Occurrence of thermolabile and regulatory NAD-linked glutamate dehydrogenase in Pseudomonas fluorescens.

NAD-linked glutamate dehydrogeanse [EC 1.4.1.2] was detected together with NADP-linked glutamate dehydrogenase [EC 1.4.1.4] and aspartase [EC 4.3.1.1] in Pseudomonas fluorescens cells. The three enzymes were distinctly separated by DEAE-Sephadex column chromatography. The NAD-linked enzyme was extremely thermolabile and was rapidly inactivated even at temperatures as low as 35--40 degrees C. The combined addition of NAD+ and glutamate, however, effectively stabilized the enzyme. The glutamate saturation profile of the NAD-linked enzyme exhibited cooperativity with a Hill coefficient (n) of 1.4. ATP inhibited the enzyme in an allosteric manner, increasing the n value to 2.2. These results suggest a novel type of metabolic regulation shared by the three enzymes in the biosynthesis and catabolism of amino acids.     

Nicotinamide-adenine dinucleotide-linked "malic" enzyme in flight muscle of the tse-tse fly (Glossina) and other insects.

1. A high activity of NAD-linked "malic" enzyme was found in homogenates of flight muscle of different species of tse-tse fly (Glossina). The activity was the same as, or higher than, that of malate dehydrogenase and more than 20-fold that of NADP-linked "malic" enzyme. A similar enzyme was found in the flight muscle of all other insects investigated, but at much lower activities. 2. ACa2+-stimulated oxaloacetate decarboxylase activity was present in all insect flight-muscle preparations investigated, in constant proportion to the NAD-linked "malic" enzyme. 3. A partial purification of the NAD-linked "malic" enzyme from Glossina was effected by DEAE-cellulose chromatography, which separated the enzyme from malate dehydrogenase and NADP-linked "malic" enzyme, but not from oxaloacetate decarboxylase. 4. The intracellular localization of the NAD-linked "malic" enzyme was predominantly mitochondrial; latency studies suggested a localization in the mitochondrial matrix space. 5. Studies on the partially purified enzyme demonstrated that it had a pH optimum between 7.6 and 7.9. It required Mg2+ or Mn2+ for activity; Ca2+ was not effective. The maximum rate was the same with either cation, but the concentration of Mn2+ required was 100 times less than that of Mg2+. Acitivity with NADP was only 1-3% of that with NAD, unless very high (greater than 10mM) concentrations of Mn2+ were present. 6. It is suggested that the NAD-linked "malic" enzyme functions in the proline-oxidation pathway predominant in tse-tse fly flight muscle.     

Two malic enzymes in Pseudomonas aeruginosa

Cell-free extract supernatant fluids of Pseudomonas aeruginosa were shown to lack malic dehydrogenase but possess a nicotinamide adenine dinucleotide (NAD)- or NAD phosphate (NADP)-dependent enzymatic activity, with properties suggesting a malic enzyme (malate + NAD (NADP) --> pyruvate + reduced NAD (NADH) (reduced NADP [NADPH] + CO(2)), in agreement with earlier findings. This was confirmed by determining the nature and stoichiometry of the reaction products. Differences in heat stability and partial purification of these activities demonstrated the existence of two malic enzymes, one specific for NAD and the other for NADP. Both enzymes require bivalent metal cations for activity, Mn(2+) being more effective than Mg(2+). The NADP-dependent enzyme is activated by K(+) and low concentrations of NH(4) (+). Both reactions are reversible, as shown by incubation with pyruvate, CO(2), NADH, or NADPH and Mn(2+). The molecular weights of the enzymes were estimated by gel filtration (270,000 for the NAD enzyme and 68,000 for the NADP enzyme) and by sucrose density gradient centrifugation (about 200,000 and 90,000, respectively).     

Studies on regulatory functions of malic enzymes. VI. Purification and molecular properties of NADP-linked malic enzyme from Escherichia coli W.

