Sequence comparisons betweenAgrobacterium tumefaciens T-DNA-encoded octopine and nopaline dehydrogenases and other nucleotide-requiring enzymes: Structural and evolutionary implications

Summary The nucleotide sequences of the two T-DNA-encoded crown gall imino acid dehydrogenases octopine dehydrogenase and nopaline dehydrogenase were compared with each other and with the sequences of other dehydrogenases. A multistep strategy comprising computer sequence analysis and secondary- and antigenic-structure predictions was used. An alignment of octopine and nopaline dehydrogenase was obtained in which a 20-amino-acid N-terminal arm and six fairly long gaps in the C-terminal moiety were introduced. The aligned sequences have identities of 26% at the amino acid level and 38% at the nucleotide level. They appear to contain two domains. The N-terminal coenzyme-binding domains are similar to those of the well-characterized NAD(P) dehydrogenases. Conserved fragments were found in the C-terminal catalytic domains that likely contain essential residues for catalysis. Comparison of the sequences with those of two other 2-keto acid dehydrogenases, lactate and malate dehydrogenase, suggests that as in those enzymes, histidine, aspartic acid, and arginine residues are located at the octopine and nopaline dehydrogenase active sites. The crown gall enzymes could not be classified with any known family of dehydrogenases. Their evolutionary origin remains unknown. However, predictions concerning their internal organization may provide new insight into protein evolution.     

Malate dehydrogenase: a model for structure, evolution, and catalysis.

Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3-dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3-dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications.     

The quantitative histochemistry of a chemically induced ependymoblastoma. I. Enzymes

Enzymes from several metabolic pathways were studied quantitatively in homogenates and in homogeneous areas of frozen-dried cryostat sections of an experimental, mouse ependymoblastoma, mouse mammary carcinoma, and mouse melanoma, growing as transplants in mouse brain. Micro analyses were performed in fiveto sixfold replicates on portions of tumour with a dry weight of 0·03-0·2 μg. A close resemblance of the enzyme spectrum of the ependymoblastoma to that of immature brain was noted. Hexokinase and malate dehydrogenase were lower and lactate, glucose-6-phosphate, and NADP⁺-linked isocitrate dehydrogenases, and β-glucuronidase higher in the ependymoblastoma than in whole, adult mouse brain. Mouse mammary carcinoma had a higher level of hexokinase and lower levels of lactate and glucose-6-phosphate dehydrogenases and β-glucuronidase than ependymoblastoma. The concentration of malate dehydrogenase was lower and that of lactate, glucosed-6-phosphate, and NADP⁺-linked isocitric dehydrogenases was higher in the melanoma than in the ependymoblastoma. β-Glucuronidase levels were similar in these two neoplasms. It is suggested that the relatively high levels of several NADP⁺-linked enzymes in the ependymoblastoma may be related to increased capacity for lipid synthesis.     

Both d- and l-specific Lactate Dehydrogenases Co-exist in Individual Cephalopods

Both d-lactate-specific and l-lactate-specific lactate dehydrogenases coexist in individual cephalopods, contrary to the commonly held view that invertebrate species may contain one or other, but not both. We describe the tissue distribution of these lactate dehydrogenases and of octopine dehydrogenase, the major pyruvate reductase activity in cephalopods, in three species: common squid (Loligo vulgaris), cuttlefish (Sepia officinalis) and lesser octopus (Eledone cirrhosa). The l-specific lactate dehydrogenase of squid is shown to be a dimer of 36,000 dalton subunits.     

Determination of the hydride transfer stereospecificity of nicotinamide adenine dinucleotide linked oxidoreductases by proton magnetic resonance.

A facile proton magnetic resonance technique is described for the determination of the coenzyme stereospecificity during hydride transfer reactions catalyzed by pyridine nucleotide dependent oxidoreductases. The reliability of this technique was demonstrated by examining the coenzyme stereospecificity of lactate, malate, and 3-phosphoglycerate dehydrogenases, which are known to be A-stereospecific enzymes, as well as triosephosphate and octopine dehydrogenases, which are known to be B-stereospecific enzymes. Furthermore, by applying this technique, it was shown that the previously unstudied enzymes D-beta-hydroxybutyrate and 4-aminobutanal dehydrogenases are B- and A-stereospecific enzymes, respectively. In addition, the nicotinamide adenine dinucleotide linked reaction of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be B stereospecific, like the reaction of the nicotinamide adenine dinucleotide phosphate linked yeast enzyme.     

