A novel mode of lactate metabolism in strictly anaerobic bacteria

Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E₀′ = ?190 mV excludes direct NAD⁺ reduction (E₀′ = ?320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron‐transferring flavoprotein (Etf) that exhibited NAD⁺ reduction only when reduced ferredoxin (Fd^2?) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin‐based electron confurcation to drive endergonic lactate oxidation with NAD⁺ as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E₀′ ≈ –500 mV) to NAD⁺ according to: lactate + Fd^2? + 2 NAD⁺ → pyruvate + Fd + 2 NADH. The reduced Fd^2? is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical and finally redox energy (Fd^2? from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes.     

低温冷藏提高家蚕滞育卵NAD含量和胞质苹果酸脱氢酶活性

为了调查5℃低温处理是否改变家蚕Bombyx mori卵滞育NAD代谢, 本研究利用HPLC和分光光度法测定了经25℃和5℃分别处理的滞育卵中NADH 含量、 NAD⁺含量、 乳酸脱氢酶（LDH）活性和胞质苹果酸脱氢酶（cMDH）活性。结果表明： 5℃处理的NAD（NADH + NAD⁺）含量和cMDH活性分别增加了106%和53%, 并且显著高于25℃处理（P< 0.01）; 但是两种处理的NADH/NAD⁺比值和LDH活性没有显著差异（P> 0.05）。据此推测, 5℃低温处理加强了家蚕滞育卵NAD⁺合成和再生能力。     

The Role of Oxidized Nicotinamide Adenine Dinucleotide in Fluoride Inhibition of Active Sodium Transport in Human Erythrocytes

The rate coefficient for ²²Na release from previously labeled human erythrocytes was determined in the presence of 0.1–10 mM sodium fluoride (F). The oxidized nicotinamide adenine dinucleotide (NAD⁺) level at the end of 2 hr of incubation in tris(hydroxymethyl)aminomethane (Tris)-Ringer medium was also measured. Both parameters decreased proportionately as F concentration was raised. Both F-induced changes were immediate and were reversed by 10 mM pyruvate. The decrease in NAD⁺ concentration following enolase inhibition by F is attributed to a diminished rate of formation in the reaction catalyzed by lactic dehydrogenase (LDH) with undiminished continued utilization in the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It is postulated that the NAD⁺ lowering limited the GAPDH step, resulting in proportionate decreases in the rates of phosphoglycerate kinase (PGK) and Na,K-dependent adenosine triphosphatase (Na,K-ATPase), a reaction sequence thought to link glycolysis with active Na extrusion. Adding pyruvate with F increased NAD⁺ production at the LDH step, thus reactivating GAPDH, PGK, and Na,K-ATPase and leading to the observed restoration of ²²Na release. The results suggest, therefore, that F inhibits active Na transport in intact human erythrocytes indirectly through a lowering of NAD⁺, although, direct inhibition of the Na,K-ATPase by F may possibly occur simultaneously.     

A pH-Dependent Kinetic Model of Dihydrolipoamide Dehydrogenase from Multiple Organisms

Dihydrolipoamide dehydrogenase is a flavoenzyme that reversibly catalyzes the oxidation of reduced lipoyl substrates with the reduction of NAD⁺ to NADH. In vivo, the dihydrolipoamide dehydrogenase component (E3) is associated with the pyruvate, α-ketoglutarate, and glycine dehydrogenase complexes. The pyruvate dehydrogenase (PDH) complex connects the glycolytic flux to the tricarboxylic acid cycle and is central to the regulation of primary metabolism. Regulation of PDH via regulation of the E3 component by the NAD⁺/NADH ratio represents one of the important physiological control mechanisms of PDH activity. Furthermore, previous experiments with the isolated E3 component have demonstrated the importance of pH in dictating NAD⁺/NADH ratio effects on enzymatic activity. Here, we show that a three-state mechanism that represents the major redox states of the enzyme and includes a detailed representation of the active-site chemistry constrained by both equilibrium and thermodynamic loop constraints can be used to model regulatory NAD⁺/NADH ratio and pH effects demonstrated in progress-curve and initial-velocity data sets from rat, human, Escherichia coli, and spinach enzymes. Global fitting of the model provides stable predictions to the steady-state distributions of enzyme redox states as a function of lipoamide/dihydrolipoamide, NAD⁺/NADH, and pH. These distributions were calculated using physiological NAD⁺/NADH ratios representative of the diverse organismal sources of E3 analyzed in this study. This mechanistically detailed, thermodynamically constrained, pH-dependent model of E3 provides a stable platform on which to accurately model multicomponent enzyme complexes that implement E3 from a variety of organisms.     

