In vitro rates of oxidation and gluconeogenesis from L(+)- and D(-)lactate in bovine tissues

Slices of bovine kidney cortex, liver, heart and sternomandibularis muscle actively metabolized D- and L-lactate. Rates of D-lactate oxidation were greatest in kidney cortex followed by heart and liver with muscle exhibiting the lowest rates. L-lactate oxidation was greatest in kidney cortex followed by heart with liver and muscle exhibiting similar rates. Rates of oxidation of gluconeogenesis were similar for D- and L-lactate at 0.1 mm lactate but D utilization, as a percent of L, decreased as substrate concentrations increased to 50 mM. Bovine tissues appear to possess significant potential for D(-)lactate utilization. Estimates of this and possible interactions are discussed.     

Utilization of Saccharomyces cerevisiae recombinant strain incapable of both ethanol and glycerol biosynthesis for anaerobic bioproduction

The yeast Saccharomyces cerevisiae produces ethanol and glycerol as major unwanted byproducts, unless ethanol and glycerol are the target compounds. Minimizing the levels of these byproducts is important for bioproduction processes using yeast cells. In this study, we constructed a yeast strain in which both ethanol and glycerol production pathways were disrupted and examined its culture characteristics. In wild-type yeast strain, metabolic pathways that produce ethanol and glycerol play an important role in reoxidizing nicotinamide adenine dinucleotide (NADH) generated during glycolysis, particularly under anaerobic conditions. Strains in which both pathways were disrupted therefore failed to grow and consume glucose under anaerobic conditions. Introduction of desired metabolic reaction(s) coupled with NADH oxidation enabled the engineered strain to consume substrate and produce target compound(s). Here we introduced NADH-oxidization-coupled L-lactate production mechanisms into a yeast strain incapable of ethanol and glycerol biosynthesis, based on in silico simulation using a genome-scale metabolic model of S. cerevisiae. From the results of in silico simulation based on flux balance analysis, a feasible anaerobic non-growing metabolic state, in which L-lactate yield approached the theoretical maximum, was identified and this phenomenon was verified experimentally. The yeast strain incapable of both ethanol and glycerol biosynthesis is a potentially valuable host for bioproduction coupled with NADH oxidation under anaerobic conditions.     

Relationship between hydroxypyruvate and the production of oxalate in vitro.

Chicken liver lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC1.1.1.27) catalyses the reversible reduction reaction of hydroxypyruvate to L-glycerate. It also catalyses the oxidation reaction of the hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form to glycolate. At pH 8, these latter two reactions are coupled. The coupled system equilibrium is attained when the NAD+/NADH ratio is greater than unity. Hydroxypyruvate binds to the enzyme at the same site as the pyruvate. When there are substances with greater affinity to this site in the reaction medium and their concentration is very high, hydroxypyruvate binds to the enzyme at the L-lactate site. In vitro and with purified preparation of lactate dehydrogenase, hydroxypyruvate stimulates the production of oxalate from glyoxylate-hydrated form and from NAD; the effect is due to the fact that hydroxypyruvate prevents the binding of non-hydrated form of glyoxylate to the lactate dehydrogenase in the pyruvate binding site. At pH 8, THE L-glycerate stimulates the production of glycolate from glyoxylate-non-hydrated form and NADH since hydroxypyruvate prevents the binding of glyoxylate-hydrated form to the enzyme     

Identification of lactaldehyde dehydrogenase in Methanocaldococcus jannaschii and its involvement in production of lactate for F420 biosynthesis

One of the early steps in the biosynthesis of coenzyme F(420) in Methanocaldococcus jannaschii requires generation of 2-phospho-L-lactate, which is formed by the phosphorylation of L-lactate. Preliminary studies had shown that L-lactate in M. jannaschii is not derived from pyruvate, and thus an alternate pathway(s) for its formation was examined. Here we report that L-lactate is formed by the NAD(+)-dependent oxidation of l-lactaldehyde by the MJ1411 gene product. The lactaldehyde, in turn, was found to be generated either by the NAD(P)H reduction of methylglyoxal or by the aldol cleavage of fuculose-1-phosphate by fuculose-1-phosphate aldolase, the MJ1418 gene product.     

