Presence Of L-lactate Research Articles

Enhancement of the response of a lactate oxidase/peroxidase-based fiberoptic sensor by compartmentalization of the enzyme layer

A bienzyme fiberoptic sensor for the chemiluminescent flow injection analysis of L-lactate was developed. Lactate oxidase and peroxidase were covalently immobilized on preactivated polyamide membrane either separately on different membranes or randomly coimmobilized on the same membrane. Hydrogen peroxide, generated by the lactate oxidase reaction in the presence of L-lactate, was the substrate of the second reaction, catalyzed by peroxidase, in the course of which light was emitted in the presence of luminol. Compartmentalization of the enzyme layer was obtained by stacking a peroxidase membrne on a lactate oxidase membrane at the sensing tip of the fiberoptic sensor. When using such a sensing tip, the response of the fiberoptic sensor for lactate was 22 times higher than the response obtained when using a membrane bearing the two enzymes randomly coimmobilized. With the compartmentalized sensor tip, the detection limit was 250 pmol lactate and the coefficient of variation was 1.7% for 10 replicates of 6.25 nmol lactate. The method was applied for lactate determination in reconstituted whey solutions, and the results were in good agreement with the analysis by an enzyme electrode.

Specific effects of thiosulphate and L-lactate on hemocyanin-O2 affinity in a brachyuran hydrothermal vent crab

The hydrothermal vent crab Bythograea thermydron Williams (Brachyura: Bythograeidae) is exposed to high environmental concentrations of hydrogen sulphide. Hydrogen sulphide has previously been shown to be oxidized to a non-toxic form, thiosulphate (S2O3 2-), that accumulates in the hemolymph (to concentrations>1 mmoll-1). Hemocyanin-oxygen (Hc-O2) affinity was determined in dialysed, fresh or frozen hemolymph samples from B. thermydron. Although freezing is known to alter the affinity or cooperativity of some decapod crustacean hemocyanins, neither Hc-O2 affinity nor cooperativity was significantly altered in B. thermydron hemolymph samples that had been frozen, consistent with previous findings. Oxygen affinity of B. thermydron hemocyanin was significantly increased by the presence of L-lactate. Likewise, Hc-O2 affinity was significantly increased by the presence of 1.5 mmol S2O3 2- I-1. The magnitude of this effect was the same as for similar concentrations of L-lactate. This specific effect of S2O3 2- does not appear to be a general property of crustacean hemocyanins, as there was no significant effect of S2O3 2- on Hc-O2 affinity or cooperativity in dialysed hemolymph samples from the brachyuran crabs Cancer anthonyi Rathbun and C. antennarius Stimpson, or the thalassinid ghost shrimp Callianassa californiensis Dana. In the context of high environmental sulphide concentrations coupled with low PO 2, and the subsequent accumulation of S2O3 2- in crab hemolymph, the increase in Hc-O2 affinity due to thiosulphate appears to be an adaptive response in B. thermydron.

A microelectrode study of the mechanisms of L-lactate entry into and release from frog sartorius muscle.

1. Changes in intracellular pH and intracellular anion levels were monitored in frog sartorius muscle fibres during exposure to extracellular L-lactate, using ion-sensitive microelectrodes. 2. Resting intracellular pH (pHi) in 20 mmol l-1 HEPES buffer was 7.18 +/- 0.015 (S.E. of mean, n = 62). Exposure to an extracellular solution at pH 6.5 buffered with 20 mmol l-1 3-(N-morpholino)propanesulphonic acid (MOPS) resulted in a slow intracellular acidification. 3. A reversible decrease in pHi and an increase in intracellular anion levels was observed when L-lactate replaced chloride in equimolar amounts. The increase in intracellular anion level is consistent with intracellular accumulation of L-lactate ion. 4. The rate and steady-state change in pHi and anion level was a function of both extracellular pH and L-lactate concentration, providing evidence for the coupled movement of lactate and proton equivalents. 5. The initial rate of uptake of L-lactate, as measured by the change of pHi, was a non-linear function of the extracellular L-lactate concentration at extracellular pH 6.8 and 7.35. 6. No saturation was observed with concentrations of L-lactate between 5 and 60 mmol l-1 at pH 7.35 and 2.5 and 40 mmol l-1 at pH 6.8. 7. The non-linear relationship between the initial rate of change in pHi and extracellular L-lactate was well fitted by a curve defining uptake as the sum of a carrier process displaying Michaelis-Menten kinetics and a passive diffusion component. The apparent Km of the carrier was 10 mmol l-1 at pHo 7.35 and 4 mmol l-1 at pHo 6.8. 8. The initial rate of change of pHi in the presence of L-lactate was significantly inhibited 39.1 +/- 6.2% by 2-5 mmol l-1 alpha-cyano-4-hydroxycinnamate (n = 9; P less than 0.05, paired t test). 9. alpha-Cyano-4-hydroxycinnamate had no detectable effect on the initial rate of change of pHi induced by propionate exposure. 10. The initial rate of change of pHi induced by L-lactate was not affected by 20-100 mumol l-1 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS). 11. We conclude that L-lactate crosses the membrane of the frog sartorius muscle with proton equivalents via (1) a carrier-mediated process, and (2) passive diffusion of lactic acid. In the physiological range of L-lactate concentrations and pH the transport process dominates.