Ethanol exposure modulates hepatic S-adenosylmethionine and S-adenosylhomocysteine levels in the isolated perfused rat liver through changes in the redox state of the NADH/NAD system

Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD⁺ ratio, and the concentrations of SAM and SAH in the liver tissue were determined by high-performance liquid chromatography. The increase in the NADH/NAD⁺ ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-Methylpyrazole, an inhibitor the NAD⁺-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD⁺ ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADH-dependent enzyme lactate dehydrogenase, restored the NADH/NAD⁺ ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD⁺ redox couple and the effects of ethanol on methionine metabolism in the liver.     

Effect of NAD on Malate Oxidation in Intact Plant Mitochondria

Potato tuber mitochondria oxidizing malate respond to NAD⁺ addition with increased oxidation rates, whereas mung bean hypocotyl mitochondria do not. This is traced to a low endogenous content of NAD⁺ in potato mitochondria, which prove to take up added NAD⁺. This mechanism concentrates NAD⁺ in the matrix space. Analyses for oxaloacetate and pyruvate (with pyruvate dehydrogenase blocked) are consistent with regulation of malate oxidation by the internal NAD⁺/NADH ratio.     

Genetically encoded fluorescent indicator for imaging NAD/NADH ratio changes in different cellular compartments

Background

The ratio of NAD⁺/NADH is a key indicator that reflects the overall redox state of the cells. Until recently, there were no methods for real time NAD⁺/NADH monitoring in living cells. Genetically encoded fluorescent probes for NAD⁺/NADH are fundamentally new approach for studying the NAD⁺/NADH dynamics.

Methods

We developed a genetically encoded probe for the nicotinamide adenine dinucleotide, NAD(H), redox state changes by inserting circularly permuted YFP into redox sensor T-REX from Thermus aquaticus. We characterized the sensor in vitro using spectrofluorometry and in cultured mammalian cells using confocal fluorescent microscopy.

Results

The sensor, named RexYFP, reports changes in the NAD⁺/NADH ratio in different compartments of living cells. Using RexYFP, we were able to track changes in NAD⁺/NADH in cytoplasm and mitochondrial matrix of cells under a variety of conditions. The affinity of the probe enables comparison of NAD⁺/NADH in compartments with low (cytoplasm) and high (mitochondria) NADH concentration. We developed a method of eliminating pH-driven artifacts by normalizing the signal to the signal of the pH sensor with the same chromophore.

Conclusion

RexYFP is suitable for detecting the NAD(H) redox state in different cellular compartments.

General significance

RexYFP has several advantages over existing NAD⁺/NADH sensors such as smallest size and optimal affinity for different compartments. Our results show that normalizing the signal of the sensor to the pH changes is a good strategy for overcoming pH-induced artifacts in imaging.     

Purification and characterization of pyruvate dehydrogenase complex from borccoli floral buds.

The pyruvate dehydrogenase complex (PDC) was purified from Brassica oleracea var. italica floral buds to a specific activity of approximately 6 μmol of NADH formed/min/ mg of protein. The PDC had cofactor requirements for NAD⁺, thiamine pyrophosphate, coenzyme A, and a divalent cation (Mg²⁺, Ca²⁺, or Mn²⁺). The enzyme catalyzed the oxidative decarboxylation of pyruvate at a rate threefold faster than 2-oxobutyrate but was inactive toward 2-oxoglutarate. The PDC was competively inhibited by acetyl-CoA against CoA and NADH against NAD⁺. The enzyme was shown to be more sensitive to regulation by NADH than acetyl-CoA.     