Malate dehydrogenase and lactate dehydrogenase in trematodes and turbellarians

Studies have been made on the activity and properties of malate and lactate dehydrogenases from the cattle rumen trematodes Eurytrema pancreaticum, Calicophoron ijimai and the turbellarian Phagocata sibirica which has a common free-living ancestor with the trematodes. All the species studied have a highly active malate dehydrogenase, its activity in the reaction of reducing oxaloacetate being 6-14 times higher than in the reaction of malate oxidation. The affinity of malate dehydrogenase to oxaloacetate was found to be higher than that to malate. The activity of lactate dehydrogenase (reducing the pyruvate) was lower than the activity of malate dehydrogenase, the difference being 50 times for C. ijimai, 4 times for E. pancreaticum and 10 times for P. sibirica.     

Anaerobic metabolism of the common cockle, Cardium edule. II. Partial purification and properties of lactate dehydrogenase and octopine dehydrogenase. A comparative study.

1. Octopine dehydrogenase and lactate dehydrogenase were purified 190-fold and 10-fold respectively from the adductor muscle of the marine bivalve Cardium edule by gel filtration on Sephadex G-100 and chromatography on DEAE-Sephadex A-50. 2. Lactate dehydrogenase was capable to convert D- and L-lactate, had a molecular weight of about 70 000 and 280 000 daltons, exhibits no distinct pH optimum and was not inhibited by lactate. The enzyme showed apparent Km values of 0.16 mM for pyruvate and 16 mM and 48 mM for D- and L-lactate respectively. 3. In comparison to the purified enzymes from other species, octopine dehydrogenase from Cardium edule showed similar biochemical properties : pH optima of 6.8 and 8.7 respectively, Km values of 0.9 mM (for pyruvate) and 2.0 mM (for arginine), a molecular weight of 37 000 daltons and inhibition by octopine. Electrophoretic studies on standard polyacrylamide gels showed five isoenzymes. 4. The biochemical properties of both dehydrogenases are compared to the conditions in vivo of these animals and the biological role of the octopine dehydrogenase is discussed.     

Anaerobic energy metabolism of goldfish,Carassius auratus (L.)

Summary The concentrations of pyruvate, lactate, oxalo-acetate, aceto-acetate -hydroxybutyrate, -ketoglutarate, glutamate, NH ₄ ⁺ , NAD⁺ and NADH were measured in goldfish tissues after previous conditioning to normal and anoxic (12h) conditions. For 11 different metabolites efficiency of different extraction methods was tested by means of internal standards. The recoveries were generally over 80%. The substrate/product couples of the reactions catalysed by lactate dehydrogenase, malate dehydrogenase, -hydroxybutyrate dehydrogenase and glutamate dehydrogenase were used as redox parameters. In the lateral red muscle the redox state did not change during 12 h of anoxia. In the dorsal white muscle only the cytoplasmic redox state underwent a change, as indicated by the increase of the lactate/pyruvate ratio from 20 to 110. In liver both cytoplasm and mitochondria were reduced during anoxia. From the measured values the NAD⁺/NADH ratio was found to change only in white muscle, while the calculated free NAD⁺/NADH ratios were reduced in anoxic white muscle cytoplasm, anoxic liver mitochondria, and anoxic liver cytoplasm. Oxalo-acetate concentrations calculated from the equilibrium constants of lactate dehydrogenase and malate dehydrogenase were at least one order of magnitude smaller than the measured values. The data obtained from anoxic goldfish are in contrast to available data on other animals and support earlier reports which indicate that this animal has a special anaerobic metabolism. The results are discussed especially with respect to the role of ethanol as a sink for reducing equivalents.Abbreviations LDH lactate dehydrogenase - MDH malate dehydrogenase - HBDH -hydroxybutyrate dehydrogenase - GIDH glutamate dehydrogenase     

