dehydrogenase was selectively removed by this affinity-chromatographic method from a microsomal carboxylation system. The system, which was solubilized by detergent and is dependent on vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone or analogues with other side chains), lost its activity on the removal of the enzyme. The activity can be completely restored to the system by adding purified cytoplasmic NAD(P)H dehydrogenase or by using the quinol form of vitamin K1 (2-methyl-3-phytyl-1,4-naphthaquinol).     

Intramembraneous localization of rat liver microsomal hexose-6-phosphate dehydrogenase and membrane permeability to its substrates.

A method for purifying hexose-6-phosphate dehydrogenase (beta-D-glucose: NAD(P) -oxidoreductase, EC 1.1.1.47) from rat liver microsomes is described. The purified enzyme was shown to be homogeneous by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. It is shown that the enzyme is bound to the inner surface of microsomal membranes, and that glucose 6-phosphate, but not NADP, penetrates almost freely into the membranes at 37 degrees C.     

NAD+-dependent retinol dehydrogenase in liver microsomes

A microsomal NAD+-dependent retinol dehydrogenase is being described with optimal activity at physiological pH. The enzyme was present in liver microsomes of rats and also in a strain of deermice which lacks the cytosolic retinol dehydrogenase. Unlike the latter enzyme, the microsomal retinol dehydrogenase was not inhibited by either ethanol or 4-methylpyrazole; its activity was insensitive to CO and not oxygen dependent, in contradistinction with that of the microsomal cytochrome P-450 and NADPH-dependent retinol oxidase. Chronic ethanol consumption resulted in an increased activity of the microsomal retinol dehydrogenase which may contribute to hepatic retinol depletion, especially in view of the insensitivity of the enzyme to ethanol inhibition.     

Effect of SH-reagents on aldehyde dehydrogenase activity of the rat liver

SH-reagents: tetraethylthiuram disulphide (TETD), 5,5'-dithiobisnitrobenzoic acid (DTNB), p-chloromercurybenzoate (p-ChMB), N-ethylmaleimide (NEM) were studied for their effect on the aldehyde dehydrogenase activity of mitochondrion (isoenzymes I and II) and microsome (isoenzyme II) fractions of the rat liver. TETD is established to inhibit isoenzyme I and isoenzyme II activity of mitochondrial aldehyde dehydrogenase by 100 and 50%, respectively, and the microsomal enzyme activity by 20%. DTNB and NEM inhibit 30-50% of the activity in two isoforms of mitochondrial aldehyde dehydrogenase having no effect on the enzymic activity in microsomes; p-ChMB inhibits completely the activity of the enzyme under study both in the mitochondrial and microsomal fractions. A conclusion is drawn that SH-groups are very essential for manifestation of the catalytic activity in the NAD+-dependent aldehyde dehydrogenase from mitochondrial and microsomal fractions.     

Characterization of a novel type of human microsomal 3alpha -hydroxysteroid dehydrogenase: unique tissue distribution and catalytic properties

We report characterization of a novel member of the short chain dehydrogenase/reductase superfamily. The 1513-base pair cDNA encodes a 319-amino acid protein. The corresponding gene spans over 26 kilobase pairs on chromosome 2 and contains five exons. The recombinant protein produced using the baculovirus system is localized in the microsomal fraction of Sf9 cells and is an integral membrane protein with cytosolic orientation of its catalytic domain. The enzyme exhibits an oxidoreductase activity toward hydroxysteroids with NAD(+) and NADH as the preferred cofactors. The enzyme is most efficient as a 3alpha-hydroxysteroid dehydrogenase, converting 3alpha-tetrahydroprogesterone (allopregnanolone) to dihydroprogesterone and 3alpha-androstanediol to dihydrotestosterone with similar catalytic efficiency (V(max) values of 13-14 nmol/min/mg microsomal protein and K(m) values of 5-7 microm). Despite approximately 44-47% sequence identity with retinol/3alpha-hydroxysterol dehydrogenases, the enzyme is not active toward retinols. The corresponding message is abundant in human trachea and is present at lower levels in the spinal cord, bone marrow, brain, heart, colon, testis, placenta, lung, and lymph node. Thus, the new short chain dehydrogenase represents a novel type of microsomal NAD(+)-dependent 3alpha-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution.     