Redox involvement in acid secretion in the amphibian gastric mucosa

Summary Gastric fundic metabolism was studied by spectroscopic observation in frog mucosa during transitions of secretory status in vitro and by direct measurement of pyridine nucleotides and associated metabolites in biopsies of dog fundic mucosa also during secretory oxidation of the redox components from flavin adenine dinucleotide (FAD) to cytochromea ₃. Addition of histamine resulted in reduction of these components with onset of secretion by about 50%. In contrast, the effect of apparently, burimamide and subsequently histamine on the ratio of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced (NAD⁺/NADH) was relatively slight. Further, the presence of burimamide substantially reduces the effect of amytal on the pyridine nucleotide spectrum and abolishes the effect of amytal on FAD and the cytochromes. Measurements of lactate, pyruvate, -ketoglutarate, NH₃ and glutamate in the dog showed that whereas the calculated NAD⁺/NADH ratio in the cytoplasm declined with onset of secretion, the calculated mitochondrial ratio rose. No change was noted in the nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate, reduced (NADP⁺/NADPH) ratio. It is concluded that (1) H₂ antagonists act by blocking substrate flow into the mitochondrial respiratory chain, (2) conversely, histamine stimulation acts at the level of substrate mobilization, and (3) there may be a cross-over in the mitochondrial chain between NAD⁺ and FAD.     

Tumor growth of neurofibromin-deficient cells is driven by decreased respiration and hampered by NAD+ and SIRT3

Neurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion, decreasing both respiration and intracellular NAD⁺. Expression of the alternative NADH dehydrogenase NDI1 raises NAD⁺/NADH ratio, enhances the activity of the NAD⁺-dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. The antineoplastic effect of NDI1 is mimicked by administration of NAD⁺ precursors or by rising expression of the NAD⁺ deacetylase SIRT3 and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes. These findings shed light on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD⁺/NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss.Subject terms: Cancer metabolism, Cell biology     

Efficient Whole-Cell Biocatalyst for Acetoin Production with NAD+ Regeneration System through Homologous Co-Expression of 2,3-Butanediol Dehydrogenase and NADH Oxidase in Engineered Bacillus subtilis

Acetoin (3-hydroxy-2-butanone), an extensively-used food spice and bio-based platform chemical, is usually produced by chemical synthesis methods. With increasingly requirement of food security and environmental protection, bio-fermentation of acetoin by microorganisms has a great promising market. However, through metabolic engineering strategies, the mixed acid-butanediol fermentation metabolizes a certain portion of substrate to the by-products of organic acids such as lactic acid and acetic acid, which causes energy cost and increases the difficulty of product purification in downstream processes. In this work, due to the high efficiency of enzymatic reaction and excellent selectivity, a strategy for efficiently converting 2,3-butandiol to acetoin using whole-cell biocatalyst by engineered Bacillus subtilis is proposed. In this process, NAD⁺ plays a significant role on 2,3-butanediol and acetoin distribution, so the NADH oxidase and 2,3-butanediol dehydrogenase both from B. subtilis are co-expressed in B. subtilis 168 to construct an NAD⁺ regeneration system, which forces dramatic decrease of the intracellular NADH concentration (1.6 fold) and NADH/NAD⁺ ratio (2.2 fold). By optimization of the enzymatic reaction and applying repeated batch conversion, the whole-cell biocatalyst efficiently produced 91.8 g/L acetoin with a productivity of 2.30 g/(L·h), which was the highest record ever reported by biocatalysis. This work indicated that manipulation of the intracellular cofactor levels was more effective than the strategy of enhancing enzyme activity, and the bioprocess for NAD⁺ regeneration may also be a useful way for improving the productivity of NAD⁺-dependent chemistry-based products.     

Lactate dehydrogenase isoenzymes of sperm cells and testes

1. The kinetic and metabolic properties of lactate dehydrogenase isoenzyme LDH_x from human sperm cells and rat testes were studied. 2. LDH_x shows a sensitivity to inhibition by stilboestrol diphosphate, urea and guanidinium chloride different from that of the LDH-H₄ and LDH-M₄ isoenzymes. 3. About 10 and 20% of the total lactate dehydrogenase activity of testes and sperm cells respectively were associated with particulate fractions. In sperm cells 11% was localized in the middle piece and 18·8% in the head fraction. LDH_x was found in all particulate fractions of sperm cells. The middle piece contained 41·0% of total LDH_x activity and showed high succinate dehydrogenase activity. 5. The pH-dependence of lactate/pyruvate and NAD⁺/NADH concentration ratios were estimated. Lactate dehydrogenase in sperm cells has maximal activity with NADH as coenzyme at pH7·5 and with NADPH as coenzyme at pH6·0. At pH6·0 a 10% greater oxidation of NADPH than of NADH was found. At acid pH lactate hydrogenase may function as an enzyme bringing about transhydrogenation from NADPH to NAD⁺. 6. In agreement with the stoicheiometry of the lactate de- hydrogenase reaction, the lactate/pyruvate concentration ratio decreased with increasing pH. 7. The lactate/pyruvate and NAD⁺/NADH concentration ratios were estimated with glucose, fructose and sorbitol as substrates and as a function of time after addition of these substrates. During a 20min. period after the addition of the substrates, changes in lactate/pyruvate and NAD⁺/NADH concentration ratios were noticed. Increasing concentration of the substrates mentioned gave rise to asymptotic increases in lactate and pyruvate. 8. Sorbitol did not act as a substrate for LDH_x. 9. The findings described are consistent with the idea that LDH_x is different from other known lactate dehydrogenase isoenzymes, but that it has a metabolic function similar to that of the isoenzymes of other tissues.     

