Mitochondrial aldehyde dehydrogenase from higher plants

Aldehyde dehydrogenase has been purified to homogeneity from mitochondria of potato tubers and pea epicotyls. Although the enzyme had a high affinity for glycolaldehyde it also had a high affinity for a number of other aliphatic and arylaldehydes. It is proposed that the codification glycolaldehyde dehydrogenase (EC 1.2.1.22) should be abandoned in favour of mitochondrial aldehyde dehydrogenase (EC 1.2.1.3). The purified enzyme showed esterase activity and had properties similar to those reported for the mammalian mitochondrial aldehyde dehydrogenase. Although the natural substrate(s) for the enzyme is not known, the kinetic properties of the enzyme are consistent with it playing a role in the oxidation of acetaldehyde, glycolaldehyde and indoleacetaldehyde.     

Insulin-like effect of vanadate on malic enzyme and glucose-6-phosphate dehydrogenase activities in streptozotocin-induced diabetic rat liver

The effect of oral administration of sodium orthovanadate on hepatic malic enzyme (EC 1.1.1.40) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activities was investigated in nondiabetic and diabetic rats. Streptozotocin-induced diabetic rats were characterized by 4.7-fold increase in plasma glucose and 82% decrease in plasma insulin levels. The activities of hepatic malic enzyme and glucose-6-phosphate dehydrogenase were also diminished (P less than 0.001). Vanadate treatment in diabetic rats led to a significant decrease (P less than 0.001) in plasma glucose levels and to the normalization of enzyme activities, but it did not alter plasma insulin levels. In nondiabetic rats vanadate decreased the plasma insulin level by 64% without altering the enzyme activities. Significant correlation was observed between plasma insulin and hepatic lipogenic enzyme activities in untreated and vanadate-treated rats. Vanadate administration caused a shift to left in this correlation suggesting improvement in insulin sensitivity.     

Steady-state kinetics of formaldehyde dehydrogenase and formate dehydrogenase from a methanol-utilizing yeast, Candida boidinii.

Initial velocity studies and product inhibition studies were conducted for the forward and reverse reactions of formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC 1.2.1.1) isolated from a methanol-utilizing yeast Candida boidinii. The data were consistent with an ordered Bi-Bi mechanism for this reaction in which NAD+ is bound first to the enzyme and NADH released last. Kinetic studies indicated that the nucleoside phosphates ATP, ADP and AMP are competitive inhibitors with respect to NAD and noncompetitive inhibitors with respect to S-hydroxymethylglutathione. The inhibitions of the enzyme activity by ATP and ADP are greater at pH 6.0 and 6.5 than at neutral or alkaline pH values. The kinetic studies of formate dehydrogenase (formate:NAD oxidoreductase, EC 1.2.1.2) from the methanol grown C. boidinii suggested also an ordered Bi-Bi mechanism with NAD being the first substrate and NADH the last product. Formate dehydrogenase the last enzyme of the dissimilatory pathway of the methanol metabolism is also inhibited by adenosine phosphates. Since the intracellular concentrations of NADH and ATP are in the range of the Ki values for formaldehyde dehydrogenase and formate dehydrogenase the activities of these main enzymes of the dissimilatory pathway of methanol metabolism in this yeast may be regulated by these compounds.     

Bioelectrochemical fuel cell and sensor based on a quinoprotein,alcohol dehydrogenase

A biofuel cell, yielding a stable and continuous low-power output, based on the enzymatic oxidation of methanol to formic acid has been designed and investigated. The homogeneous kinetics of the electrochemically-coupled enzymatic oxidation reaction were investigated and optimized. The biofuel cell also functioned as a sensitive method for the detection of primary alcohols. A method for medium-scale preparation of the enzyme alcohol dehydrogenase [alcohol:(acceptor) oxidoreductase, EC 1.1.99.8] is described.     