NADP-linked malic enzyme [EC 1.1.1.40] was highly purified from Escherichia coli W cells. The purified enzyme was homogeneous as judged by ultracentrifugation and gel electrophoresis. The apparent molecular weights obtained by sedimentation equilibrium analysis, from diffusion and sedimentation constants, and by disc electrophoresis at various gel concentrations were 471,000, 438,000, and 495,000, respectively. The subunit molecular weights obtained by sedimentation equilibrium analysis in the presence of 6 M guanidine hydrochloride and gel electrophoresis in the presence of sodium dodecyl sulfate were 76,000 and 82,000, respectively. The sedimentation coefficient (S(0)20, W) was 13.8S, and the molecular activity was 44,700 min-1 at 30 degrees C. The amino acid composition of the enzyme was determined, and the results were compared with those of NAD-linked malic enzyme from the same organism and those of pigeon liver NADP-linked malic enzyme. The partial specific volume was calculated to be 0.738 ml/g. The Km value for L-malate was 2.3 mM at pH 7.4. Malonate, tartronate, glutarate, and DL-tartrate competitively inhibited the activity. The saturation profile for L-malate exhibited a marked cooperativity in the presence of both chloride ions and acetyl-CoA. However, acetyl-CoA alone did not show cooperativity or produce inhibition in the absence of chloride ions. Vmax and Km were determined as a function of pH. The optimum pH for the reaction was 7.8. Inspection of the Dixon plots suggested that three ionizable groups of the enzyme are essential for the enzyme activity. In addition to the oxidative decarboxylase activity, the enzyme preparation exhibited divalent metal ion-dependent oxaloacetate decarboxylase and alpha-keto acid reductase activities. Based on the above results, the molecular properties of the enzymatic reaction are discussed.     

Characterization of two members of a novel malic enzyme class.

The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes. We report the purification of the two isozymes to homogeneity, and their in vitro characterization. Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa. The NAD(P)(+) specific malic enzyme [EC 1.1.1.39] exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD(+) or NADP(+). The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP(+) dependent malic enzymes. Its activity is positively regulated by succinate and fumarate. In contrast to the NAD(P)(+) specific malic enzyme, the NADP(+) dependent malic enzyme [EC 1.1.1.40] shows Michaelis-Menten type behavior with respect to malate and NADP(+). Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism. Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme. Our characterization of the two R. meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far.     

Regulation of coenzyme utilization by mitochondrial NAD(P)-dependent malic enzyme

1. Skeletal muscle mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1. 39, L-malate:NAD+ oxidoreductase (decarboxylating)] from herring could use both coenzymes, NAD and NADP, in a similar manner. 2. The coenzyme preference of mitochondrial NAD(P)-dependent malic enzyme was probed using dual wavelength spectroscopy and pairing the natural coenzymes, NAD or NADP with their respective thionicotinamide analogues, s-NADP or s-NAD, that have absorbance maxima in reduced forms at 400 nm. 3. s-NAD and s-NADP were found to be good alternate substrates for NAD(P)-dependent malic enzyme, the apparent Km values for the thioderivatives were similar to those of the corresponding natural coenzymes. 4. ATP produced greater inhibition of the NAD or s-NAD linked reactions than of the NADP or s-NADP-linked reactions of skeletal muscle mitochondrial NAD(P)-dependent malic enzyme. 5. At 5 mM malate concentration and in the presence of 2 mM ATP the NADP-linked reaction is favoured and the activity ratios, V(s-NADP)/V(NAD) or V(NADP)/V(s-NAD), are 6 and 26, respectively.     

NADP+ -dependent malic enzyme of Rhizobium meliloti.

The bacterium Rhizobium meliloti, which forms N2-fixing root nodules on alfalfa, has two distinct malic enzymes; one is NADP+ dependent, while a second has maximal activity when NAD+ is the coenzyme. The diphosphopyridine nucleotide (NAD+)-dependent malic enzyme (DME) is required for symbiotic N2 fixation, likely as part of a pathway for the conversion of C4-dicarboxylic acids to acetyl coenzyme A in N2-fixing bacteroids. Here, we report the cloning and localization of the tme gene (encoding the triphosphopyridine nucleotide [NADP+]-dependent malic enzyme) to a 3.7-kb region. We constructed strains carrying insertions within the tme gene region and showed that the NADP+ -dependent malic enzyme activity peak was absent when extracts from these strains were eluted from a DEAE-cellulose chromatography column. We found that NADP+ -dependent malic enzyme activity was not required for N2 fixation, as tme mutants induced N2-fixing root nodules on alfalfa. Moreover, the apparent NADP+ -dependent malic enzyme activity detected in wild-type (N2-fixing) bacteroids was only 20% of the level detected in free-living cells. Much of that residual bacteroid activity appeared to be due to utilization of NADP+ by DME. The functions of DME and the NADP+ -dependent malic enzyme are discussed in light of the above results and the growth phenotypes of various tme and dme mutants.     