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     

Kinetic Parameters and Lactate Dehydrogenase Isozyme Activities Support Possible Lactate Utilization by Neurons

Lactate is potentially a major energy source in brain, particularly following hypoxia/ischemia; however, the regulation of brain lactate metabolism is not well understood. Lactate dehydrogenase (LDH) isozymes in cytosol from primary cultures of neurons and astrocytes, and freshly isolated synaptic terminals (synaptosomes) from adult rat brain were separated by electrophoresis, visualized with an activity-based stain, and quantified. The activity and kinetics of LDH were determined in the same preparations. In synaptosomes, the forward reaction (pyruvate + NADH + H⁺ → lactate + NAD⁺), which had a V _max of 1,163 μmol/min/mg protein was 62% of the rate in astrocyte cytoplasm. In contrast, the reverse reaction (lactate + NAD⁺ → pyruvate + NADH + H⁺), which had a V _max of 268 μmol/min/mg protein was 237% of the rate in astrocytes. Although the relative distribution was different, all five isozymes of LDH were present in synaptosomes and primary cultures of cortical neurons and astrocytes from rat brain. LDH1 was 14.1% of the isozyme in synaptic terminals, but only 2.6% and 2.4% in neurons and astrocytes, respectively. LDH5 was considerably lower in synaptic terminals than in neurons and astrocytes, representing 20.4%, 37.3% and 34.8% of the isozyme in these preparations, respectively. The distribution of LDH isozymes in primary cultures of cortical neurons does not directly reflect the kinetics of LDH and the capacity for lactate oxidation. However, the kinetics of LDH in brain are consistent with the possible release of lactate by astrocytes and oxidative use of lactate for energy in synaptic terminals. Special issue dedicated to John P. Blass.     

Effect of micromolar Ca2+ on NADH inhibition of bovine kidney α-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification

Summary NADH inhibition of bovine kidney -ketoglutarate dehydrogenase complex was compared at 10 m free Ca²⁺ or in the absence of Ca²⁺ (i.e., < 1.0 nM free Ca²⁺). In the presence of Ca^2–, NADH inhibition was appreciably decreased for a wide range of NADH : NAD⁺ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 m free Ca²⁺ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca²⁺ on the S_0.5 for -ketoglutarate which was decreased by Ca²⁺ with a half-maximal effect at a similar concentration. The effect of Ca²⁺ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the -ketoglutarate dehydrogenase complex is required to elicit the effect of Ca²⁺ on NADH inhibition.At a fixed -ketoglutarate concentration (50 m), removal of Ca² reduced the activity of the -ketoglutarate dehydrogenase complex by 8,5-fold (due to an increase in S_0.5 for -ketoglutarate) and, in the presence of different NADH : NAD⁺ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxP_i) or a combination of the phosphate potential and NADH :NAD⁺ ratio are also described. The possibility that the level of intramitochondrial free Ca²⁺ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.     

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD⁺]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD⁺]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD⁺]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD⁺]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD⁺]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.     