Parameters controlling opine formation during muscular activity and environmental hypoxia

Summary In order to elucidate the regulatory parameters which determine multiple opine formation in marine invertebrates, anaerobiosis was induced in 25 species from several phyla by stimulating the animals to vigorous muscular activity or by subjecting them to environmental hypoxia. The quantity of glycolytic end products and the corresponding amino acids were measured. In a second set of experiments the amounts of substrates and products of the opine dehydrogenase reactions in the isolated introvert retractor muscle (IRM) ofSipunculus nudus were determined in both situations.During environmental hypoxia opines accumulated according to the contents of the corresponding amino acids. Mass action ratios (MAR) of the opine dehydrogenase reactions in the isolated IRM were in the range of control values (octopine dehydrogenase 1.9·10¹¹ mol^–2·l², strombine dehydrogenase 2.2·10¹⁰ mol^–2·l²). During muscular activity those opines accumulated preferentially which corresponded to the highest opine dehydrogenase activities. In the isolated IRM only octopine accumulated during contractile activity; the MAR of the octopine dehydrogenase reaction was near the control value while the MAR of the strombine dehydrogenase reaction deviated by a factor of 9.The results indicate that during environmental hypoxia the opine dehydrogenases present in a tissue catalyze near equilibrium and the relative amount of opines accumulated is dictated by the concentration of the corresponding amino acids. During muscular activity only those opine dehydrogenases catalyze near equilibrium which are present in sufficiently high activities to keep pace with an increased glycolytic flux. Therefore, different opines may accumulate in the same animal during muscular activity and during environmental hypoxia.     

Lactate and malate dehydrogenase binding to the microsomal fraction from chicken liver

Chicken liver microsomal fractions show lactate and malate dehydrogenase activities which behave differently with respect to successive extractions by sonication in 0.15 M NaCl, 0.2% Triton X-100 and 0.15 M NaCl, respectively. The Triton X-100-treated pellet did not show malate dehydrogenase activity but exhibited a 10-fold increase in lactate dehydrogenase activity with respect to the sonicated pellet. Total extracted lactate and malate dehydrogenase activities were, respectively, 7.5 and 1.7 times higher than that in the initial pellet. Different isoenzyme compositions were observed for cytosoluble and microsomal extracted lactate and malate dehydrogenases. When the ionic strength (0-500 mM) or the pH values (6.1-8.7) of the media were increased, an efficient release of lactate dehydrogenase was found at NaCl 30-70 mM and pH 6.6-7.3. Malate dehydrogenase solubilization under the same conditions was very small, even at NaCl 500 mM, but it attained a maximum in the 7.3-8.7 pH range. Cytosoluble lactate dehydrogenase bound in vitro to 0.15 M NaCl-treated (M2) and sonicated (M3) microsomal fractions but not to the crude microsomal fraction (M1). Particle saturation by lactate dehydrogenase occurred with M2 and M3, which contained binding sites with different affinities. Cytosoluble malate dehydrogenase did not bind to M1, M2 and M3 fractions, however, a little binding was found when purified basic malate dehydrogenase was incubated with M2 or M3 fractions.     

Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.     

