Improving the thermal stability of lactate oxidase by directed evolution

Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 °C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution. Received 15 September 2006; received after revision 21 October 2006; accepted 2 November 2006     

The effects of salicylate and aspirin on the activity of phosphorylase a in perfused hearts of rats

Summary Sodium salicylate and aspirin are known to have a glycogenolytic effect as judged by either the glycogen level or lactate production in perfused hearts of rats. In this work it was possible to demonstrate that phosphorylase a level was increased in the hearts subjected to the action of these drugs.     

β-Blockade of morphine-induced hyperlactacidemia in rabbits

Summary In morphinized rabbits blood lactate levels are elevated. Hyperlactacidemia perists after cessation of morphine injections. This morphine-induced lactate accumulation is completely abolished by simultaneous propranolol treatment. Phentolamine does not modify the action of morphine.ERA CNRS No. 412.Acknowledgement. The authors express their appreciation to MissD. Abadie for her help with the lactate determinations.     

Beta-blockade of morphine-induced hyperlactacidemia in rabbits.

In morphinized rabbits blood lactate levels are elevated. Hyperlactacidemia persists after cessation of morphine injections. This morphine-induced lactate accumulation is completely abolished by simultaneous propranolol treatment. Phentolamine does not modify the action of morphine.     

Role of lipid peroxidation in ferric lactate-enhanced calcium uptake by Ehrlich carcinoma cells

Comparison of Ca²⁺ uptake by Ehrlich carcinoma cells in presence of ferric lactate or aluminum lactate, and formation of thiobarbituric acid-reactive substances, suggests that lipid peroxidation is associated with but not the cause of calcium overload that can lead to cell injury and death.     

Effects of aresenite and oxamate on the in vitro functional activity of estrogen-dominated rate uterine horns.

Arsenite but not oxamate produce in vitro a distinct depression of estrogen-dominated uterine motility, both in the absence of substrate as well as in the presence of exogenous glucose or lactate. The addition of oxamate to preparations suspended in a medium with lactate as the sole external substrate ameliorates the depression of uterine motility elicited by arsenic.     

Increased mitochondrial palmitoylcarnitine/carnitine countertransport by flavone causes oxidative stress and apoptosis in colon cancer cells

Cancer cell metabolism is characterized by limited oxidative phosphorylation in order to minimize oxidative stress. We have previously shown that the flavonoid flavone in HT-29 colon cancer cells increases the uptake of pyruvate or lactate into mitochondria, which is followed by an increase in O₂^−.. production that finally leads to apoptosis. Similarly, a supply of palmitoylcarnitine in combination with carnitine induces apoptosis in HT-29 cells by increasing the mitochondrial respiration rate. Here we show that flavone-induced apoptosis is increased more than twofold in the presence of palmitoylcarnitine due to increased mitochondrial fatty acid transport and the subsequent metabolic generation of O₂^−. in mitochondria is the initiating factor for the execution of apoptosis. Received 12 August 2005; received after revision 12 October 2005; accepted 14 October 2005     

Lactate dehydrogenase and glutamate dehydrogenase activities in the circumventricular organs of rat brain following neonatal monosodium glutamate

Glutamate (glu) an excitatory neurotransmitter amino acid, is present in high concentrations in the mammalian central nervous system and is the most abundant amino acid in our daily diet. In the present study the activities of lactate dehydrogenase (LDH) and glutamate dehydrogenase (GDH) were evaluated in the circumventricular organs (CVO) of the brain in 25-day-old rats following MSG administration at a dose of 4 mg/g b.wt during the first ten days of life. The results show the LDH activity increased to 265% of that in the control (p<0.001), whereas GDH activity was significantly decreased (p<0.05), The great elevation in LDH, a cytoplasmic marker enzyme, is apparently due to cytoskeletal changes brought about as a consequence of glu toxicity, whereas lowered GDH activity indicates altered glu homostasis in the blood-brain-barrier deficient areas following neonatal exposure to glu.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8-7.4) and ionic strength (20-50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO4(2-) and cations (Mg2+, Ca2+) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD+ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0-37 degrees C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

Evolution of myocardial glycolysis in perinatal rats]

Glycolysis was assessed in the Langendorff perfused rat heart during the perinatal period. Glucose uptake, lactate production, 14C-glucose incorporation into glycogen, tissue content content of glycogen, lactate and high energy phosphates were measured in the foetus of 21.5 days post co?tum and in the newborn of 1 and 7 days post partum. Glycogen synthesis and anaerobic glycolysis were found to be more active in the foetal heart and to decrease during the first neonatal week.     

Effect of hypoxia on glycolysis in isolated fetal rat heart]

Glycolysis was assessed in the isolated foetal Rat in hypoxia. Measurements were made of glucose uptake, lactate output, C14 glucose incorporation into glycogen and tissular levels of ATP, PCr, glycogen and lactate. Glycolysis was stimulated by hypoxia to a greater extent in the young foetal heart of 16.5 days post coitum than in the foetal heart at term. Thus high energy phosphates were maintained at a higher level in the younger heart. The results are discussed in relation to the high resistance to hypoxia of the foetal heart.     