L-lactate oxidation by skeletal muscle mitochondria

1. Mitochondria isolated from rat skeletal muscle possess lactate dehydrogenase which is involved in direct oxidation of L-lactate in the presence of external NAD. 2. L-lactate oxidation can be stimulated in a reversible manner by ADP. 3. Mitochondrial lactate oxidation is sensitive to oxamate-inhibitor of LDH, alpha-cyano-3-hydroxy-cinnamate-pyruvate translocase inhibitor and respiratory chain inhibitors (rotenone, antimycin A, KCN). 4. In the same conditions the mitochondria did not oxidize pyruvate in the absence of malate, whereas, oxidize pyruvate plus external NADH in an uncoupling manner.     

Kinetic formulations for the oxidation and the reduction of glyoxylate by lactate dehydrogenase.

Chicken liver lactate dehydrogenase (L-lactate : NAD+ oxidoreductase, EC 1.1.1.27) irreversibly catalyses the oxidation of glyoxylate (hydrated form) (I) to oxalate (pH = 9.6) and the reduction of (non-hydrated form) (II) to glycolate (pH = 7.4). (I) attaches to the enzyme in the pyruvate binding site and (II) attaches to the enzyme at the L-lactate binding site. The oxidation of (I) (pH = 9.6) is adapted to the following mechanism: (see book). The abortive complexes, E-NADH-I and E-NAD+-II, are responsible for the inhibition by excess substrate in the reduction and oxidation systems, respectively. When lactate dehydrogenase and NAD+ are preincubated, E-NAD+- NAD+ appears and causes inhibition by excess NAD+ in the glyoxylate-lactate dehydrogenase-NAD+ and L-lactate-lactate dehydrogenase-NAD+ systems; the second NAD+ molecule attaches to the enzyme at the L-lactate binding site.     

Electrochemical evaluation of lactate dehydrogenase immobilized in polyacrylamide gels

Lactate dehydrogenase (EC 1.1.1.27) has been immobilized in polyacrylamide gels over a platinum grid matrix. The immobilized enzyme is used to oxidize L-lactate in the presence of nicotinamide adenine dinucleotide (NAD+) and ferricyanide. The NADH produced is then chemically oxidized back to NAD+ by ferricyanide. The coupled reduction of ferricyanide ions to ferrocyanide ions results in a measurable electrochemical potential. This measurable zero-current potential is found to be Nernstian in nature and directly proportional to the logarithm values of L-lactate concentration over the range of 2 X 10(-5) to 5 X 10(-2)M. The results indicate that immobilized lactate dehydrogenase can be incorporated into a system to detect L-lactate acid in aqueous solutions.     

L-Lactate or pyruvate stimulated growth of Novikoff rat hepatoma cells

Novikoff rat hepatoma cells (subline N1S1-67) grew when 30 mM L-lactate or pyruvate was substituted for D-glucose in Swim's medium 67 supplemented with dialyzed calf bovine serum. A 2.6-fold increase in cell number (1.34 generations) was obtained. RNA, DNA, protein and dry weight increased in proportion to the cell number. In control medium lacking L-lactate, pyruvate or D-glucose, cell growth of 0.42 generation was obtained. Growth with L-lactate was dependent on the L-lactate concentration up to 30 mM at which the greatest increase in cell number occurred. Significant growth did not occur when D-lactate, glycerol, acetate, alpha-ketoglutarate, succinate or malate, each at 30 mM, was substituted for D-glucose. Growth in the medium containing L-lactate was not due to the utilization of D-glucose or some other substrate carried into the culture with the inoculum. Medium contamination by D-glucose was insufficient to explain the growth obtained in the medium containing L-lactate, but could have accounted for growth in the control medium. Throughout growth, the concentration of L-lactate in the medium remained unchanged. The increase in cell number cannot be explained by L-lactate triggering the utilization of glycogen, nor by oxidation and degradation of protein, amino acids, fatty acids, or carbohydrate moieties of glycoprotein in the medium. L-Lactate does not serve as a significant carbon or energy source in the growth of these cells.     