Cytochrome b2, an electron carrier between flavocytochrome b2 and cytochrome c. Rapid kinetic characterization of the electron-transfer parameters with ionic-strength-dependence.

The oxidation-reduction properties of free cytochrome b2 isolated by controlled proteolysis from flavocytochrome b2, i.e. the flavodehydrogenase-bound cytochrome b2, were investigated by using stopped-flow spectrophotometry. The rapid kinetics of the reduction of cytochrome b2 by flavocytochrome b2 in the presence of L-lactate are reported. The self-exchange rate constant between reduced cytochrome b2 bound to the flavodehydrogenase and free cytochrome b2 was determined to be 10(5) M-1 X S-1 at 5 degrees C, I 0.2 and pH 7.0. The specific electron-transfer reaction between reduced cytochrome b2 and cytochrome c was also studied, giving an apparent second-order rate constant of 10(7) M-1 X S-1 at 5 degrees C, I 0.2 and pH 7.0. This electron-exchange rate is slightly modulated by ionic strength, following the Debye-Hückel relationship with a charge factor Z1Z2 = -1.9. Comparison of these data with those for the reduction of cytochrome c by flavodehydrogenase-bound cytochrome b2 [Capeillère-Blandin (1982) Eur. J. Biochem. 128, 533-542] leads to the conclusion that the intramolecular electron exchange between haem b2 and haem c within the reaction complex occurs at a rate very similar to that determined experimentally in presence of the flavodehydrogenase domain. The low reaction rate observed with free cytochrome b2 is ascribed to the low stability of the reaction complex formed between free cytochrome b2 and cytochrome c.

Complex formation and intermolecular electron transfer between flavocytochrome b2 in the crystal and cytochrome c.

The present study addresses the question whether tetrameric flavocytochrome b2 in the crystal is catalytically competent and, if so, whether it is possible to prepare a functional complex of the crystalline enzyme with its physiological electron acceptor cytochrome c. By single crystal microspectrophotometry we show that the native reduced enzyme can be oxidized by oxygen or ferricyanide and that the oxidized enzyme can be reduced by the electron donor L-lactate. Reduced cytochrome c appears to diffuse through the liquid channels of flavocytochrome b2 crystals and, at low ionic strength, to accumulate in amounts stoichiometrically equivalent to the enzyme protomers. Both cytochromes can be oxidized by ferricyanide. In the presence of L-lactate, both cytochromes become reduced. Since reduction of cytochrome c by L-lactate requires the catalytic action of flavocytochrome b2, it is concluded that the structure of the crystalline enzyme not only allows for electron transfer from L-lactate to flavin and intramolecular electron transfer from flavin to heme, but also for the formation of a productive complex with cytochrome c.

Reduction of respiratory-chain cytochrome b by lactate in Saccharomyces cerevisiae.

Cytochrome b of yeast mitochondria can be reduced by a part of the electrons resulting from the oxidation of lactate enantiomers. 1. The respiration of D-lactate and L-lactate is 30-40% inhibited by antimycin A. 2. Reduction of cytochrome b is observed in submitochondrial particles in the presence of low concentration of D-lactate and L-lactate (half-optimal concentration of 4.7 mM and 2.4 mM respectively) in the presence of different bc1 inhibitors. 3. Reduction of cytochrome b and c1 occurs in purified complex III of yeast in the presence of L-lactate and added L-lactate: NAD+ oxidoreductase. 4. In the particles obtained from yeast grown in lactate the oxidation of L-lactate involves the reduction of a pigment absorbing at 558 nm.

Metabolite-controlled phosphorylation of phosphofructokinase in rat hepatocytes.