Activation Kinetics of NAD-Dependent Malic Enzyme of Cauliflower Bud Mitochondria

NAD-dependent malic enzyme (EC 1.1.1.39) was obtained from isolated mitochondria of cauliflower buds (Brassica oleracea L., var. botrytis). The NAD-linked activity is accompanied by a minor NADP-linked activity. Some contaminant NADP-malic enzyme from the supernatant and the plasma membrane is usually present in crude mitochondrial preparations. NAD-dependent malic enzyme has been purified 38-fold by ammonium sulfate fractionation and gel permeation chromatography, to a specific activity up to 2 micromoles per minute per milligram.     

Distinct effects of pyridoxal phosphate on NAD- and NADP- linked malic enzymes of Escherichia coli

NADP-linked malic enzyme from Escherichia coli W was inactivated by pyridoxal 5'-phosphate (PLP) following pseudo-first order kinetics. The inactivation was, however, reversed upon addition of an aminothiol, such as penicillamine and cysteamine, whereas the activity was not restored, when the PLP-inactivated enzyme was treated with NaBH4 prior to the addition of aminothiol. The inactivating effect was specific to PLP and no other structural analogs of PLP tested inactivated the enzyme, except that pyridoxal exhibited a similar effect, though to a lesser extent. In contrast, NAD-linked malic enzyme from the same micro-organism was insensitive to PLP, even in the presence of 0.8 M guanidine hydrochloride.     

Histochemical localization of oxidative enzymes in the hepatopancreas of scylla serrata (forskål) (brachyura: decapoda)

A histochemical study on the distribution of mitochondrial and oxidative enzymes viz., succinic, NAD and NADP-linked isocitrate and malic dehydrogenases and cytochrome oxidase in the hepatopancreas of Scylla serrata (Forskål) shows that the R cells possess the highest concentration of them at the apical parts; F and B cells showed poor staining reactions whereas E cells and connective tissue exhibited trace staining reactions. A moderate staining for these was obtained in the cells lining the main ducts.

The bilateral removal of eyestalks resulted in the stimulation of succinic and NAD-linked malic dehydrogenases and cytochrome oxidase, observed 4 h after the operation; after 24 h, however, these enzymes showed a slight reduction in the activity. A significant increase in the activity of NADP-linked isocitrate and malic dehydrogenases was noted 24 h after eyestalk ablation. The major alterations were noticed in the R cells.

After injecting eyestalk extract into destalked or intact crabs, a fall in the staining intensity of succinic, NAD-linked malic and isocitrate dehydrogenases in the R cells was apparent 2–4 h after the treatment but was subsequently re-established after 24 h.

It seems from the present results that there may be a factor(s) in the eyestalk of S. serrata which regulates the oxidative metabolism in the hepatopancreas. The physiological significance of oxidative enzymes in various cytologic elements is discussed.     

Changes of the NADP-linked malic enzyme in the developing rat skeletal muscle

The activities of NADP-linked malic enzyme, hexose monophosphate shunt dehydrogenases and NADP-linked isocitrate dehydrogenase were studied during development of skeletal muscle and compared with those in the liver. The variation patterns of malic enzyme activity in the liver and in the skeletal muscle were very similar, however the amplitude of the changes was different. The enzyme activity increased approx 16-fold in the liver and about 2-fold in skeletal muscle at the same stage of development. In skeletal muscle the increase of the malic enzyme activity was only slightly higher than of lactic dehydrogenase and citrate synthase. Studies on the intracellular distribution of malic enzyme in skeletal muscle showed that both mitochondrial and extramitochondrial enzymes increased between 20th and 37th day of life, the increase of the extramitochondrial enzyme being more pronounced. The hexose monophosphate shunt dehydrogenases activity showed an increase in the liver but no change was observed in the skeletal muscle at the weaning time. Changes in the activity of the liver and skeletal muscle isocitrate dehydrogenase were not significant between 10th and 80th day of life. The results suggest that the malic enzyme in the liver is playing a different physiological role than in the skeletal muscle.     