L-lactate metabolism in potato tuber mitochondria

We investigated the metabolism of L-lactate in mitochondria isolated from potato tubers grown and saved after harvest in the absence of any chemical agents. Immunologic analysis by western blot using goat polyclonal anti-lactate dehydrogenase showed the existence of a mitochondrial lactate dehydrogenase, the activity of which could be measured photometrically only in mitochondria solubilized with Triton X-100. The addition of L-lactate to potato tuber mitochondria caused: (a) a minor reduction of intramitochondrial pyridine nucleotides, whose measured rate of change increased in the presence of the inhibitor of the alternative oxidase salicyl hydroxamic acid; (b) oxygen consumption not stimulated by ADP, but inhibited by salicyl hydroxamic acid; and (c) activation of the alternative oxidase as polarographically monitored in a manner prevented by oxamate, an L-lactate dehydrogenase inhibitor. Potato tuber mitochondria were shown to swell in isosmotic solutions of ammonium L-lactate in a stereospecific manner, thus showing that L-lactate enters mitochondria by a proton-compensated process. Externally added L-lactate caused the appearance of pyruvate outside mitochondria, thus contributing to the oxidation of extramitochondrial NADH. The rate of pyruvate efflux showed a sigmoidal dependence on L-lactate concentration and was inhibited by phenylsuccinate. Hence, potato tuber mitochondria possess a non-energy-competent L-lactate/pyruvate shuttle. We maintain, therefore, that mitochondrial metabolism of L-lactate plays a previously unsuspected role in the response of potato to hypoxic stress.     

The effect of different dissolved oxygen tensions on growth and enzyme activities of Campylobacter sputorum subspecies bubulus

Campylobacter sputorum subspecies bubulus was grown in batch cultures in which the dissolved oxygen tension (d.o.t) was maintained at various constant levels. At a range of d.o.t. from 0.002 to 0.05 atm, which allowed good growth (mean generation time approximately 1.5 h), L-lactate was preferentially consumed before D-lactate. L-lactate oxidation was accompanied by equimolar acetate production during exponential growth. A value for YL-lactate (g dry weight bacteria per mol L-lactate) of 54 was determined. Net acetate production stopped when C. sputorum started to use D-lactate after consumption of L-lactate. When a culture growing exponentially at the expense of L-lactate was shifted from a d.o.t. of 0.02 atm to a d.o.t. of 0.15 atm, growth was impaired, and L-lactate consumption and corresponding acetate production diminished. This decrease correlated with a loss of lactate dehydrogenase activity after the shift. Campylobacter sputorum appeared to possess cytochromes of the b- and c-type and a carbon monoxide-binding pigment. Evidence is given that the principal site of oxygen damage is lactate dehydrogenase rather than the cytochrome chain.     

D- and L-lactate catabolism to CO2 in rat tissues

The current study was initiated in order to compare the rates of oxidative catabolism of D- and L-lactate in various rat tissues. Uniformly labeled D- or L-[14C]lactate was incubated at 37 degrees C in a closed system with tissue homogenates in Krebs-Ringer phosphate buffer. Evolved 14CO2 was trapped in a center well containing a fluted filter paper saturated with strong base and the radioactivity determined. The ratio of L-lactate to D-lactate oxidation was greatest in brain, followed by kidney, heart, and liver. In liver the rate of oxidation of D-lactate exceeded that of L-lactate, in heart the rates were not significantly different and in the other two tissues L-lactate was oxidized more rapidly than D-lactate. These results indicate that the rate of D-lactate catabolism is considerable and is relatively greater than had been reported previously.     

Pyridine Nucleotide-Linked Lactate Dehydrogenase of Tetrahymena: Evidence for D- and L-Enzymes in the Mitochondria

An NAD-linked lactate dehydrogenase (LDH) in a crude mitochondrial fraction obtained from Tetrahymena homogenates was previously reported by this laboratory. This fraction contains the NADH and succinate oxidase system as well as the mitochondrial cytochromes and carries out oxidative phosphorylation. The preparation catalyzes the oxidation of D- and L-lactate linked only to certain analogs of NAD; it has not been possible to demonstrate NAD-dependent D- or L-lactate oxidation nor is there any evidence that either of these enzymes is a flavoprotein as indicated by their inability to reduce directly certain artificial electron acceptors. A lactate racemase is not present.     

Glyceraldehyde metabolism in human erythrocytes in comparison with that of glucose and dihydroxyacetone

Metabolism of D-glyceraldehyde in human erythrocytes in comparison with that of glucose and dihydroxyacetone was studied. Both trioses were metabolized to produce L-lactate at rates comparable to that of L-lactate formation from glucose. Almost complete inactivation of glyceraldehyde-3-phosphate dehydrogenase by treatment of cells with iodoacetate resulted in a 95% decrease in L-lactate formation from the ketotriose as well as from glucose, whereas L-lactate formation from the aldotriose was only partially reduced (60%). D-Lactate was produced faster from either the aldotriose or the ketotriose than from glucose, but the ability of the two trioses to produce D-lactate was far lower than that to produce L-lactate. Almost complete inhibition of aldehyde dehydrogenase by disulfiram and of both aldose reductase and aldehyde reductase II by sorbinil, had no effect on L-lactate formation from D-glyceraldehyde. The present study suggests that D-glyceraldehyde is metabolized via two or more pathways including the glycolytic pathway after its phosphorylation by triokinase, and that neither oxidation to D-glyceric acid nor reduction to glycerol is a prerequisite for D-glyceraldehyde metabolism.     