Purification and characterization of human liver "high Km" aldehyde dehydrogenase and its identification as glutamic gamma-semialdehyde dehydrogenase

The enzyme previously considered as an isozyme (E4, ALDH IV) of human liver aldehyde dehydrogenase (NAD+) (EC 1.2.1.3) has been purified to homogeneity by the use of ion exchange chromatography on CM-Sephadex and affinity chromatography on Blue Sepharose CL-6B and 5'-AMP Sepharose 4B and identified as glutamic gamma-semialdehyde dehydrogenase, or more precisely 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12). Glutamic gamma-semialdehyde dehydrogenase was never previously purified to homogeneity from any mammalian species. The homogeneous enzyme is seen on isoelectric focusing gels as two fine bands separated by 0.12 pH units: pI = 6.89 and 6.77. In addition, the enzyme also appears as two bands in gradient gels; however, in polyacrylamide gels containing sodium dodecyl sulfate the enzyme migrates as one band, indicating that its subunits are of identical size. Because the enzyme molecule is considerably smaller (Mr approximately 142,000-170,000) than that of aldehyde dehydrogenases (EC 1.2.1.3) (Greenfield, N. J., and Pietruszko, R. (1977) Biochim. Biophys. Acta 483, 35-45; Mr approximately 220,000) and its subunit weight is different (70,600 versus approximately 54,000 for E1 and E2 isozymes), the enzyme is not an isozyme of aldehyde dehydrogenase previously described. The Michaelis constants for glutamic gamma-semialdehyde dehydrogenase with acetaldehyde and propionaldehyde are in the millimolar range. Its substrate specificity within the straight chain aliphatic aldehyde series is essentially confined to that of acetaldehyde and propionaldehyde with butyraldehyde and longer chain length aldehydes being considerably less active. Other substrates include succinic, glutaric, and adipic semialdehydes in addition to glutamic gamma-semialdehyde. The reaction velocity with glutamic gamma-semialdehyde is at least an order of magnitude larger than with carboxylic acid semialdehydes. Aspartic beta-semialdehyde is not a substrate. The reaction catalyzed appears to be irreversible. Although NADP can be used, NAD is the preferred coenzyme. The enzyme also exhibits an unusual property of being subject to substrate inhibition by NAD.     

The metabolism of ethanol-derived acetaldehyde by alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (EC 1.2.1.3) in Drosophila melanogaster larvae.

Both aldehyde dehydrogenase (ALDH, EC 1.2.1.3) and the aldehyde dehydrogenase activity of alcohol dehydrogenase (ADH, EC 1.1.1.1) were found to coexist in Drosophila melanogaster larvae. The enzymes, however, showed different inhibition patterns with respect to pyrazole, cyanamide and disulphiram. ALDH-1 and ALDH-2 isoenzymes were detected in larvae by electrophoretic methods. Nonetheless, in tracer studies in vivo, more than 75% of the acetaldehyde converted to acetate by the ADH ethanol-degrading pathway appeared to be also catalysed by the ADH enzyme. The larval fat body probably was the major site of this pathway.     

Time-dependence of the effect of X-irradiation on the formation of glycerol phosphate dehydrogenase and other dehydrogenases in the developing rat brain

X-irradiation (100-1500 r) administered to the heads of rats 8-30 days of age inhibited the development of glycerol phosphate dehydrogenase (l-glycerol 3-phosphate-NAD oxidoreductase, EC 1.1.1.8) in the brain stem and cerebral hemispheres. At 40 days of age and older no effect was observed. This inhibition was a delayed phenomenon, dose-dependent and with no recovery. It is proposed that the inhibition of enzyme formation is related to radiation damage caused to DNA. Actinomycin D inhibited the development of glycerol phosphate dehydrogenase in a manner similar to ionizing radiation. Four other dehydrogenases also showed age-dependent radiosensitivities. ;Malic enzyme' (EC 1.1.1.40), lactate dehydrogenase (EC 1.1.1.27) and malate dehydrogenase (EC 1.1.1.37) ceased to be radiosensitive at about 8 days of age and isocitrate dehydrogenase (NADP) (EC 1.1.1.42) at 16 days. The correlation between developmental increase in enzyme activity and radiosensitivity held closely for glycerol phosphate dehydrogenase and isocitrate dehydrogenase and to a smaller extent for the others.     

