Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, sorbitol dehydrogenase and xanthine oxidase from mouse tissues.

1. Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase, sorbitol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase extracted from tissues of inbred mice were examined. 2. ADH isozymes were differentially distributed in mouse tissues: A2--liver, kidney, adrenals and intestine; B2--all tissues examined; C2--stomach, adrenals, epididymis, ovary, uterus, lung. 3. Two NAD+-specific aldehyde dehydrogenase isozymes were observed in liver and kidney and differentially distributed in other tissues. Alcohol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase were also stained when aldehyde dehydrogenase was being examined. 4. Two aldehyde oxidase isozymes exhibited highest activities in liver. 5. "Phenazine oxidase" was widely distributed in mouse tissues whereas xanthine oxidase exhibited highest activity in intestine and liver extracts. 6. Genetic variants for ADH-C2 established its identity with a second form of sorbitol dehydrogenase observed in stomach and other tissues. The major sorbitol dehydrogenase was found in high activity in liver, kidney, pancreas and male reproductive tissues.     

A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae a nuclear recessive mutation, lpd1, which simultaneously abolishes the activities of lipoamide dehydrogenase, 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase has been identified. Strains carrying this mutation can grow on glucose or poorly on ethanol, but are unable to grow on media with glycerol or acetate as carbon source. The mutation does not prevent the formation of other tricarboxylic acid cycle enzymes such as fumarase, NAD⁺-linked isocitrate dehydrogenase or succinate-cytochrome c oxidoreductase, but these are produced at about 50%–70% of the wild-type levels. The mutation probably affects the structural gene for lipoamide dehydrogenase since the amount of this enzyme in the cell is subject to a gene dosage effect; heterozygous lpd1 diploids produce half the amount of a homozygous wild-type strain. Moreover, a yeast sequence complementing this mutation when present in the cell on a multicopy plasmid leads to marked overproduction of lipoamide dehydrogenase. Homozygous lpd1 diploids were unable to sporulate indicating that some lipoamide dehydrogenase activity is essential for sporulation to occur on acetate.     

Interaction between NAD-dependent isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex, and NADH:ubiquinone oxidoreductase

Interaction between the alpha-ketoglutarate dehydrogenase complex and NAD+-dependent isocitrate dehydrogenase was detected with a variety of techniques including polyethylene glycol precipitation, ultracentrifugation, and centrifugal gel filtration on a Sepharose 6B column. The interaction was specific in that citrate synthase, cytosolic malate dehydrogenase, and NADP-dependent isocitrate dehydrogenase did not interact with alpha-ketoglutarate dehydrogenase complex. The interaction was not inhibited by either 0.1 M KCl or 0.4 M (NH4)2SO4, but was completely prevented by 5% glycerol. A new method for the preparation of NADH: ubiquinone oxidoreductase resulted in an enzyme having a protein subunit composition similar to that of classical complex I preparation. Evidence is given for the existence of ternary complexes containing NADH:ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-NAD-dependent isocitrate dehydrogenase and NADH: ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-succinate thiokinase. These data suggest that a part of the citric acid cycle may be located in the vicinity of NADH: ubiquinone oxidoreductase. These complexes may facilitate the transport of metabolites among these enzymes without their equilibrating with the whole compartment.     

Glycerol dehydrogenase. structure, specificity, and mechanism of a family III polyol dehydrogenase