Chemo-enzymatic D-enantiomerization of DL-lactate

We investigated the total conversion of racemic lactate, L-lactate, and pyruvate into D-lactate, which is very useful as a starting material for the synthesis of chiral compounds and much more valuable than the L-enantiomer by means of coupling of L-specific oxidation of the racemate with L-lactate oxidase and non-enantiospecific reduction of pyruvate to DL-lactate with sodium borohydride. In this one-pot system, L-lactate was enantiospecifically oxidized to an achiral product, pyruvate, which was chemically reduced to DL-lactate leading to a turnover. Consequently, either DL-lactate, L-lactate, or pyruvate was fully converted to the D-enantiomer. We optimized the reaction conditions: DL-lactate was converted to D-lactate in 99% of the theoretical yield and with more than 99% enantiomeric excess. DL-alpha-Hydroxybutyrate and alpha-ketobutyrate were converted also to D-alpha-hydroxybutyrate in the same way, though slowly.     

Cloning of a Neisseria meningitidis gene for L-lactate dehydrogenase (L-LDH): evidence for a second meningococcal L-LDH with different regulation.

We report the cloning of lldA, a Neisseria meningitidis gene for L-lactate dehydrogenase (L-LDH). Escherichia coli contains a single L-LDH gene (lldD) in the lld operon (previously lct). E. coli grown in complex media does not have L-LDH activity, but the activity is induced by growth in defined medium with L-lactate as the carbon source. In contrast, meningococci contain at least one L-LDH in addition to the lldA gene product. These enzymes are active in meningococci grown in complex media and are not dependent on growth in L-lactate. The predicted amino acid sequence of lldA is homologous to that of E. coli lldD and of other prokaryotic and eukaryotic flavin mononucleotide-containing enzymes that catalyze the oxidation of L-lactate and other small alpha-hydroxy acids. A mutant with a deletion in lldA was found to have reduced L-LDH activity. However, this mutant was able to grow on L-lactate, indicating that a second L-LDH must exist. Activity of the lldA enzyme was affected by growth conditions, being increased by growth on a defined medium with either L-lactate or pyruvate as the carbon source. For meningococci grown on a complex medium, activity of the lldA enzyme was increased by growth on plates or in well-aerated broth. A second L-lactate-oxidizing activity was seen in bacteria grown in poorly aerated broth. Neisseria gonorrhoeae contains a homolog of lldA. As for meningococci, mutation of the gonococcal lldA reduced L-LDH activity but did not affect growth on L-lactate.     

L-lactate dehydrogenase from leaves of Capsella bursa-pastoris (L.) Med.

By ammonium sulfate fractionation and gel filtration an enzyme preparation which catalyzed NAD⁺-dependent L-lactate oxidation (10^-4 kat kg^-1 protein), as well as NADH-dependent pyruvate reduction (10^-3 kat kg^-1 protein), was obtained from leaves of Capsella bursa-pastoris. This lactate dehydrogenase activity was not due to an unspecific activity of either glycolate oxidase, glycolate dehydrogenase, hydroxypyruvate reductase, alcohol dehydrogenase, or a malate oxidizing enzyme. These enzymes could be separated from the protein displaying lactate dehydrogenase activity by gel filtration and electrophoresis and distinguished from it by their known properties. The enzyme under consideration does not oxidize D-lactate, and reduces pyruvate to L-lactate (the configuration of which was determined using highly specific animal L-lactate dehydrogenase). Based on these results the studied Capsella leaf enzyme is classified as L-lactate dehydrogenase (EC 1.1.1.27). It has a K_m value of 0.25 mmol l^-1 (pH 7.0, 0.3 mmol l^-1 NADH) for pyruvate and of 13 mmol l^-1 (pH 7.8, 3 mmol l^-1 NAD⁺) for L-lactate. Lactate dehydrogenase activity was also detected in the leaves of several other plants.Abbreviation FMN flavin adenine mononucleotide     