5-keto-D-gluconate production is catalyzed by a quinoprotein glycerol dehydrogenase,major polyol dehydrogenase,in gluconobacter species

Acetic acid bacteria, especially Gluconobacter species, have been known to catalyze the extensive oxidation of sugar alcohols (polyols) such as D-mannitol, glycerol, D-sorbitol, and so on. Gluconobacter species also oxidize sugars and sugar acids and uniquely accumulate two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, in the culture medium by the oxidation of D-gluconate. However, there are still many controversies regarding their enzyme systems, especially on D-sorbitol and also D-gluconate oxidations. Recently, pyrroloquinoline quinone-dependent quinoprotein D-arabitol dehydrogenase and D-sorbitol dehydrogenase have been purified from G. suboxydans, both of which have similar and broad substrate specificity towards several different polyols. In this study, both quinoproteins were shown to be identical based on their immuno-cross-reactivity and also on gene disruption and were suggested to be the same as the previously isolated glycerol dehydrogenase (EC 1.1.99.22). Thus, glycerol dehydrogenase is the major polyol dehydrogenase involved in the oxidation of almost all sugar alcohols in Gluconobacter sp. In addition, the so-called quinoprotein glycerol dehydrogenase was also uniquely shown to oxidize D-gluconate, which was completely different from flavoprotein D-gluconate dehydrogenase (EC 1.1.99.3), which is involved in the production of 2-keto-D-gluconate. The gene disruption experiment and the reconstitution system of the purified enzyme in this study clearly showed that the production of 5-keto-D-gluconate in G. suboxydans is solely dependent on the quinoprotein glycerol dehydrogenase.     

Role of glycerol 3-phosphate dehydrogenase in glyceride metabolism. Effect of diet on enzyme activities in chicken liver.

1. The metabolic role of hepatic NAD-linked glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) was investigated vis-a-vis glyceride synthesis, glyceride degradation and the maintainence of the NAD redox state. 2. Five-week-old chickens were placed on five dietary regimes: a control group, a group on an increased-carbohydrate-lowered-fat diet, a group on a high-fat-lowered-carbohydrate diet, a starved group and a starved-refed group. In each group the specific activity (mumol/min per g wet wt. of tissue) of hepatic glycerol 3-phosphate dehydrogenase was compared with the activities of the beta-oxoacyl-(acyl-carrier protein) reductase component of fatty acid synthetase, glycerol kinase (EC 2.7.1.30) and lactate dehydrogenase (EC 1.1.1.27). 3. During starvation, the activities of glycerol 3-phosphate dehydrogenase, glycerol kinase and lactate dehydrogenase rose significantly. After re-feeding these activities returned to near normal. All three activities rose slightly on the high-fat diet. Lactate dehydrogenase activity rose slightly, whereas those of the other two enzymes fell slightly on the increased-carbohydrate-lowered-fat diet. 4. The activity of the beta-oxoacyl-(acyl-carrier protein) reductase component of fatty acid synthetase, a lipid-synthesizing enzyme, contrasted strikingly with the other three enzyme activities. Its activity was slightly elevated on the increased-carbohydrate diet and significantly diminished on the high-fat diet and during starvation. 5. The changes in activity of the chicken liver isoenzyme of glycerol 3-phosphate dehydrogenase in response to dietary stresses suggest that the enzyme has an important metabolic role other than or in addition to glyceride biosynthesis.     

Purification and properties of soybean nodule xanthine dehydrogenase

Xanthine dehydrogenase (EC 1.2.1.37), an essential enzyme for ureide metabolism was purified from the cytosol fraction of soybean nodules. The purified xanthine dehydrogenase was shown to be homogeneous by electrophoresis and a pI of 4.7 was determined by isoelectric focusing. The enzyme had a molecular weight of 285,000 and two subunits of molecular weight 141,000 each. The holoenzyme contained 1.7 (±0.7) mol Mo and 8.1 (±2.0) mol Fe/mol enzyme and the enzyme also contained FMN and is thus a molybdoironflavoprotein. Soybean xanthine dehydrogenase is the second enzyme in plants demonstrated to contain Mo and the first xanthine-oxidizing enzyme reported to contain FMN, rather than FAD as the flavin cofactor.     

Effect of phenylpyruvate on pyruvate dehydrogenase activity in rat brain mitochondria

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of (14)CO(2) from [1-(14)C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K(i) of 100mum. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli.

The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.     

Interaction of mitochondrial malate dehydrogenase monomer with phospholipid vesicles.

