Oxidation of lactate in isolated rat heart mitochondria

In unwashed mitochondria the oxidation of L-lactate (with NAD+) proceeds in presence of the added lactate dehydrogenase. The respiration is characterized by the high rate in state 4 and is stimulated by ADP. This process takes place in unwashed mitochondria and homogenate of the heart in absence of added lactate dehydrogenase. Oxidation of lactate with NAD+ is inhibited by rotenone. It has been also revealed that the oxidation of glutamate is insufficiently altered in presence of lactate (with NAD+) in unwashed mitochondria as compared with the washed ones. It is supposed that the stimulating effect of lactate with NAD+ on the mitochondria respiration is not so much a result of the membrane-damaged action as a result of oxidation of lactate dehydrogenase reaction products: phosphorylative oxidation of pyruvate and nonconjugated oxidation of NADH. Utilization of these products takes place in the main respiratory chain, including its first stage.     

NAD recycling in the collagen membrane.

NAD recycling in the collagen membrane was investigated as follows: (1) Alcohol dehydrogenase and lactate dehydrogenase were co-immobilized in the collagen membrane and the rate of lactate production by immobilized enzymes was compared with that of free enzymes by using free NAD. An increased rate was observed in the case of immobilized enzyme. (2) The soluble high molecular weight derivatives of NAD (dextran-NAD) were immobilized in the collagen membrane with the two dehydrogenases and recycling of dextran-NAD in the membrane was examined. Lactate was produced by the membrane without adding free NAD. The interaction between the high molecular weight NAD derivatives and enzymes are also discussed.     

Characterisation of a highly hydrophobically modified lactate dehydrogenase

1. Lysine residues of porcine H4 lactate dehydrogenase (L-lactate:NAD+ oxidoreductase EC 1.1.1.27) were modified with methyl-epsilon-(N-2,4-dinitrophenyl)aminocaproimidate - HCl. With increasing incorporation of the reagent a linear decrease of enzymatic activity was noticed. No essential lysyl group with an extraordinary reactivity was modified. 2. The active forms of the modified enzyme with different incorporation values were separated from denatured material by fractional precipitation and gel chromatography. An epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase was obtained with an average incorporation of 38 groups per tetramer and a residual activity of 42%. This material proved to be homogenous in cellulose electrophoresis. 3. The epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase is soluble only in glycine buffer at pH 8 and can be stabilized as ternary complex with NAD+ and sodium sulfite. Gel chromatography and ORD measurements show no strong conformational change. 4. epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase has similar Km values for pyruvate, NADH, lactate and NAD+ as the native enzyme, and shows a lower thermostability due to a diminished stabilization by the hydrate layer on the surface.     

Substrate-induced conformational changes in lactate dehydrogenase. Proteolysis of the immobilized enzyme in the presence of specific substrates.

We report here a new approach to the study of the conformation of enzymes in the presence of specific substrates. Rabbit muscle lactate dehydrogenase was attached to CL-Sepharose via a cleavable spacer arm (-NH-(CH2)6NHCO(CH2)2SS(CH2)2CO-). The bound lactate dehydrogenase was digested with subtilisin BPN' in the presence of substrates of lactate dehydrogenase. The use of a flow system permits the maintenance of saturating levels of substrates. Proteolysis was followed by loss of activity of the enzyme column. The time course of proteolysis in the presence of either NADH, NAD+, or pyruvate alone did not differ from the control. However, when NADH and pyruvate were present simultaneously, the enzyme became more susceptible to proteolysis. The initial rate of proteolysis was increased by 40%. The abortive ternary complex (lactate dehydrogenase - NAD+ - pyruvate) also showed an increase in susceptibility to proteolysis. These findings clearly show that the productive ternary complex (lactate dehydrogenase - NADH - pyruvate) is conformationally different from the apoenzyme and binary complexes under optimal catalytic conditions.     

Lactate dehydrogenase isoenzymes of human semen

1. A lactate dehydrogenase isoenzyme present in human spermatozoa and semen was isolated and characterized biochemically in term of its pH for optimum activity and by means of K(m) values for lactate, NAD(+) and NAD analogues. The results were compared with those obtained with the human heart-type and the liver-type lactate dehydrogenase isoenzymes. 2. The enzyme was characterized by its resistance to digestion with different proteolytic enzymes. The time for 50% digestion in terms of residual dehydrogenase activity was compared with times obtained for the H(4)- and M(4)-types.     