Purification and characterization of 20-hydroxy-leukotriene B4 dehydrogenase in human neutrophils

Leukotriene B4 (LTB4) is converted to 20-hydroxy-LTB4 (20-OH-LTB4) which is subsequently oxidized to 20-carboxy-LTB4 (20-COOH-LTB4). The oxidation of the hydroxy LTB4 to the carboxy LTB4 by human neutrophils was associated with the reduction of NAD+ and required both cytosolic and microsomal fractions. We isolated a cytosolic protein which oxidized the hydroxy LTB4 in the presence of NAD+ and the microsomal fraction. It was homogeneous on SDS/PAGE, with a subunit molecular mass of 37 kDa, and may be a dimeric protein with two identical or similar subunits because its molecular mass, estimated by Sephadex G-100 column chromatography, was about 80 kDa. The protein was an alcohol dehydrogenase with high affinity for omega-hydroxy fatty acids, such as 12-hydroxylaurate and 16-hydroxypalmitate. We conclude that the cytosolic protein oxidizes 20-OH-LTB4 to 20-oxo-LTB4 and the microsomal fraction oxidizes the oxo-LTB4 to the carboxy-LTB4, based on the finding that the activity which oxidizes omega-hydroxy fatty acids is present only in the cytosol fraction, while that which oxidizes hydrophobic aldehydes is found only in the microsomal fraction and that the stoichiometry of the formation of 20-COOH-LTB4 to the reduction of NAD+ was 1:2. The affinity of the dehydrogenase for 20-OH-LTB4 may be higher than that for 12-hydroxylaurate (Km = 70 microM), because the latter inhibited the oxidation of the former by only 40%, at a concentration of 12-hydroxylaurate 10 times higher than that of 20-OH-LTB4. The enzyme activity was not affected by pyrazole and 4-methylpyrazole at millimolar concentrations. These characteristics indicate that the dehydrogenase is a unique type of alcohol dehydrogenase.     

Enzymatic in situ analysis by 1H-NMR of the hydrogen transfer stereospecificity of NAD(P)+-dependent dehydrogenases

We have established a simple procedure for the in situ analysis of stereospecificity of an NAD(P)-dependent dehydrogenase for C-4 hydrogen transfer of NAD(P)H by means of glutamate racemase [EC 5.1.13] and glutamate dehydrogenase [EC 1.4.1.3]. Glutamate racemase inherently catalyzes the exchange of alpha-H of glutamate with 2H during racemization in 2H2O. When the reactions of glutamate racemase and glutamate dehydrogenase, which is pro-S specific for the C4-H transfer of NAD(P)H, are coupled in 2H2O, [4S-2H]-NAD(P)H is exclusively produced. Therefore, if 1H is fully retained at C-4 of NAD(P)+ after incubation of a reaction mixture containing both the enzymes and a dehydrogenase to be tested, the stereospecificity of the dehydrogenase is the same as that of glutamate dehydrogenase. When the C4-H of NAD(P)+ is exchanged with 2H, the enzyme to be examined is different from glutamate dehydrogenase in stereospecificity. Thus, we can readily determine the stereospecificity by 1H-NMR measurement of NAD(P)+ without isolation of the coenzymes and products.     

Properties and synthesis regulation of NAD(P)H dehydrogenases from Rhodobacter capsulatus