The association between bovine and porcine mitochondrial malate dehydrogenase (EC 1.1.1.37) and phospholipid vesicles was investigated. At concentrations at which malate dehydrogenase exists as a dimer, entrapment within the aqueous compartment but not binding of the 14C-labelled enzyme was observed. The dissociated enzyme was labile to moderate heat and to p-chloromercuribenzoate, but in both cases inactivation was decreased by incubation with suspensions of charged phospholipid vesicles. This suggested an interaction between enzyme subunits and phospholipid, and this was confirmed by direct binding measurements and by studies that followed changes in the fluorescein-labelled enzyme. The circular-dichroism spectra of the enzyme indicated a high alpha-helix content, and suggested that a small conformational change occurred when the enzyme dissociated. Fluorescence data also suggested less-rigid molecules after dissociation. A possible mechanism, based on the flexibility of enzyme monomer and its interaction with phospholipids, by which mitochondrial matrix enzymes are specifically localized in cells, is discussed.     

Glutamate dehydrogenase and aspartate aminotransferase in Trypanosoma cruzi.

1. The cultured, epimastigote-form of Trypanosoma cruzi contains NADP-linked glutamate dehydrogenase (EC 1.4.1.4), with a molecular weight of about 280,000, similar to the enzyme from Plasmodium chabaudi and different from the enzymes from higher animal sources. 2. T. cruzi also contains aspartate aminotransferase (EC 2.6.1.1), with properties similar to those of the enzyme from mammals. 3. The concerted action of the transaminase and glutamate dehydrogenase might be responsible for the production of NH3 which characterizes the protein catabolism in T. cruzi.     

Further characterization of L-beta-hydroxyacid dehydrogenase from Drosophila

L-beta-Hydroxyacid dehydrogenase (L-beta-hydroxyacid-NAD-oxidoreductase, EC 1.1.1.45) of Drosophila is composed of two, identical subunits with a molecular weight of approx. 33 300. The enzyme was purified 938-fold from Drosophila melanogaster. An isoelectric point of 8.6 was determined for L-beta-hydroxyacid dehydrogenase. An amino acid analysis was conducted of the purified enzyme. A single subunit was obtained by SDS-gel electrophoresis of the purified enzyme. Translation of larval and adult mRNA in a mRNA-dependent reticulocyte lysate, followed by immune precipitation using anti-L-beta-hydroxyacid dehydrogenase IgG revealed a single L-beta-hydroxyacid dehydrogenase subunit of 33 300. Larval and adult proteins were the same size. The enzyme does not appear to be subjected to substantial post-translational modifications.     

Rat liver microsomal 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase: solubilization, separation and partial purification

Rat liver microsomes contain 3 alpha-hydroxysteroid dehydrogenase (HSD) (EC 1.1.1.50) and dihydrodiol dehydrogenase (DHD) (EC 1.3.1.20) activities. The two enzyme activities were solubilized by 10% Triton X-100 or 0.4% sodium deoxycholate. Unlike the cytosolic enzyme (Penning & Talalay (1983) Proc. Natl. Acad. Sci. U.S.A., 80, 4505), the microsomal HSD and DHD activities were not inhibited by indomethacin. Chromatography of the microsomal Triton X-100 extract on Affigel Blue and then on Phenyl-Sepharose gave an HSD preparation containing no detectable (less than 3 - 5%) DHD activity, whereas chromatography of the deoxycholate extract on Phenyl-Sepharose provided a DHD preparation that lacked measurable HSD activity. These results are in sharp contrast to the cytosolic enzyme where both HSD and DHD activities could be copurified to homogeneity (Penning et al. (1984) Biochem. J. 222, 601).     

The reaction of choline dehydrogenase with some electron acceptors.

1. The choline dehydrogenase (EC 1.1.99.1) WAS SOLUBILIZED FROM ACETONE-DRIED POWDERS OF RAT LIVER MITOCHONDRIA BY TREATMENT WITH Naja naja venom. 2. The kinetics of the reaction of enzyme with phenazine methosulphate and ubiquinone-2 as electron acceptors were investigated. 3. With both electron acceptors the reaction mechanism appears to involve a free, modified-enzyme intermediate. 4. With some electron acceptors the maximum velocity of the reaction is independent of the nature of the acceptor. With phenazine methosulphate and ubiquinone-2 as acceptors the Km value for choline is also independent of the nature of the acceptor molecule. 5. The mechanism of the Triton X-100-solubilized enzyme is apparently the smae as that for the snake venom solubilized enzyme.     