Affinity chromatography of lactate dehydrogenase on immobilized nucleotides

The interaction of two isoenzymes of lactate dehydrogenase from pig heart muscle (H(4)) and rabbit skeletal muscle (M(4)), with immobilized nucleotides was examined: the effects of pH and temperature on the binding of lactate dehydrogenase were studied with immobilized NAD(+) matrices. The influence of substrate, product and sulphite on the binding of heart muscle lactate dehydrogenase to immobilized NAD(+) was investigated. The interaction of both lactate dehydrogenase isoenzymes with immobilized pyridine and adenine nucleotides and their derivatives were measured. The effects of these parameters on the interaction of lactate dehydrogenase with immobilized nucleotides were correlated with the known kinetic and molecular properties of the enzymes in free solution.     

Structure of lactate dehydrogenase inhibitor generated from coenzyme.

Two inhibitors of lactate dehydrogenase generated during NADH storage have been isolated by chromatography. One is a dimer of the dinucleotide where the AMP moiety is unmodified. The other is also generated from NAD+ in the presence of a high concentration of phosphate ions at alkaline pH. This inhibitor was proved to be the addition compound of one phosphate group to position C-4 of the nicotinamide ring of NAD+ by NMR spectroscopy, enzymatic cleavage, and dissociation to NAD+ at neutral pH. This compound is a competitive inhibitor with respect to NAD+ in the presence of the lactate dehydrogenase with a Ki of 2 X 10(-7) M. The interaction of this inhibitor with lactate dehydrogenase is discussed relative to the structure of this enzyme.     

Alteration of coenzyme specificity of lactate dehydrogenase from Thermus thermophilus by introducing the loop region of NADP(H)-dependent malate dehydrogenase

Previously we found that replacement of seven amino acid residues in a loop region markedly shifted the coenzyme specificity of malate dehydrogenase from NAD(H) toward NADP(H). In the present study, we replaced the seven amino acid residues in the corresponding region of an NAD(H)-dependent lactate dehydrogenase with those of NADP(H)-dependent malate dehydrogenase, and examined the coenzyme specificity of the resulting mutant enzyme. Coenzyme specificity was significantly shifted by 399-fold toward NADPH when k cat/Km(coenzyme) was used as the measure of coenzyme specificity. The effect of the replacements on coenzyme specificity is discussed based on in silico simulation of the three-dimensional structure of the lactate dehydrogenase mutant.     

Mouse intracellular immunoglobulin M. Structure and identification of a free thiol group

1. The formation of the non-enzymic adduct of NAD(+) and sulphite was investigated. In agreement with others we conclude that the dianion of sulphite adds to NAD(+). 2. The formation of ternary complexes of either lactate dehydrogenase or malate dehydrogenase with NAD(+) and sulphite was investigated. The u.v. spectrum of the NAD-sulphite adduct was the same whether free or enzyme-bound at either pH6 or pH8. This suggests that the free and enzyme-bound adducts have a similar electronic structure. 3. The effect of pH on the concentration of NAD-sulphite bound to both enzymes was measured in a new titration apparatus. Unlike the non-enzymic adduct (where the stability change with pH simply reflects HSO(3) (-)=SO(3) (2-)+H(+)), the enzyme-bound adduct showed a bell-shaped pH-stability curve, which indicated that an enzyme side chain of pK=6.2 must be protonated for the complex to form. Since the adduct does not bind to the enzyme when histidine-195 of lactate dehydrogenase is ethoxycarbonylated we conclude that the protein group involved is histidine-195. 4. The pH-dependence of the formation of a ternary complex of lactate dehydrogenase, NAD(+) and oxalate suggested that an enzyme group is protonated when this complex forms. 5. The rate at which NAD(+) binds to lactate dehydrogenase and malate dehydrogenase was measured by trapping the enzyme-bound NAD(+) by rapid reaction with sulphite. The rate of NAD(+) dissociation from the enzymes was calculated from the bimolecular association kinetic constant and from the equilibrium binding constant and was in both cases much faster than the forward V(max.). No kinetic evidence was found that suggested that there were interactions between protein subunits on binding NAD(+).     

Inhibition of lipogenesis by halothane in isolated rat liver cells.