Three NAD(P)H dehydrogenases were found and purified from a soluble fraction of cells of the purple non-sulfur bacterium Rhodobacter capsulatus, strain B10. Molecular mass of NAD(P)H, NADPH and NADH dehydrogenases are 67 000 (4 · 18 000), 35 000 and 39 000, and the isoelectric points are 4.6, 4.3 and 4.5, respectively. NAD(P)H dehydrogenase is characterized by a higher sensitivity to quinacrine, NADPH dehydrogenase by its sensitivity to p-chloromercuribenzoate and NADH dehydrogenase by its sensitivity to sodium arsenite. In contrast to the other two enzymes, NAD(P)H dehydrogenase is capable of oxidizing NADPH as well as NADH, but the ratio of their oxidation rates depends on the pH. All NAD(P)H dehydrogenases reacted with ferricyanide, 2,6-dichlorophenolindophenol, benzoquinone and naphthoquinone, but did not exhibit transhydrogenase, reductase or oxidase activity. Moreover, NADH dehydrogenase was also capable of reducing FAD and FMN. NAD(P)H and NADH dehydrogenases possessed cytochrome-c reductase activity, which was stimulated by menadione and ubiquinone Q₁. The activity of NAD(P)H and NADH dehydrogenases depended on culture-growth conditions. The activity of NAD(P)H dehydrogenase from cells grown under chemoheterotrophic aerobic conditions was the lowest and it increased notably under photoheterotrophic anaerobic conditions upon lactate or malate growth limitation. The activity of NADH dehydrogenase was higher from the cells grown under photoheterotrophic anaerobic conditions upon nitrate growth limitation and under chemoheterotrophic aerobic conditions. NADPH dehydrogenase synthesis dependence on R. capsulatus growth conditions was insignificant.     

Superoxide-independent reduction of vanadate by rat liver microsomes/NAD(P)H: vanadate reductase activity.

It has been reported that vanadate-stimulated oxidation of NAD(P)H by microsomal systems can proceed anaerobically, in contrast to the general notion that the oxidation proceeds exclusively by an O(2-)-dependent free radical chain mechanism. The current study indicates that microsomal systems are endowed with a vanadate-reductase property, involving a NAD(P)H-dependent electron transport cytochrome P450 system. Our ESR measurements demonstrated the formation of a vanadium(IV) species in a mixture containing vanadate, rat liver microsomes, and NAD(P)H. This vanadium(IV) species was identified as the vanadyl ion (VO2+) by comparison with the ESR spectrum of VOSO4. The initial rate of vanadium(IV) formation depends linearly on the concentration of microsomes. The Michaelis-Menten constants were found to be: km = 1.25 mM and Vmax = 0.066 mumol (min)-1 (mg microsomes)-1, respectively. Pretreatment of the microsomes with carbon monoxide or K3Fe(CN)6 reduced vanadium(IV) generation, suggesting that the NAD(P)H-dependent electron transport cytochrome P450 system plays a significant role in the microsomal reduction of vanadate. Measurements under argon or in the presence of superoxide dismutase caused only minor (less than 10%) reductions in vanadium(IV) generation. The VO2+ species was also detected in NAD(P)H oxidation by fructose plus vanadate, a reaction known to proceed via an O(2-)-mediated chain mechanism. However, the amount of vanadium(IV) generated by this reaction was an order of magnitude smaller than that by the microsomal system and was inhibitable by superoxide dismutase, affirming the conclusion that the microsomal/NAD(P)H system is endowed with the (O(2-)-independent) vanadium(V) reductase property.     

Glucose dehydrogenase of bovine heart: activity, purification and properties of the enzyme

The activity of glucose dehydrogenase (EC 1.1.1.47; GDH; glucose NAD(P) oxidoreductase) was demonstrated in the supernatant obtained after centrifugation (1500 g) of bovine heart homogenate. More than 50% of GDH activity was found in the microsomal fraction. The optimum pH for the microsomal enzyme was 8.9. About 200-fold purification of GDH was achieved by successive application of ultracentrifugation in 0.25 M mannitol, treatment with solid ammonium sulphate, and CM-32 cellulose chromatography. The purified preparation oxidized not only glucose but also D-glucosamine, N-acetylglucosamine and xylose in 87, 63 and 23%, respectively. Purified GDH was inhibited by p-chloromercuribenzoate and glucose 6-phosphate.     