Uridine diphosphate D-glucose dehydrogenase of Aerobacter aerogenes

Uridine diphosphate d-glucose dehydrogenase (EC 1.1.1.22) from Aerobacter aerogenes has been partially purified and its properties have been investigated. The molecular weight of the enzyme is between 70,000 and 100,000. Uridine diphosphate d-glucose is a substrate; the diphosphoglucose derivatives of adenosine, cytidine, guanosine, and thymidine are not substrates. Nicotinamide adenine dinucleotide (NAD), but not nicotinamide adenine dinucleotide phosphate, is active as hydrogen acceptor. The pH optimum is between 9.4 and 9.7; the K(m) is 0.6 mm for uridine diphosphate d-glucose and 0.06 mm for NAD. Inhibition of the enzyme by uridine diphosphate d-xylose is noncooperative and of mixed type; the K(i) is 0.08 mm. Thus, uridine diphosphate d-glucose dehydrogenase from A. aerogenes differs from the enzyme from mammalian liver, higher plants, and Cryptococcus laurentii, in which uridine diphosphate d-xylose functions as a cooperative, allosteric feedback inhibitor.     

Oestradiol-17 alpha dehydrogenase from chicken liver.

The NADP+-linked oestradiol-17 alpha dehydrogenase (EC 1.1.1.148) present in cell-free extracts of chicken liver was investigated with the aim of separating it from a closely related oestradiol-17 beta dehydrogenase (EC 1.1.1.62) found in the same subcellular fraction. However, its chromatographic behaviour on CM-cellulose and DEAE-cellulose was almost identical with that previously reported for the latter enzyme, including resolution into two peaks on the anion-exchanger. Both peaks contained oestradiol-17 alpha dehydrogenase and oestradiol-17 beta dehydrogenase activity. Further attempts to separate the putative enzymes by dye-ligand chromatography with the use of the dyes Procion Yellow, Reactive Red and Cibachron Blue linked to Sepharose were unsuccessful, and they behaved identically on affinity columns of adenosine 2',5'-bisphosphate-agarose and 17 beta-oestradiol 3-hemisuccinate bound to Sepharose. A previous report of partial separation on Sephadex G-200 was not confirmed. Slab gel electrophoresis of enzyme preparations after affinity chromatography on adenosine 2',5'-bisphosphate-agarose revealed multiple bands in systems containing sodium dodecyl sulphate, whereas analysis by rod gel electrophoresis gave two major and one minor bands that stained coincidently for oestradiol-17 alpha dehydrogenase, oestradiol-17 beta dehydrogenase, epitestosterone dehydrogenase and testosterone dehydrogenase activities. Isoelectric focusing gave four enzymically active peaks that each oxidized oestradiol-17 alpha and -17 beta. Apparent Km values for the two forms of oestradiol-17 alpha dehydrogenase obtained by DEAE-cellulose chromatography were 17 and 23 microM for oestradiol-17 alpha, and 8.7 and 11.0 microM for NADP+. Limited kinetic studies with oestradiol-17 alpha and -17 beta with the use of the mixed-substrate method showed that the total velocity was equal to the sum of the separate velocities. The active-site inhibitor-alkylating agent 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one did not cause time- or temperature-dependent inhibition, in contrast with the reported case of the oestradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities of the human placental oestradiol dehydrogenase. NADP+ appeared to afford some protection against inhibition. Investigation of substrate specificity with a limited range of steroids suggests that the enzyme(s) from chicken liver differs substantially from the oestradiol-17 beta dehydrogenase from human placenta, and although the evidence is not conclusive it suggests the existence of one enzyme.     

Purification and properties of NADP-dependent glutamate dehydrogenase from yeast nuclear fractions.

1. NADP-dependent glutamate dehydrogenase (EC 1.4.1.4) extracted from nuclear fractions of Saccharomyces cerevisiae was partially purified. The final purification achieved was over 100-fold over the initial extract. 2. Cellulose acetate electrophoresis shows that the preparation is close to homogeneity and that the enzyme is slightly more anionic than cytoplasmic glutamate dehydrogenase. 3. The response of the nuclear activity to variation of pH, of inorganic phosphate and other electrolyte concentration and of the concentration of the reaction substrates has been investigated. Several differences were detected in comparison with cytoplasmic glutamate dehydrogenase.