The role of 6-phosphogluconate dehydrogenase in Rhizobium.

A nicotinamide adenine dinucleotide (NAD) linked 6-phosphogluconate (6-PG)dehydrogenase has been detected in Rhizobium. The enzyme activity is similar in both slow- and fast-growing rhizobia. The nicotinamide adenine dinucleotide phosphate (NADP) dependent 6-PG dehydrogenase was detected only in the fast growers and was more than twice as active as the NAD-linked enzyme. Partial characterization of the products of 6-PG oxidation in Rhizobium suggests that the NADP-linked enzyme is the decarboxylating enzyme of the pentose phosphate (PP) pathway (EC 1.1.1.44) whereas a phosphorylated six-carbon compound, containing ketonic group(s), is the product of the oxidation catalyzed by the NAD-linked enzyme.     

Malic enzymes of salmon trout heart mitochondria: separation and some physicochemical properties of NAD-preferring and NADP-specific enzymes

Mitochondria isolated from the heart of the Baltic salmon trout Salmo trutta contain two distinct malic enzymes. One of these enzymes (NAD-preferring malic enzyme) catalyses the oxidative decarboxylation of malate in the presence of either NAD or NADP. The specific activity with NAD was six times that with NADP as coenzyme. The second enzyme is specific for NADP. These two malic enzymes have been separated by: ion exchange chromatography of DEAE-Sephacel, affinity chromatography on 2',5'ADP-Sepharose 4B, gel filtration on Sephacryl S-300 and polyacrylamide gel electrophoresis. The mol. wts of the two native malic enzymes determined by gel filtration were found to be 280,000 and 190,000 for NAD-preferring and NADP-specific malic enzyme, respectively. Chromatofocusing revealed the isoelectric points of the two enzymes at pH 5.45 and 5.85 for NAD-preferring and NADP-specific malic enzyme, respectively.     

Reverse reaction of malic enzyme for HCO3- fixation into pyruvic acid to synthesize L-malic acid with enzymatic coenzyme regeneration

Malic enzyme [L-malate: NAD(P)(+) oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)(+)). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO(3)(-) fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD(+), and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO(3)(-) and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 degrees C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD(+).     

Dehydrogenation of androsterone by purified 3α-hydroxy steroid-dependent nicotinamide–adenine dinucleotide (phosphate)-transhydrogenating enzyme of rat liver

1. An enzyme from rat liver, catalysing 3alpha-hydroxy steroid-dependent NAD(P) transhydrogenation and NAD-linked and NADP-linked dehydrogenation of 3alpha-hydroxy steroids, has been purified 100-fold by chromatography on DEAE-cellulose and calcium phosphate gel. 2. No separation of these activities into different protein fractions has been achieved. 3. The properties of the enzyme in catalysing NAD-linked and NADP-linked dehydrogenation have been compared, with androsterone as substrate. Differences were found in pH optima, affinity for coenzyme and steroid, equilibrium constants and effects of salts. 4. NAD-linked dehydrogenation is inhibited by NADPH(2) but is protected from this inhibition by chloride, which alone is itself an inhibitor. 5. The relevance of these findings to the problem of the number of enzymes involved in catalysis of 3alpha-hydroxy steroid-dependent transhydrogenation is discussed.     

Separation and properties of the NAD-linked and NADP-linked isozymes of succinic semialdehyde dehydrogenase in Euglena gracilis z.

Euglena gracilis z contained two succinic semialdehyde dehydrogenases (EC 1.2.1.16), one requiring NAD and the other NADP, and these isozymes were separated from each other and partially purified. The NAD-linked isozyme was relatively stable on storage at 5 degrees C whereas the NADP-linked one was extremely unstable unless 30% glycerol or ethyleneglycol was added. The optimum pH was 8.7 and optimum temperature 35-45 degrees C for both isozymes. They were inhibited by Zn2+ and activated, particularly the NAD-linked enzyme, by K+. Sulfhydryl reagents activated both isozymes. The Km values for succinic semialdehyde were 1.66 - 10(-4) M with the NAD-linked isozyme and 1.06 - 10(-3) M with the NADP-linked one. The NADP-linked isozyme was induced by glutamate while the NAD-linked one was not. Probable roles of these isozymes in the physiology of Euglena gracilis are discussed.     