The activation of glucose dehydrogenase by p-chloromercuribenzoate

Summary P-Chloromercuribenzoate alters various reactions of rat liver glucose (hexose phosphate) dehydrogenase differently. The reagent has little effect on the glucose: NAD or the glucose: NADP oxidoreductases, doubles the rates of oxidations of galactose-6-phosphate and glucose-6-phosphate by NADP and greatly stimulates the oxidations of glucose-6-phosphate and galactose-6-phosphate by NAD. The reagent appears to react with a sulfhydryl group of the enzyme since activation is reversed and prevented by mercaptoethanol. The direct reaction of the reagent with the enzyme is indicated by its lower thermal stability in the presence of the p-chloromercuribenzoate. The size of the enzyme appears to be the same when determined by sucrose gradient centrifugation in the presence or absence of p-chloromercuribenzoate. In microsomes, the oxidation of NADH or NADPH hampers measurements of glucose dehydrogenase. Since p-chloromercuribenzoate inhibits microsomal oxidation of reduced nicontinamide nucleotides, it is possible to assay for glucose dehydrogenase accurately in the presence of the mercurial in microsomes and microsomal extracts and thus measure the effectiveness of a detergent in extracting the enzyme from microsomes.Abbreviation pcMB p-chloromercuribenzoic acid     

Hydride transfer stereospecificity of rat liver aldehyde dehydrogenases

The stereospecificity of hydride transfer to NAD+ by several forms of rat liver aldehyde dehydrogenase was determined by a nuclear magnetic resonance method. The forms included several mitochondrial and microsomal isozymes from normal liver, as well as isozymes from xenobiotic-treated and tumor cells. The proton added to NAD+ comes exclusively from the aldehyde substrate and in all cases was A (pro-R)-stereospecific.     

Enzymatic lipid peroxidation and oxidative metabolism of aminazine in brain microsomal fractions]

NAD (P) H-dependent enzymic systems, both of lipid peroxidation and chlorpromazine oxidative metabolism are shown to be localized in the microsomal fractions from human and rat brain. Hydroxy-derivatives of chlorpromazine (e.g. 7-OH-chlorpromazine) formed in the course of enzymic NADPH-dependent metabolism possess antioxidant activity and inhibit lipid peroxidation in the brain microsomes. The properties of enzymic NAD (P) H-dependent oxigenase systems in the membranes of the microsomal reticulum of the liver and brain are compared.     

Succinate Dehydrogenase and Other Respiratory Pathways in Thylakoid Membranes of Synechocystis sp. Strain PCC 6803: Capacity Comparisons and Physiological Function

Respiration in cyanobacterial thylakoid membranes is interwoven with photosynthetic processes. We have constructed a range of mutants that are impaired in several combinations of respiratory and photosynthetic electron transport complexes and have examined the relative effects on the redox state of the plastoquinone (PQ) pool by using a quinone electrode. Succinate dehydrogenase has a major effect on the PQ redox poise, as mutants lacking this enzyme showed a much more oxidized PQ pool. Mutants lacking type I and II NAD(P)H dehydrogenases also had more oxidized PQ pools. However, in the mutant lacking type I NADPH dehydrogenase, succinate was essentially absent and effective respiratory electron donation to the PQ pool could be established after addition of 1 mM succinate. Therefore, lack of the type I NADPH dehydrogenase had an indirect effect on the PQ pool redox state. The electron donation capacity of succinate dehydrogenase was found to be an order of magnitude larger than that of type I and II NAD(P)H dehydrogenases. The reason for the oxidized PQ pool upon inactivation of type II NADH dehydrogenase may be related to the facts that the NAD pool in the cell is much smaller than that of NADP and that the NAD pool is fully reduced in the mutant without type II NADH dehydrogenase, thus causing regulatory inhibition. The results indicate that succinate dehydrogenase is the main respiratory electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases regulate the reduction level of NADP and NAD, which, in turn, affects respiratory electron flow through succinate dehydrogenase.     