The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver

1. The concentrations of the oxidized and reduced substrates of the lactate-, beta-hydroxybutyrate- and glutamate-dehydrogenase systems were measured in rat livers freeze-clamped as soon as possible after death. The substrates of these dehydrogenases are likely to be in equilibrium with free NAD(+) and NADH, and the ratio of the free dinucleotides can be calculated from the measured concentrations of the substrates and the equilibrium constants (Holzer, Schultz & Lynen, 1956; Bücher & Klingenberg, 1958). The lactate-dehydrogenase system reflects the [NAD(+)]/[NADH] ratio in the cytoplasm, the beta-hydroxybutyrate dehydrogenase that in the mitochondrial cristae and the glutamate dehydrogenase that in the mitochondrial matrix. 2. The equilibrium constants of lactate dehydrogenase (EC 1.1.1.27), beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and malate dehydrogenase (EC 1.1.1.37) were redetermined for near-physiological conditions (38 degrees ; I0.25). 3. The mean [NAD(+)]/[NADH] ratio of rat-liver cytoplasm was calculated as 725 (pH7.0) in well-fed rats, 528 in starved rats and 208 in alloxan-diabetic rats. 4. The [NAD(+)]/[NADH] ratio for the mitochondrial matrix and cristae gave virtually identical values in the same metabolic state. This indicates that beta-hydroxybutyrate dehydrogenase and glutamate dehydrogenase share a common pool of dinucleotide. 5. The mean [NAD(+)]/[NADH] ratio within the liver mitochondria of well-fed rats was about 8. It fell to about 5 in starvation and rose to about 10 in alloxan-diabetes. 6. The [NAD(+)]/[NADH] ratios of cytoplasm and mitochondria are thus greatly different and do not necessarily move in parallel when the metabolic state of the liver changes. 7. The ratios found for the free dinucleotides differ greatly from those recorded for the total dinucleotides because much more NADH than NAD(+) is protein-bound. 8. The bearing of these findings on various problems, including the following, is discussed: the number of NAD(+)-NADH pools in liver cells; the applicability of the method to tissues other than liver; the transhydrogenase activity of glutamate dehydrogenase; the physiological significance of the difference of the redox states of mitochondria and cytoplasm; aspects of the regulation of the redox state of cell compartments; the steady-state concentration of mitochondrial oxaloacetate; the relations between the redox state of cell compartments and ketosis.     

Polarographic Study of Dicarboxylic-Acid-dependent Export of Reducing Equivalents from Illuminated Chloroplasts

Isolated pea chloroplasts, prepared by differential centrifugation, catalyzed O₂ evolution in the light in the presence of 0.03 to 3 millimolar malate, 0.12 to 1.2 millimolar NAD, 4 millimolar pyruvate and exogenous NAD-malate dehydrogenase and lactate dehydrogenase. The reaction, which did not occur in the absence of any one of these factors, was accompanied by the consumption of pyruvate; the ratio of O₂ evolved to pyruvate consumed was <0.5. When 0.1 millimolar [¹⁴C]malate was supplied most of the ¹⁴C label was recovered as malate. At low concentrations of malate (<0.1 millimolar), the ratio of O₂ evolved to malate supplied was greater than 0.5.     

Affinity chromatography of nicotinamide nucleotide-dependent dehydrogenases on immobilized nucleotide derivatives

A series of chemically-defined adenosine phosphate ligands attached to Sepharose 4B were used as active-site probes in studying the interaction of enzymes with their coenzymes and substrates and to test the suitability of these matrices for `general ligand' affinity chromatography. Nicotinamide nucleotide-dependent dehydrogenases were used as models to test this methodology. Elution from these columns by NAD⁺ and/or AMP gradients (in the presence or the absence of substrates and/or nicotinamide mononucleotide) was consistent with: (1) the compulsory ordered addition of substrates to lactate and malate dehydrogenase; (2) the necessity for the NMN moiety of NAD⁺ to bind to these enzymes before the substrate; and illustrated: (3) that the binding of these two hydrogenases to these columns compared very well with the published three-dimensional models for these enzymes and (4) that separation of mixtures of dehydrogenases depended on the choice of matrix and displacing ion and whether any additions (e.g. substrates) were made to the gradients used. These techniques were used to purify UDP-glucose dehydrogenase from a crude starting material on a phosphate-linked UDP (or ADP) matrix. The binding of this enzyme to these two columns was not consistent with either an ordered or random addition of substrates and suggested a more complex mechanism.     

Several dehydrogenases and kinases compete for endocytosis from plasma by rat tissues.