NAD-linked formate dehydrogenase from methanol-grown Pichia pastoris NRRL-Y-7556

An NAD-linked formate dehydrogenase (EC 1.2.1.2.) from methanol-grown Pichia pastoris NRRL Y-7556 has been purified. The purification procedure involved ammonium sulfate fractionation, hollow-fiber H1P10 filtration, ion-exchange chromatography, and gel filtration. Both dithiothreitol (10 mm) and glycerol (10%) were required for stability of the enzyme during purification. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and by sedimentation pattern in an ultracentrifuge. The enzyme has a molecular weight of 94,000 and consists of two subunits of identical molecular weight. Formate dehydrogenase catalyzes specifically the oxidation of formate. No other compounds tested can replace NAD as the electron acceptor. The Michaelis constants were 0.14 mm for NAD and 16 mm for formate (pH 7.0, 25 °C). Optimum pH and temperature for formate dehydrogenase activity were around 6.5–7.5 and 20–25 °C, respectively. Amino acid composition of the enzyme was also studied. Antisera prepared against the purified enzyme from P. pastoris NRRL Y-7556 form precipitin bands with isofunctional enzymes from different strains of methanol-grown yeasts, but not bacteria, on immunodiffusion plates. Immunoglobulin fraction prepared against the enzyme from yeast strain Y-7556 inhibits the catalytic activity of the isofunctional enzymes from different strains of methanol-grown yeasts.     

Glucose-6-phosphate dehydrogenase from a tetracycline producing strain of Streptomyces aureofaciens: some properties and regulatory aspects of the enzyme

Glucose-6-phosphate dehydrogenase from Streptomyces aureofaciens exhibited activity with both NAD and NADP, the maximum reaction rate being 1.6 times higher for NAD-linked activity than for the NADP-linked one. The KM values for NAD-linked activity were 2.5 mM for glucose-6-phosphate and 0.27 mM for NAD, and for NADP-linked activity 0.8 mM for glucose-6-phosphate and 0.08 mM for NADP. NAD- and NADP-linked activities were inhibited by both NADH and NADPH. (2'-phospho-)adenosinediphospho-ribose inhibited only NAD-linked activity. The inhibition was competitive with respect to NAD and noncompetitive with respect to glucose-6-phosphate.     

NAD-linked glutamate dehydrogenase in Trypanosoma cruzi.

1. Epimastigotes of Trypanosoma cruzi, Tulahuén strain, contained a NAD-linked glutamate dehydrogenase (EC 1.4.1.3), in addition to the already known NADP-linked enzyme enzyme (EC 1.4.1.4). 2. The partially purified NAD-linked enzyme had a higher molecular weight and was much more labile than the NADP-linked enzyme, and was inhibited by purine nucleotides. 3. These results further emphasize the difference in glutamate metabolism between the parasite and its mammalian host.     

Association of NADP- and NAD-linked malic enzyme acitivities in Zea mays: relation to C4 pathway photosynthesis.

Two of the three metabolic subtypes of species utilizing C₄-pathway photosynthesis are defined by high activities of either NADP malic enzyme (NADP malic enzyme type) or a coenzyme A (CoA)- and acetyl-CoA-activated NAD malic enzyme (NAD malic enzyme type). These enzymes function to decarboxylate malate as an integral part of the photosynthetic process. Leaves of NADP malic enzyme-type species also contain significant NAD-dependent malic enzyme activity. The purpose of the present study was to examine the nature and photosynthetic role of this activity. With Zea mays, this NAD-dependent activity was found to vary widely in fresh leaf extracts. Incubating extracts at 25 °C resulted in a disproportionate increase in NAD activity so that the final ratio of NADP to NAD activity was always about 5. Strong evidence was provided that the NADP and NAD malic enzyme activities in Z. mays extracts were catalyzed by the same enzyme. These activities remained associated during purification and were coincident after polyacrylamide gel electrophoresis. The pH optimum for NAD-dependent activity was about 7.1, compared with 8.3 for NADP malic enzyme activity. Other properties of the NAD-dependent activity are described, a particularly notable feature being the inhibition of this activity by less than 1 μm NADP and NADPH. Evidence is provided that the NADP malic enzyme of several other NADP malic enzyme-type C₄ species also has associated activity toward NAD. We concluded that the NAD-dependent malic enzyme activity would have no significant function in photosynthesis.