A quantitative histochemical study of NADPH-ferrihemoprotein reductase activity

Summary A quantitative histochemical assay for NADPH-ferrihemoprotein (P450) reductase had been developed. For optimal activity, it is necessary to use a relatively electropositive tetrazolium salt such as neotetrazolium chloride as the final acceptor. The apparentK _m of the reaction is 0.83 mM. Its specificity has been proven in two ways: (i) activity is increased selectively in the pericentral zone of liver from rats treated with phenobarbitone, an inducer of the reductase, though not in liver of rats injected with 3-methylcholanthrene, which induces NAD(P)H dehydrogenase; (ii) it is competitively inhibited by NADP⁺ (K _i=1.50mm) though unaffected by dicumarol, an inhibitor of NAD(P)H dehydrogenase activity. An NADP⁺ concentration ten times greater than the substrate concentration inhibits the histochemical reaction and the reaction in a microsomal fraction assayed biochemically to the same degree (70% inhibition). The amount of inhibition is independent of temperature, of the zone of the acinus and of the treatment of the animal.Continuous microdensitometric monitoring of the reaction product as it is formed has shown that the specific reaction is linear with incubation up to 10 min, thus allowing end-point measurements to be used for cytophotometric analysis.     

Aldehyde dehydrogenase heterogeneity in rat hepatic cells

In normal rat liver, aldehyde dehydrogenase (Aldehyde:NAD+ oxidoreductase, EC 1.2.1.3; ALDH) is found primarily in mitochondrial and microsomal fractions. During hepatocarcinogenesis, an additional tumor-associated aldehyde dehydrogenase (T-ALDH) is detectable in the cytosol of preneoplastic and neoplastic cells. We report here differences in the ALDH distribution pattern in different rat hepatoma cell lines compared to normal rat hepatocytes. Of the four basal ALDH enzymes, one mitochondrial ALDH and one microsomal ALDH account for 96% of total ALDH molecules detectable with our probes in normal hepatocytes. The other two mitochondrial and microsomal ALDH enzymes are only detectable in the appropriate subcellular fraction from large populations of cells. The tumor-associated ALDH is not detectable in normal hepatocytes. In addition to varying amounts of T-ALDH in the six different rat hepatoma cell lines examined, differences in the amounts of mitochondrial and microsomal ALDHs also occur in both high and low T-ALDH activity hepatoma cell lines. Each of five ALDH enzymes examined has a characteristic half-life varying from 45 min to 95 h.     

The proton-translocating nicotinamide–dinucleotide (phosphate) transhydrogenase of rat liver mitochondria

1. The NAD(P) transhydrogenase activity of the soluble fraction of sonicated rat liver mitochondrial preparations was greater than the NAD-linked isocitrate dehydrogenase activity, and the NAD-linked and NADP-linked isocitrate dehydrogenase activities were not additive. The NAD-linked isocitrate dehydrogenase activity was destroyed by an endogenous autolytic system or by added nucleotide pyrophosphatase, and was restored by a catalytic amount of NADP. 2. We concluded that the isocitrate dehydrogenase of rat liver mitochondria was exclusively NADP-specific, and that the oxoglutarate/isocitrate couple could therefore be used unequivocally as redox reactant for NADP in experiments designed to operate only the NAD(P) transhydrogenase (or loop 0) segment of the respiratory chain in intact mitochondria. 3. During oxidation of isocitrate by acetoacetate in intact, anaerobic, mitochondria via the rhein-sensitive, but rotenone- and arsenite-insensitive, NAD(P) transhydrogenase, measurements of the rates of carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive pH change in the presence of various oxoglutarate/isocitrate concentration ratios gave an -->H(+)/2e(-) quotient of 1.94+/-0.12 for outward proton translocation by the NAD(P) transhydrogenase. 4. Measurements with a K(+)-sensitive electrode confirmed that the electrogenicity of the NAD(P) transhydrogenase reaction corresponded to the translocation of one positive charge per acid equivalent. 5. Sluggish reversal of the NAD(P) transhydrogenase reaction resulted in a significant inward proton translocation. 6. The possibility that isocitrate might normally be oxidized via loop 0 at a redox potential of -450mV, or even more negative, is discussed, and implies that a P/O quotient of 4 for isocitrate oxidation might be expected.