BACKGROUND: Bacillus stearothermophilus glycerol dehydrogenase (GlyDH) (glycerol:NAD(+) 2-oxidoreductase, EC 1.1.1.6) catalyzes the oxidation of glycerol to dihydroxyacetone (1,3-dihydroxypropanone) with concomitant reduction of NAD(+) to NADH. Analysis of the sequence of this enzyme indicates that it is a member of the so-called iron-containing alcohol dehydrogenase family. Despite this sequence similarity, GlyDH shows a strict dependence on zinc for activity. On the basis of this, we propose to rename this group the family III metal-dependent polyol dehydrogenases. To date, no structural data have been reported for any enzyme in this group. RESULTS: The crystal structure of B. stearothermophilus glycerol dehydrogenase has been determined at 1.7 A resolution to provide structural insights into the mechanistic features of this family. The enzyme has 370 amino acid residues, has a molecular mass of 39.5 kDa, and is a homooctamer in solution. CONCLUSIONS: Analysis of the crystal structures of the free enzyme and of the binary complexes with NAD(+) and glycerol show that the active site of GlyDH lies in the cleft between the enzyme's two domains, with the catalytic zinc ion playing a role in stabilizing an alkoxide intermediate. In addition, the specificity of this enzyme for a range of diols can be understood, as both hydroxyls of the glycerol form ligands to the enzyme-bound Zn(2+) ion at the active site. The structure further reveals a previously unsuspected similarity to dehydroquinate synthase, an enzyme whose more complex chemistry shares a common chemical step with that catalyzed by glycerol dehydrogenase, providing a striking example of divergent evolution. Finally, the structure suggests that the NAD(+) binding domain of GlyDH may be related to that of the classical Rossmann fold by switching the sequence order of the two mononucleotide binding folds that make up this domain.     

Activities of formylmethanofuran dehydrogenase,methylenetetrahydromethanopterin dehydrogenase,methylenetetrahydromethanopterin reductase,and heterodisulfide reductase in methanogenic bacteria

The activities of formylmethanofuran dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin reductase, and heterodisulfide reductase were tested in cell extracts of 10 different methanogenic bacteria grown on H₂/CO₂ or on other methanogenic substrates. The four activities were found in all the organisms investigated: Methanobacterium thermoautotrophicum,M. wolfei, Methanobrevibacter arboriphilus, Methanosphaera stadtmanae, Methanosarcina barkeri (strains Fusaro and MS), Methanothrix soehngenii, Methanospirillum hungatei, Methanogenium organophilum, and Methanococcus voltae. Cell extracts of H₂/CO₂ grown M. barkeri and of methanol grown M. barkeri showed the same specific activities suggesting that the four enzymes are of equal importance in CO₂ reduction to methane and in methanol disproportionation to CO₂ and CH₄. In contrast, cell extracts of acetate grown M. barkeri exhibited much lower activities of formylmethanofuran dehydrogenase and methylenetetrahydromethanopterin dehydrogenase suggesting that these two enzymes are not involved in methanogenesis from acetate. In M. stadtmanae, which grows on H₂ and methanol, only heterodisulfide reductase was detected in activities sufficient to account for the in vivo methane formation rate. This finding is consistent with the view that the three other oxidoreductases are not required for methanol reduction to methane with H₂.     

NAD-linked, factor-dependent formaldehyde dehydrogenase or trimeric, zinc-containing, long-chain alcohol dehydrogenase from Amycolatopsis methanolica.

NAD-linked, factor-dependent formaldehyde dehydrogenase (FD-FA1DH) of the Gram-positive methylotrophic bacterium, Amycolatopsis methanolica, was purified to homogeneity. It is a trimeric enzyme with identical subunits (molecular mass 40 kDa) containing 6 atoms Zn/enzyme molecule. The factor is a heat-stable, low-molecular-mass compound, which showed retention on an Aminex HPX-87H column. Inactivation of the factor occurred during manipulation, but activity could be restored by incubation with dithiothreitol. The identity of the factor is still unknown. It could not be replaced by thiol compounds or cofactors known to be involved in metabolism of C1 compounds. Of the aldehydes tested, only formaldehyde was a substrate. However, the enzyme showed also activity with higher aliphatic alcohols and the presence of the factor was not required for this reaction. Methanol was not a substrate, but high concentrations of it could replace the factor in the conversion of formaldehyde. Presumably, a hemiacetal of formaldehyde is the genuine substrate, which, in the case of methanol, acts as a factor leading to methylformate as the product. This view is supported by the fact that formate could only be detected in the reaction mixture after acidification. Inhibition studies revealed that the enzyme contains a reactive thiol group, being protected by the binding of NAD against attack by heavy-metal ions and aldehydes. Studies on the effect of the order of addition of coenzyme and substrate suggested that optimal catalysis required NAD as the first binding component. Substrate specificity and the induction pattern clearly indicate a role of the enzyme in formaldehyde oxidation. However, since FD-FA1DH was also found in A. methanolica grown on n-butanol, but not on ethanol, it may have a role in the oxidation of higher aliphatic alcohols as well. FD-FA1DH and the factor from A. methanolica are very similar to a combination already described for Rhodococcus erythropolis [Eggeling, L. & Sahm, H. (1985) Eur. J. Biochem. 150, 129-134]. NAD-linked, glutathione-dependent formaldehyde dehydrogenase (GD-FA1DH) resembles FD-FA1DH in many respects. Since glutathione has so far not been detected in Gram-positive bacteria, FD-FA1DH could be the counterpart of this enzyme in Gram-positive bacteria. Alignment of the N-terminal sequence (31 residues) of FD-FA1DH with that of GD-FA1DH from rat liver indeed showed similarity (30% identical positions). However, comparable similarity was found with class I alcohol dehydrogenase from this organism and with cytosolic alcohol dehydrogenase from Saccharomyces cerevisiae, isozyme 1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of the carbon source and cyclic AMP on isocitrate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase in Klebsiella pneumoniae C3