Oxidation of NADH via an "external" pathway in skeletal-muscle mitochondria and its possible role in the repayment of lactacid oxygen debt

Mitochondria isolated from skeletal muscle of rat catalyse oxidation of the external NADH (in the presence of rotenone, antimycin A and cytochrome c) at a rate of 15 natoms O2/min/mg protein by a pathway sensitive to mersalyl. In a medium supplemented with commercial lactate dehydrogenase, or when mitochondria were incubated in the presence of a cytoplasm, the NADH oxidation could be arrested by pyruvate. The inhibitory effect of pyruvate could be released by lactate. In the presence of NAD and cytochrome c, the reconstructed system containing skeletal muscle mitochondria plus cytoplasmic fraction was active in oxidation of L-lactate despite of the presence of rotenone and antimycin A. The lactate oxidation was sensitive to mersalyl and cyanide.     

Inhibition of lactate glucogneogenesis in rat kidney by dichloroacetate.

1. Sodium dichloroacetate (1mM) inhibited glucose production from L-lactate in kidney-cortex slices from fed, starved or alloxan-diabetic rates. In general gluconeogenesis from other substrates was no inhibited. 2. Sodium dichloracetate inhibited glucose production from L-lactate but no from pyruvate in perfused isolated kidneys from normal or alloxan-diabetic rats. 3. Sodium dichloroacetate is an inhibitor of the pyruvate dehydrogenase kinase reaction and it effected conversion of pyruvate dehydrogenase into its its active (dephosphorylated) form in kidney in vivo. In general, pyruvate dehydrogenase was mainly in the active form in kidneys perfused or incubated with L-lactate and the inhibitory effect of dichloroacetate on glucose production was not dependent on activation of pyruvate dehydrogenase. 4. Balance data from kidney slices showed that dichloroacetate inhibits lactate uptake, glucose and pyruvate production from lactate, but no oxidation of lactate. 5. The mechanism of this effect of dichloroactetate on glucose production from lactate has not been fully defined, but evidence suggests that it may involve a fall in tissue pyruvate concentration and inhibition of pyruvate carboxylation.     

Sodium ion/L-lactate co-transport in rabbit small-intestinal brush-border-membrane vesicles.

Uptake of L-lactate into rabbit jejunal brush-border-membrane vesicles prepared by a Ca2+-precipitation procedure was studied by a rapid filtration technique with L-[14C]-lactate as tracer. Transport of L-lactate into an intravesicular (osmotically reactive) space could be established. An inwardly directed NaCl gradient (outside 21 mM/inside 0mM) stimulated the uptake of L-lactate at 15 s 2-4-fold compared with that observed with an equal KCl gradient. A transient accumulation of L-lactate inside the vesicles (overshoot) was observed in the presence of an NaCl gradient. Gradients of LiCl, RbCl, CsCl or choline chloride were not able to replace NaCl in the stimulation of L-lactate uptake. L-Lactate uptake was saturable only in the presence of Na+. D-Lactate, DL-thiolactate (2-DL-mercaptopropionate), pyruvate and propionate inhibited the Na+-stimulated L-lactate uptake; D-lactate, thiolactate and pyruvate provoked trans-stimulation of L-lactate uptake. Artificially imposed diffusion potentials (inside negative) did not exert any effect on the Na+-dependent L-lactate uptake. The results are consistent with the existence of an electroneutral Na+/L-lactate co-transport system in the brush border of rabbit small intestine.     

Relationship of lactate dehydrogenase specificity and growth rate to lactate metabolism by Selenomonas ruminantium.

A lactate-fermenting strain of Selenomonas ruminantium (HD4) and a lactatenonfermenting strain (GA192) were examined with respect to the stereoisomers of lactate formed during glucose fermentation, the stereoisomers of lactate fermented by HD4, and the characteristics of the lactate dehydrogenases of the strains. GA192 formed L-lactate and HD4 formed L-lactate and small amounts of D-lactate from glucose. HD4 fermended L- but not D-lactate. Both strains contain nicotinamide adenine dinucleotide (NAD)-specific lactate dehydrogenases, and no NAD-independent lactate oxidation was detected. Continuous cultures of both strains grown with limiting glucose produced mainly propionate and acetate and little lactate at dilution rates less than 0.4/h, with shifts to increasing amounts of lactate and less acetate and propionate as the dilution rate was increased from 0.4/h to approximately 1/h.