α-Acetolactate overexpression from glucose in the diacetyl producer Lactococcus lactis ssp. lactis bv. diacetylactis B2103, a natural mutant lacking α-acetolactate decarboxylase

We investigated the effects of the oxygen supply rate on the activity of pyruvate metabolic pathways and their end products, the lactatedehydrogenase (LDH), pyruvateformiatelyase (PFL), pyruvatedehydrogenase (PDH) and acetolactatesynthase (ALS) pathways, in the Lactococcus lactis ssp. lactis bv. diacetylactis strain B2103/74. We found that this culture, apart from inactivated α-acetyldecarboxylase, also possesses a unique natural capacity to overexpress α-acetolactate (AL) up to 25–28 mM. Our search for similar properties among the diacetilicus bv. strains showed that this ability is quite rare. We identified a single additional strain, 7590 from the National Russian Collection of Industrial Microorganisms (NRCIM-7590), which displayed a similar capacity. However, unlike B2103/74, NRCIM-7590 has an active α-acetolactate decarboxylase and therefore can only produce acetoin. AL overexpression took place under conditions of intense aeration (K _L a ≥ 90–120 h^?1), and the composition of the medium played a decisive role in AL productivity. We found that AL overproduction is determined by a diversion of a portion of pyruvate flow from the LDH to the PDH and ALS pathways. We further found that all additional pyruvate, supplied from LDH, is utilized exclusively by the ALS pathway because of the restricted capacity of the PDH pathway. This shift in pyruvate metabolism in the B2103/74 strain, from LDH to PDH and ALS pathways, is associated with the initiation of an oxidation reaction that reduces oxygen to H₂O and sequesters NADH from the LDH pathway in the process. A specific manifestation of this reaction in B2103/74 and NRCIM-7590 cultures, which results in a profound shift of the pyruvate metabolism towards the production of α-acetolactate, is due to the function of a potent oxidative system that shifts 75–80% of NADH flow from LDH to the oxidative pathway, resulting in the regeneration of NAD⁺. The nature of this oxidative system is not known. Based on our studies, we propose that the structure of the newly discovered oxidative system is similar to a simple transmembrane electron transport chain.     

Activity of NAD-dependent glucose-6-phosphate dehydrogenase in yeast-like organisms

Summary From tested yeast-like organisms, onlyGeotrichum candidum showed the same activity of glucose-6-phosphate dehydrogenase with both NAD⁺ and NADP⁺. i. e. 0.017–0.019 mol NADH/min. mg dry weight of cell free extracts. Omission of Mg⁺⁺ in the reaction mixture did not influence the activity of the enzyme in the presence of NAD⁺. Cell free extracts ofEndomyces magnusii showed only low activity of this enzyme and the ratio of its activity in the presence of NAD⁺ and NADP⁺, respectively, varied in individual cultures.Rhodotorula glutinis showed only an NADP⁺-dependent activity.     

The role of enzymes of pyruvate and citrate metabolism in control of gluconeogenesis in oocytes and embryos of the loach,Misgurnus fossilis L.

Summary Cessation of gluconeogenesis during oocyte maturation inMisgurnus fossilis L. is accompanied by an increase of pyruvate dehydrogenase activity (EC 1.2.4.1). The activity of other enzymes of citrate and pyruvate metabolism (citrate synthetase, EC 4.1.3.7, pyruvate carboxylase, EC 6.4.1.1., malate dehydrogenase, EC 1.1.1.37) remains constant during oocyte maturation and early embryogenesis.In the course of oocyte maturation the levels of acetyl-CoA, pyruvate and citrate remained unchanged, but the level of malate and oxaloacetate underwent drastic increase. The level of phosphoenolpyruvate increased about two-fold. The mitochondrial (NAD⁺)/(NADH) ratio was calculated by measurement of intermediates of the glutamate dehydrogenase reaction and it was found to increase six-fold during oocyte maturation. The lower mitochondrial (NAD⁺)/(NADH) ratio in oocytes compared to that in the embryos is likely to be responsible for the transfer of reducing equivalents from mitochondria to cytoplasm, while in embryos transfer in the opposite direction takes place.     

Alanine dehydrogenase of the N2-fixing blue-green alga,Anabaena cylindrica

The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD⁺ specific and oxaloacetate can partially substitute for pyruvate. The K _m ^app for NAD⁺ is 14 M and 60 M at low and high NAD⁺ concentrations, respectively. The K _m ^app for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K _m ^app for NH ₄ ⁺ varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N₂-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N₂-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.Abbreviations ADH alanine dehydrogenase - DEAE diethylamino ethyl cellulose - EDTA ethylenediamine tetraacetic acid - GDH glutamic dehydrogenase - GS glutamine synthetase - GOT aspartate-glutamate aminotransferase - NAD⁺ nicotinamide adenine dinucleotide - NADH reduced nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH reduced nicotinamide adenine dinucleotide phosphate - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl) aminomethane