Plasma contains many enzymes that are probably derived from damaged cells. These enzymes are cleared at characteristic rates. We showed previously that in rats the rapid clearance of alcohol dehydrogenase, lactate dehydrogenase M4 and the mitochondrial and cytosolic isoenzymes of malate dehydrogenase is largely due to endocytosis by macrophages in liver, spleen and bone marrow. We now demonstrate that uptake of each of the enzymes by these tissues is in general decreased by simultaneous injection of a high dose of one of the other dehydrogenases or a high dose of adenylate kinase or creatine kinase. A similar dose of colloidal albumin did not significantly decrease uptake of the four dehydrogenases. Nor was uptake of colloidal albumin, apo-peroxidase from horseradish or multilamellar liposomes influenced by a high dose of mitochondrial malate dehydrogenase. These results indicate that the four dehydrogenases and the two kinases are specifically endocytosed via the same receptor. We suggest that this receptor contains a group, possibly a nucleotide, with affinity for the nucleotide-binding sites of the enzymes.     

A comparative study of NAD+ binding sites in dehydrogenases by circular polarization of fluorescence.

The spectra of the circular polarization of luminescence of a number of dehydrogenases with the fluorescent coenzyme nicotinamide-1,-N⁶-ethenoadenine dinucleotide were measured. By use of this technique it is demonstrated that there is a difference in structure between the adenine subsite in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase on the one hand and pig heart lactate dehydrogenase, horse liver alcohol dehydrogenase, beef liver glutamate dehydrogenase, and pig heart malate dehydrogenase on the other hand. It is concluded that the non-co-operative dehydrogenases have similar, if not identical, adenine subsites whereas in glyceraldehyde-3-phosphate dehydrogenase, a strongly co-operative enzyme, a different structure of the adenine subsite has evolved.     

A new family of 2-hydroxyacid dehydrogenases

The NADH-dependent hydroxypyruvate reductase from cucumber and the pdxB gene product of E. coli display significant homology to E. coli D-3-phosphoglycerate dehydrogenase. In contrast, these proteins do not display much similarity with other oxidoreductases or with other 2-hydroxyacid dehydrogenases in particular. On the basis of their relatedness and the structure of their substrates, these three enzymes constitute a new family of 2-hydroxyacid dehydrogenases distinct from malate and lactate dehydrogenase.     

Activity of liver mitochondrial Krebs cycle NAD<Superscript>+</Superscript>-dependent dehydrogenases in rats with hepatitis induced by acetaminophen under conditions of alimentary protein deficiency

Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD⁺/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deficiency. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD⁺/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein deficiency caused a more pronounced decrease in the activity of studied Krebs cycle NAD⁺-dependent dehydrogenases and a 2.2-fold increase of the mitochondrial NAD⁺/NADН ratio.     

Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Corynebacterium glutamicum

Like many other bacteria, Corynebacterium glutamicum possesses two types of L-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 1.1.99.16) and a cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum. A mutant with a site-directed deletion in the mqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicum obscure.     

ELECTROCONVULSIVE SEIZURE: AN INVESTIGATION INTO THE VALIDITY OF CALCULATING THE CYTOPLASMIC FREE [NAD+]/[NADH] [H+] RATIO FROM SUBSTRATE CONCENTRATIONS OF BRAIN

Abstract— This study is an investigation into the applicability of redox calculations to brain. At six intervals following electroconvulsive scizure, multiple metabolites were measured in freeze-blown brain from unanesthetized rats. From substrate ratios, the time course of the rapid changes in the cytoplasmic free [NAD⁺]/ [NADH] [H⁺] ratio was calculated from the reactions of lactate dehydrogenase [EC 1.1.1.27], malate dehydrogenase [EC 1.1.1.37] and glycerolphosphate dehydrogenase [EC 1.1.1.8], The pattern of the redox ratios in the control animals was also compared with the same ratios determined in freeze-clamped liver, a relatively homogeneous tissue. Though some evidence for effects of compartmentation are present in the results from brain, these effects are relatively minor. There was found to be very good agreement in the direction and magnitude of change of the redox ratios calculated from lactate dehydrogenase and malate dehydrogenase, and even from glycerolphosphate dehydrogenase at points of low flux. In spite of rapid changes of metabolites, the reaction catalyzed by glutamateoxaloacetate transaminase remained very near its equilibrium position at all time periods. From the results it has been concluded that in spite of the obvious structural heterogeneity of brain, meaningful calculations of the cytoplamic redox state in brain are possible.