When strain C3 of Klebsiella pneumoniae is grown on a minimal medium with excess glucose, isocitrate dehydrogenase, malate dehydrogenase, and succinate dehydrogenase specific activities increase in the last period of the exponential growth phase and in the beginning of the stationary phase. Glucose exhaustion does not alter the development of malate dehydrogenase and succinate dehydrogenase, but specific activities are higher than those obtained with excess glucose. In contrast, glucose exhaustion can be correlated with a decrease of isocitrate dehydrogenase specific activity in the stationary phase. Induction of strain C3 isocitrate dehydrogenase by glucose in complex medium and repression by cAMP in mineral medium were observed. Glucose induction and the NADP/NADPH ratio are suggested as regulatory mechanisms controlling isocitrate dehydrogenase synthesis in the Enterobacteriaceae, but the former appears to be restricted to some Klebsiella strains.     

NAD-dependent, PQQ-containing methanol dehydrogenase: a bacterial dehydrogenase in a multienzyme complex

Cell-free extracts of methanol-grown Nocardia sp. 239 only show significant dye-linked methanol-oxidizing activity when NAD+ is added to the assay mixture. This activity resides in a multienzyme complex which could be resolved into 3 components, namely the methanol dehydrogenase, NAD-dependent aldehyde dehydrogenase and NADH dehydrogenase. In its dissociated form, the methanol dehydrogenase no longer shows dye reduction and although rises in the absorbance values around 340 nm are seen on addition of methanol plus NAD+ to the enzyme, this is not due to NADH production. However, dye reduction (NAD dependent) could be restored on incubating methanol dehydrogenase with the corresponding NADH dehydrogenase, obtained from the enzyme complex. It is concluded that this novel methanol dehydrogenase transfers the reducing equivalents, derived from methanol, directly to its associated NADH dehydrogenase via a mechanism in which NAD+ and PQQ are involved.     

Biochemical development of preimplantation mouse embryos: In vivo activities of fructose 1,6-diphosphate aldolase,glucose 6-phosphate dehydrogenase,malate dehydrogenase,and lactate dehydrogenase

The activities of four enzymes were determined during the first four days of mouse embryogenesis. Two enzymes, fructose 1,6-diphosphate aldolase and malate dehydrogenase, increase about 30% in activity, and this increase is attributed to slow but continued enzyme synthesis. The other two enzymes, glucose 6-phosphate dehydrogenase (X-linked) and lactate dehydrogenase, remain constant for the first two days and then decline exponentially with half-times of 19 and 17 hr, respectively. These declines in activity cannot be explained by the appearance of soluble inactivators or by the disappearance of soluble activators. Likewise, although temporally related to the passage of the embryos from the oviducts into the uterine horns, the changes in enzyme activity do not result from this change in embryonic environment, and specific degradative processes beginning on day 2 of embryonic development are postulated.This study was supported by USPHS Grant HD 03132 and by a grant from the School of Medicine, University of California, San Francisco Medical Center. The senior author is the recipient of USPHS Research Career Development Award HD 35,565.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde reductase, aldehyde oxidase and xanthine oxidase from horse tissues

Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (AHD), aldehyde reductase (AHR), aldehyde oxidase (AOX) and xanthine oxidase (XOX) extracted from horse tissues were examined. Five ADH isozymes were resolved: three corresponded to the previously reported class I ADHs (EE, ES and SS) (Theorell, 1969); a single form of class II ADH (designated ADH-C2) and of class III ADH (designated ADH-B2) were also observed. The latter isozyme was widely distributed in horse tissues whereas the other enzymes were found predominantly in liver. Four AHD isozymes were differentially distributed in subcellular preparations of horse liver: AHD-1 (large granules); AHD-3 (small granules); and AHD-2, AHD-4 (cytoplasm). AHD-1 was more widely distributed among the horse tissues examined. Liver represented the major source of activity for most AHDs. A single additional form of NADPH-dependent AHR activity (identified as hexonate dehydrogenase), other than the ADHs previously described, was observed in horse liver. Single forms of AOX and XOX were observed in horse tissue extracts, with highest activities in liver.     

Activity patterns of phosphofructokinase,glyceraldehydephosphate dehydrogenase,lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle

Summary Methods for standardized determination of phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in nanogram samples of microdissected single fibres of rabbit psoas and soleus muscle are described. Fast and slow fibres in soleus muscle show lower absolute activities of these enzymes than the respective fibre types in psoas muscle. Slow fibres represent a more uniform population in the two muscles according to absolute and relative activities of the enzymes investigated. Slow fibres are characterized by high activities of MDH and relatively low activities of glycolytic enzymes. Fast fibres in the soleus muscle represent a population with high activities of MDH and glycolytic enzymes. Fast fibres in psoas muscle represent a heterogeneous population with high activities of glycolytic enzymes and extremely variable activity of MDH. More than 10-fold differences exist in the MDH activities of the extreme types of this fibre population. Differences in the activity levels of MDH in single fast type fibres but also in the activities of glycolytic enzymes between fast and slow fibres are greater than those reported between extreme white and red rabbit muscles.     

The foetal development of lactate dehydrogenase isoenzymes, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from human striated muscle

1. Human foetal skeletal muscles involved in support and in periodic contractility were studied for their content of total extractable lactate dehydrogenase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities as well as for the relative distribution of lactate dehydrogenase isoenzymes. 2. During foetal development a linear steady increase in total lactate dehydrogenase activity as well as a linear decrease in the H/M sub-unit ratio of the isoenzymes was found. 3. No significant changes were found in the activities of the enzymes of the hexose monophosphate shunt (C-6 oxidation). 4. The changes found suggest a steady increased synthesis of lactate dehydrogenase M-sub-units in human skeletal muscles during foetal development. 5. The weekly changes in the total lactate dehydrogenase activity and in lactate dehydrogenase isoenzymes are lower in muscles involved in support than in those involved in periodic contractility. 6. These findings, together with the literature available, are consistent with the morphological fact that foetal development of skeletal muscles mostly concerns the white muscle fibres and not the red muscle fibres.     

The isozymes of lactate dehydrogenase, malate dehydrogenase and glucosephosphate isomerase in Italian ictalurids

1. The isozymes of lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and glucose-phosphate isomerase (GPI) of three species of Italian ictalurids: Ictalurus sp., I. nebulosus marmoratus, and I. punctatus, were analyzed. 2. Isoelectric focusing (IEF) was applied to polyacrylamide gel plates, and the isozymes revealed by means of specific histochemical staining. 3. Species-specific monomorphic patterns were found for LDH. 4. In contrast, MDH and GPI have the same patterns in I. sp. and I. nebulosus marmoratus and different patterns in I. punctatus. 5. Comparison of the isozymatic patterns of the three species clearly showed the close relationship between I. sp. and I. nebulosus marmoratus and the relative taxonomic distance of I. punctatus, and thus the early detachment of this last species from a presumptive common ancestral lineage.