1. Halothane at clinically effective concentrations [2.5 and 4% (v/v) of the gas phase of the incubation flask] was found to inhibit significantly lipogenesis from endogenous substrates, e.g., glycogen, or from added lactate plus pyruvate. This was accompanied by a decrease in the ratio of the free [NAD+]/[NADH] of the mitochondrion and the cytoplasm, as shown by the [3-hydroxybutyrate]/[acetoacetate] ratio and the [lactate]/[pyruvate] ratio. 2. Acetoacetate or pyruvate decreased the inhibitory effect of halothane and restored lipogenesis to control rates. They were reduced rapidly by 3-hydroxybutyrate dehydrogenase or lactate dehydrogenase respectively, with the concomitant oxidation of NADH and the generation of NAD+. 3. These results suggest that the mechanism by which halothane inhibits lipogenesis from glycogen or lactate is by inhibition of the oxidation of NADH; this results in inhibition of flux of carbon through pyruvate dehydrogenase and a shortage of acetyl-CoA for fatty acid synthesis. Thus when NADH acceptors are added in the presence of halothane, the concentration of mitochondrial NAD+ is raised so that the flux of carbon through pyruvate dehydrogenase increases and lipogenesis is restored.     

Retention and regeneration of native NAD(H) in noncharged ultrafiltration membrane reactors: application to L-lactate and gluconate production

NAD(H) was retained in a noncharged ultrafiltration membrane reactor for the simultaneous and continuous production of L-lactate and gluconate with coenzyme regeneration. Polyethyleneimine (PEI), a 50-kDa cationic polymer, achieved coenzyme retentions above 0.8 for PEI/NAD(H) molar ratios higher than 5. The ionic strength of the inlet medium caused a decrease of NAD(H) retention that can be counterbalanced by an initial addition of 1% bovine serum albumin (BSA). Continuous reactor performance in the presence of PEI and BSA showed that NAD(H), glucose dehydrogenase, and lactate dehydrogenase were retained by 10-kDa ultrafiltration membranes; L-lactate and gluconate were produced at conversions higher than 95%. PEI enhanced the thermal stability of the enzymes used and increased the catalytic efficiency of glucose dehydrogenase, while no effect was found on the kinetic parameters of lactate dehydrogenase. A model that implements the kinetic equations of the two enzymes describes the reactor behavior satisfactorily. In brief, the use of PEI to retain NAD(H) is a new interesting approach to be widely applied in continuous synthesis with the large number of known dehydrogenases.     

INHIBITION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE IN MAMMALIAN NERVE BY IODOACETIC ACID

Abstract— Cat sciatic nerves were exposed to iodoacetate for a period of 5–10 min and after washing out the iodoacetate, the enzymes, glyceraldehyde-3-phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate: NAD oxidoreductase (phosphorylating); EC 1.2.1.12) and lactate dehydrogenase ( l -lactate: NAD oxidoreductase; EC 1.1.1.27) were extracted from the high-speed supernatant fraction of nerve homogenates. Concentrations of iodoacetate as low as 2.5 m m could completely block activity of glyceraldehyde-3-phosphate dehydrogenase but had no effect on lactate dehydrogenase. These findings are in accord with the classical concept shown earlier for muscle that iodoacetate blocks glycolysis by its action on glyceraldehyde-3-phosphate dehydrogenase. A complete block of activity of the enzyme was found after treatment with 2 to 5 m m -iodoacetate for a period of 10 min and such blocks were irreversible for at least 3 h. Glyceraldehyde-3-phosphate dehydrogenase activity was NAD specific, with NADP unable to substitute for NAD. The results are discussed in relation to the effect of iodoacetate in blocking glycolysis and in turn the fast axoplasmic transport of materials in mammalian nerve.     

The characteristics of lactate oxidation in the tissue mitochondria of rats of different ages]

The parameters of respiration (V3, V4) and phosphorylation (the respiration control, ADP/O) have been studied using lactate as a substrate (obligatory with NAD addition) close by meaning to pyruvate on the liver and heart mitochondrion and homogenates of newborn rats. In 20-days and adult rats the mitochondria and homogenates oxidize the lactate (with NAD) with higher rate V4 but with lower value of respiration control as compared with the newborn animals. Simultaneously, a high activity of mitochondrial NADH-oxidase, oxidizing NADH, formed in the reaction of lactate dehydrogenase not connected with ATP synthesis. The role of mitochondrial NADH-oxidase are discussed as a factor increasing lactate oxidation, removing tissue lactate and activating the age dependent energy metabolism.     