The effect of glucose on the phosphorylation of phosphofructokinase has been investigated in hepatocytes isolated froin fed and 24‐h‐starved rats by measuring the 32P radioactivity associated with the enzyme. In addition, the effects of gluconeogenic precursors and of glucagon or Bt2cAMP were followed under these conditions.The basal incorporation of phosphate into phosphofructokinase in hepatocytes from starved and fed rats was 0.2 ± 0.1 and 0.6±0.2 mol phosphate/mol phosphofructokinase (tetramer) respectively, whereas the maximal incorporation obtained was 1.03 ± 0.2 mol phosphate/mol phosphofructokinase.With 16 mM l‐lactate + 4 mM pyruvate or with 20 mM l‐alanine, the phosphorylation of the enzyme was lower than the basal incorporation found in hepatocytes from fed and starved rats.Glucose (20 mM) addition to isolated hepatocytes from fed rats resulted in an increase in the incorporation phosphate into phosphofructokinase to almost maximal value.In hepatocytes isolated from starved rats, glucose led to a 2.5‐fold increase of phosphorylation as compared to the basal incorporation. l‐Alanine (20 mM) abolished the glucose‐induced phosphorylation in hepatocytes from starved and from fed animals.Glucagon (0.1 μM) did not significantly enhance thc phosphorylation of phospliofructokinase in the presence of l‐lactate + pyruvate in hepatocytes from starved rats, but stimulated the phosphorylation to maximal value when cells had been preincubated in the presence of glucose.In contrast, in liver cells isolated from fed rats. glucagon had no additional effect on phosphofructokinase pliosphorylation in the presence of glucose, but did increase the phosphorylation to maximal value when l‐lactate + pyruvate served as substrates. l‐Alanine inhibited the glucose‐induced phosphorylation of phosphofructokinase also in the presence of glucagon.In contrast, glucagon stimulated the phosphorylation of l‐type pyruvate kinase independently from exogenous precursors and from the nutritional state of the animals.Bt2cAMP (0.1 mM) as glucagon, did not significantly enhance the phosphorylation of phosphofructokinase above the level obtained with glucose alone in hepatocytes from fed rats, but did increase the velocity of the phosphate incorporation in hepatocytes from starved rats, thus leading under both conditions to the maximal incorporation.It is concluded that the degree of phosphorylation of rat liver phosphofructokinase is affected by the metabolic state, and that glucagon (or CAMP) acts indirectly, probably by changing glucose metabolism due to increased glycogenolysis rather than by an activation of a CAMP‐dependent protein kinase.

63] l-Lactate dehydrogenase,1 FDP-activated, from Streptococcus cremoris

This chapter discusses the assay method and properties of L-lactate dehydrogenase, fructose- 1,6-diphosphate (FDP)-activated, from Streptococcus cremoris . L-lactate dehydrogenase activity is measured spectrophotometrically at 340 nm by following either the oxidation of nicotinamide adenine dinucleotide dehydrogenase (NADH) in the presence of pyruvate and FDP, or the reduction of NAD in the presence of L-lactate and FDP. The steps involved in the purification procedureof L-lactate dehydrogenase are (1) the growth of organism, (2) the preparation of cell-free extracts, (3) fractionation with streptomycin sulfate, (4) first and second fractionation with ammonium sulfate, (5) chromatography on diethylaminoethyl (DEAE)-cellulose, and (6) chromatography on DEAE-Sephadex. The stability of the enzyme is dependent on both the type and pH of the buffer in which it is stored. Cell-free extracts prepared in acidic to neutral buffers and stored at 5°C and retain more lactate dehydrogenase activity than those prepared in alkaline buffers. The activation of lactate dehydrogenase activity by FDP is inhibited by phosphate and by the treatment of the enzyme with pyridoxal phosphate followed by reduction with sodium borohydride.

Transport of D-lactate in perfused rat liver.

The transport of D-lactate across the plasma membrane was investigated in hemoglobin-free perfused rat livers, applying the multiple-indicator dilution technique (pulse labelling of D-lactate and indicator substances). The following results were obtained: 1. The steady state exchange rate at 1 mM D-lactate was 2.5 mumol x min-1 x g wet wt-1. It was proportional to the extracellular concentration in the range between 0.1 and 70 mM. 2. The transport of D-lactate was inhibited by L-lactate and pyruvate; 50% inhibition was observed at 40 mM L-lactate or 5 mM pyruvate. 3. The transport was also inhibited by alpha-cyanocinnamate and 4,4'-diisocyanostilbene-2,2'-disulfonic acid. The inhibition by cyanocinnamate was complete (with 25 mM) and fully reversible, whereas the inhibition by diisothiocyanostilbenedisulfonic acid was incomplete and irreversible; it was dependent upon the amount of diisothiocyanostilbenedisulfonic acid bound by the liver. Maximal inhibition (80%) was observed with 2 mumol diisothiocyanostilbenedisulfonic acid bound per g wet weight. 4. The intracellular concentration (ci) of D-lactate was proportional to the extracellular concentration (ce); the ratio ci/ce was 0.5 throughout the concentration range studied. It decreased in the presence of L-lactate or pyruvate. It is concluded that the transport of D-lactate is carrier-mediated, and, at least partially, electroneutral.