Limited proteolysis of lactate dehydrogenase from procine heart with trypsin: Characterization and reactivation of the fragments

Summary The ternary complex formed by native lactate dehydrogenase (LDH) from porcine heart, NAD⁺ and sulfite, was digested with trypsin over a period of 12–16 h³. After removal of the ligands and residual native lactate dehydrogenase by ion exchange chromatography dimers were obtained which were almost inactive. The dimers were lacking a hexapeptide at the N-terminus; however, the secondary structure was the same as that of native lactate dehydrogenase. The circular dichroism spectra showed a dependence on temperature which suggested an equilibrium of two different structural states.The reaction of antibodies against native porcine heart LDH with the dimers restored the catalytic activity, and subsequently the dimers behaved similarly to the native enzyme. Addition of 1 M phosphate or NAD-sulfite to the dimers restored 80–90% of the catalytic activity. It could be demonstrated that the behaviour of the reactivated dimers, in contrast to that of the inactive dimers, was similar to the behaviour of native lactate dehydrogenase. For instance, ultracentrifugal analysis showed that dimers reactivated with NAD–SO₃ ^– were associated to give tetramers.The reaction of antibodies against native LDH with the dimers reactivated with NAD–SO ₃ ^– demonstrated that the native LDH and the dimers have the same surface determinants.     

Inhibitory effect of galactose on hydrogen transfer in the hemolymph ofVespa orientalis

Summary The generation of pyruvate and lactate inVespa orientalis hemolymph is prevented by the presence of galactose. The inhibitory effect is possibly produced by a competition for NAD and occurs in concentrations of 0.05 M.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Summary Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8–7.4) and ionic strength (20–50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO ₄ ^2– ) and cations (Mg²⁺, Ca²⁺) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD⁺ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0–37°C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

Effects of a definite stress on the enzyme activities in blood plasma in mice (author's transl)

The present study was designed to analyze the effects of a short term stress on the quantitative levels of alkaline phosphatase (AP), creatine phosphokinase (CK), aldolase (ALD), lactate dehydrogenase (LDH), and lactate dehydrogenase-1-isoenzyme (LDH 1) during 24 h after stress. For all the enzymes studied higher values for the activity were found due to stress in comparison to the activity level before the stress. The period during the 24 h after stress to reach maximum activity depends on the enzyme and in part on the sex of the animal.     

Lactate dehydrogenase specificity and subunit assembly in neural tissues of the teleostPhallichthys amates

Summary The tissue specificity of lactate dehydrogenase (EC 1.1.1.27) in brain and eye of the teleostPhallichthys amates was examined by acrylamide gel electrophoresis. It is suggested that subunit association is a function of gene product accessibility superimposed upon genetic restriction of assembly.     

D(-)Lactic acid—a physiological isomer in the rat

Summary D(-)Lactate is produced in significant amounts together with L(+)lactate in the stomach of normal experimental rats. It is absorbed into the blood and constitutes a physiological isomer in this animal species.     

Mapping and quantification of biomolecules in tumor biopsies using bioluminescence

Quantitative bioluminescence and single-photon imaging have been applied for mapping concentration distributions of metabolites, such as adenosine triphosphate (ATP), glucose and lactate, in biopsies of cervical cancers in patients. Biopsies were taken before a conventional radiation treatment, and a number of clinically relevant data, such as local tumor control, patient survival, metastatic spread and so forth, were documented. There was no correlation between staging or grading and any of the metabolic parameters measured. Local correlations between ATP, glucose and lactate on a pixel-to-pixel basis were generally positive, with respective Spearman's correlation coefficients less in patients without clinically documented metastasis compared with those with metastatic spread. Lactate concentrations were significantly higher and scattered over a wider range in tumors with metastatic spread in comparison to malignancies in patients without metastasis. Thus, high local lactate levels of 20 mole/g appear to be associated with a high risk of metastasis, at least in human cervical tumors.     

Effects of arsenite and oxamate on the in vitro functional activity of estrogen-dominated rat uterine horns

Summary Arsenite but not oxamate produce in vitro a distinct depression of estrogen-dominated uterine motility, both in the absence of substrate as well as in the presence of exogenous glucose or lactate. The addition of oxamate to preparations suspended in a medium with lactate as the sole external substrate ameliorates the depression of uterine motility elicited by arsenite.This work has been supported by grants 6638 and 6294 from the Consejo Nacional de Investigaciones Cientificas y Técnicas de la República Argentina.Senior Investigator of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina.Junior Investigator of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina.     

Auswirkungen einer definierten Belastung auf die Enzymaktivitäten im Blutplasma bei der Maus Effects of a definite stress on the enzyme activities in blood plasma in mice

Summary The present study was designed to analyze the effects of a short term stress on the quantitative levels of alkaline phosphatase (AP), creatine phosphokinase (CK), aldolase (ALD), lactate dehydrogenase (LDH), and lactate dehydrogenase-1-isoenzyme (LDH 1) during 24 h after stress. For all the enzymes studied higher values for the activity were found due to stress in comparison to the activity level before the stress. The period during the 24 h after stress to reach maximum activity depends on the enzyme and in part on the sex of the animal.