A detailed investigation of the properties of lactate dehydrogenase in which the ''Essential'' cysteine-165 is modified by thioalkylation.

The reaction of pig heart lactate dehydrogenase with methyl methanethiosulphonate resulted in the modification of one thiol group per protomer, and this was located at cysteine-165 in the enzyme sequence. On reduction, both the thiomethylation of cysteine-165 and any changes in kinetic properties of the enzyme were completely reversed. Cysteine-165 has been considered essential for catalytic activity; however, cysteine-165-thiomethylated dehydrogenase possessed full catalytic activity, although the affinity of the enzyme for carbonyl-or hydroxy-containing substrates was markedly decreased. The nicotinamide nucleotide-binding capacity was unaffected, as judged by the formation of fluorescent complexes with NADH. The enzyme-mediated activation of NAD+, as judged by sulphite addition, was unaffected in thiomethylated lactate dehydrogenase. However, the affinity of oxamate for the enzyme--NADH complex was decreased by 100-fold and it was calculated that this constituted a net increase of 10.4 kJ/mol in the activation energy for binding. Thiomethylated lactate dehydrogenase was able to form an abortive adduct between NAD+ and fluoropyruvate. However, the equilibrium constant for adduct formation between pyruvate and NAD+ was too low to demonstrate this complex at reasonable pyruvate concentrations. A conformational change in the protein structure on selective thiomethylation was revealed by the decreased thermostability of the modified enzyme. The alteration of lactate dehydrogenase catalytic properties on modification depended on the bulk of the reagent used, since thioethylation resulted in an increase in Km for pyruvate (13.5 +/- 3.5 mm) and an 85% decrease in maximum catalytic activity. The implications of all these findings for the catalytic mechanism of lactate dehydrogenase are discussed.     

Acetate Utilization in Lactococcus lactis Deficient in Lactate Dehydrogenase: a Rescue Pathway for Maintaining Redox Balance

Acetate was shown to improve glucose fermentation in Lactococcus lactis deficient in lactate dehydrogenase. 13C and 1H nuclear magnetic resonance studies using [2-13C]glucose and [2-(13)C]acetate as substrates demonstrated that acetate was exclusively converted to ethanol. This novel pathway provides an alternative route for NAD+ regeneration in the absence of lactate dehydrogenase.     

Properties of glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate as an NADH-regeneration unit in enzyme reactors

Glucose-dehydrogenase-poly(ethylene glycol)-NAD conjugate (GlcDH-PEG-NAD) was prepared and its kinetic properties as an NADH-regeneration unit were investigated. The conjugate has about two molecules of active and covalently linked NAD per tetramer. The specific activity of the enzyme moiety of the conjugate in the presence of exogenous NAD is about 77% of that of the native enzyme, and this decrease is mainly due to the decrease in the V_max value. The conjugate has the same pH-stability profile as the native enzyme and an internal activity of 0.26s⁻¹ (as a monomer); its NAD moiety has similar coenzyme activity to poly(ethylene glycol)-bound NAD. These results indicate that GlcDH-PEG-NAD can be used as an NADH-regeneration unit for many dehydrogenase reactions. The coupled reaction of GlcDH-PEG-NAD and lactate dehydrogenase was then studied. The specific activity of the conjugate is 1.1 s⁻¹ (as a tetramer), the recycling rate of the active NAD moiety is 0.54 s⁻¹, and the apparent K_m value for glucose is 24 mM. Kinetically, lactate dehydrogenase behaves like a substrate with an apparent K_m value of 1.8 units·ml⁻¹ in this coupled reaction system with low coenzyme concentration. l-Lactate was continuously produced from pyruvate in a reactor with a PM10 ultrafiltration membrane, and containing GlcDH-PEG-NAD and lactate dehydrogenase. GlcDH-PEG-NAD proved to be applicable in continuous enzyme reactors as an NADH-regeneration unit with a large molecular size.     

Production of racemic lactic acid in Pediococcus cerevisiae cultures by two lactate dehydrogenases.