Energy requirement for the action of staphylococcin 1580 in Staphylococcus aureus

1. 1. Starved cells of a glucose-grown strain of Staphylococcus aureus are resistant to the action of staphylococcin 1580. Reinitiation of sensitivity is readily obtained upon the addition of glucose, but only weakly with l-lactate, although the latter induces higher ATP levels and supports l-glutamic acid uptake better than glucose does. The NADH/NAD + ratio correlates with the staphylococcin sensitivity. 2. 2. Starved pyruvate-grown cells remain partially susceptible and full sensitivity is restored both in the presence of glucose and l-lactate. 3. 3. Arsenate but not dicyclohexylcarbodiimide (DCCD) blocks the reinitiation of sensitivity in the presence of glucose. Both arsenate and DCCD block sensitivity in the presence of l-lactate. 4. 4. Aerobically grown cells are sensitive to staphylococcin 1580 under anaerobic conditions. Anaerobically grown cells are less susceptible, but sensitivity can be restored by glucose and also by l-lactate plus nitrate when cells are previously induced for nitrate reductase. 5. 5. Starved cells of a mutant strain defective in the maitenance of a high-energy state of the membrane are normally sensitive in the presence of glucose, but resistant in the presence of l-lactate. A strain lacking a functional respiratory chain (men −) is also sensitive with gucose but resistant in the presence of l-lactate. 6. 6. It is concluded that the initiation of the staphyloccin 1580 action is under control of a mechanism regulating the energy flow in the cell, and involving the presence of a high-energy phosphorylated compound.

Amino acid transport in membrane vesicles of obligately anaerobic Veillonella alcalescens

Membrane vesicles of Veillonella alcalescens, grown in the presence of L-lactate and KNO-3, actively transport amino acids under anaerobic conditions in the presence of several electron donors and the electron acceptor nitrate. The highest initial rates of uptake are obtained with L-lactate, followed by reduced nicotinamide adenine dinucleotide, glycerol-1-phosphate, formate, and L-malate.. The membrane vesicles contain the dehydrogenases for these electron donors, and these enzymes are coupled with nitrate reductase. In membrane vesicles from cells, grown in the presence of nitrate, the dehydrogenases are not coupled with fumarate reducatase, and anaerobic transport of amino acids does not occur with fumarate as electron acceptor. Under aerobic conditions none of the physiological electron donors can energize transport. However, a high rate of uptake is observed with the electron donor system ascorbate-phenazine metho-sulfate. This electron donor system also effectively energizes transport under anaerobicconditions in the presence of the electron acceptor nitrate.

Cytochrome [formula omitted] ( l-lactate déshydrogénase):équilibre et processus élémentaires de la réaction de combinaison substrat à l'enzyme

Cytochrome b 2 ( l-lactate dehydrogenase): substrate—enzyme reaction The rate constants and equilibrium dissociation constants were measured in order to study the combination of the substrate to the cytochrome b 2 ( l-lactate:ferricytochrome c oxidoreductase, EC 1.1.2.3) from yeast. A method of protection by the substrate to certain modifications of the enzyme was employed in determining the K 8 for the ES complex; denaturation of the cytochrome b 2 by urea proved suitable for this purpose. Upon this action, a non-polarized flavine fluorescence appears, synchronized with an increase of protein fluorescence. The reaction rate of this process was measured by a fluorescence spectrophotometer. The rate of modification of the cytochrome b 2 by urea decreases markedly in the presence of l-lactate or its analogue. The dissociation constant K 8 of the ES complex, which is identical to the protection constant K p , was thus determined. We obtained the values K 8 = 20–40 μ M for l-lactate and 30–2600 μM for oxalate between pH 6 and pH 8. From these results and known kinetic measurements, we can calculate the rate constants concerned with substrate fixation: K m = 1.2 mM, Kδ = 20 μ M 1 k 1 = 10 5 M −1, k −1 = 2 sec −1 . The second order velocity constant k 1 of the substrate fixation to the cytochrome b 2 thus calculated, is in good agreement with the value obtained from a direct measurement by means of a stopped flow technique.

D- and L-Lactic acid oxidase of Escherichia coli

A particulate enzyme complex from Escherichia coli catalyzing the oxidation of lactate to pyruvate has been studied. Enzyme preparations obtained from celss of E. coli, grown in synthetic medium containing DL-lactate, oxidized both isomers of lactate. An acetone powder from such preparations oxidized D-lactateonly and required methylene blue for activity. Enzyme preparations from cells of E. coli, grown in media with glucose as the source of carbon, oxidized D-lactate vigorously but showed little or no oxidative activity in the presence of L-lactate. Some of the properties of the enyzme complex have been described and the relation of the findings to adaptive enzyme formation has ben discussed.