Nicotinamide adenine dinucleotide (NAD)-dependent d(minus)-and l(plus)-lactate dehydrogenases have been partially purified 89- and 70-fold simultaneously from cell-free extracts of Pediococcus cerevisiae. Native molecular weights, as estimated from molecular sieve chromatography and electrophoresis in nondenaturing polyacrylamide gels, are 71,000 to 73,000 for d(minus)-lactate dehydrogenase and 136,000 to 139,000 for l(plus)-lactate dehydrogenase. Electrophoresis in sodium dodecyl sulfate-containing gels reveals subunits with approximate molecular weights of 37,000 to 39,000 for both enzymes. By lowering the pyruvate concentration from 5.0 to 0.5 mM, the pH optimum for pyruvate reduction by d(minus)-lactate dehydrogenase decreases from pH 8.0 to 3.6. However, l(plus)-lactate dehydrogenase displays an optimum for pyruvate reduction between pH 4.5 and 6.0 regardless of the pyruvate concentration. The enzymes obey Michaelis-Menten kinetics for both pyruvate and reduced NAD at pH 5.4 and 7.4, with increased affinity for both substrates at the acid pH. alpha-Ketobutyrate can be used as a reducible substrate, whereas oxamate has no inhibitory effect on lactate oxidation by either enzyme. Adenosine triphosphate causes inhibition of both enzymes by competition with reduced NAD. Adenosine diphosphate is also inhibitory under the same conditions, whereas NAD acts as a product inhibitor. These results are discussed with relation to the lactate isomer production during the growth cycle of P. cerevisiae.     

Involvement of pyruvate dehydrogenase in product formation in pyruvate-limited anaerobic chemostat cultures of Enterococcus faecalis NCTC 775

Enterococcus faecalis NCTC 775 was grown anaerobically in chemostat culture with pyruvate as the energy source. At low culture pH values, high in vivo and in vitro activities were found for both pyruvate dehydrogenase and lactate dehydrogenase. At high culture pH values the carbon flux was shifted towards pyruvate formate lyase. Some mechanisms possibly involved in this metabolic switch are discussed. In particular attention is paid to the NADH/NAD ratio (redox potential) and the fructose-1,6-bisphosphate-dependent lactate dehydrogenase activity as possible regulatory factors.Abbreviations PDH pyruvate dehydrogenase complex (EC 1.2.2.2) - PFL pyruvate formate lyase (EC 2.3.1.54) - LDH lactate dehydrogenase (EC 1.1.1.27) - FBP fructose-1,6-bisphosphate - MTT 3-(4,5-dimethyl-thiazoyl-2)-2,5-diphenyltetrazolium bromide - TPP thiamine pyrophosphate     

NAD(H) recycling activity of an engineered bifunctional enzyme galactose dehydrogenase/lactate dehydrogenase

A chimeric bifunctional enzyme composing of galactose dehydrogenase (galDH; from Pseudomonas fluorescens) and lactate dehydrogenase (LDH; from Bacillus stearothermophilus) was successfully constructed. The chimeric galDH/LDH possessed dual characteristics of both galactose dehydrogenase and lactate dehydrogenase activities while exhibiting hexameric rearrangement with a molecular weight of approximately 400 kDa. In vitro observations showed that the chimeric enzyme was able to recycle NAD with a continuous production of lactate without any externally added NADH. Two fold higher recycling rate (0.3 mM/h) than that of the native enzyme was observed at pH values above 8.5. Proximity effects became especially pronounced during the recycling assay when diffusion hindrance was induced by polyethylene glycol. All these findings open up a high feasibility to apply the NAD(H) recycling system for metabolic engineering purposes e.g. as a model to gain a better understanding on the molecular proximity process and as the routes for synthesizing of numerous high-value-added compounds.     

An Affinity Chromatographic Reactor for Highly Efficient Turnover of Dissociable Cofactors

A conjugated enzyme system of alcool dehydrogenase and lactate dehydrogenase was immobilized in an ultrafiltration hollow fiber tube, which was inserted in a fine nylon tube to form a hollow-fiber-capillary reactor. To this reactor, the substrates, pyruvate and ethanol, were supplied continuously. The necessary cofactor, NAD, was supplied as a pulse for a short time. The retention time of NAD in the reactor, estimated from the response curve of lactate produced, was much longer than those of the other substrates and products because of the strong adsorption of NAD to the immobilized enzymes through affinity. Therefore, the reactor could produce lactate from pyruvate for a long time without any more NAD. As a typical case, when the enzyme concentration is sufficiently high, the estimated retention time of NAD was 50 times as long as those of other materials so that the NAD turnover obtained was 412,000. The effects of NAD pulse concentration and the immobilized enzyme concentration on the retention time of NAD and NAD turnover were investigated experimentally and theoretically.