Determination of lactate dehydrogenase in human erythrocytes by capillary electrophoresis with electrochemical detection

Capillary electrophoresis (CE) was employed to analyze lactate dehydrogenase (LDH) in human erythrocytes using an amperometric detector with a carbon fiber micro-disk bundle electrode. LDH activity was measured by determining the amount of NADH generated by LDH through a enzyme-catalyzed reaction between NAD(+) and lithium lactate. The factors influencing the enzyme-catalyzed reaction, separation and detection were examined and optimized. The following conditions were suitable for the determination of LDH: running buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 7.5); separation voltage, 20.0 kV; detection potential, 1.00 V (versus saturated calomel electrode (SCE)). The conditions of enzyme-catalyzed reaction were: reaction buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 9.3); substrates, 5.0 x 10(-2)mol/l lithium lactate and 5.0 x 10(-3)mol/l NAD(+); reaction time, 10 min. The concentration limit of detection (LOD) of the method was 0.017 U/ml at a signal-to-noise (S/N) ratio of 3, which corresponded to 1.10 x 10(-10)mol/l, and the mass LOD was 2 x 10(-20)mol. The linear dynamic range was 0.039-4.65 U/ml for the injection voltage of 5.0 kV and injection time of 10s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the electrophoretic peak area. The method was applied to determine LDH in human erythrocytes. The recovery of the method was between 98 and 101%.     

Identification of 2-amino-5-aryl-pyrazines as inhibitors of human lactate dehydrogenase

A 2-amino-5-aryl-pyrazine was identified as an inhibitor of human lactate dehydrogenase A (LDHA) via a biochemical screening campaign. Biochemical and biophysical experiments demonstrated that the compound specifically interacted with human LDHA. Structural variation of the screening hit resulted in improvements in LDHA biochemical inhibition and pharmacokinetic properties. A crystal structure of an improved compound bound to human LDHA was also obtained and it explained many of the observed structure–activity relationships.     

Identification of human lactate dehydrogenase A inhibitors with anti-osteosarcoma activity through cell-based phenotypic screening

Human lactate dehydrogenase A plays a key role in the glycolytic process, the inhibition of the enzyme is therefore considered of interest in developing anticancer therapeutics. However, due to the highly polar nature of hLDHA binding pocket, it is very challenge to discover potent cellular active hLDHA inhibitor. Combined a cell-based phenotypic screening assay with a primary enzymatic assay, we discovered three cellular active hLDHA inhibitors, namely 38, 63, and 374, which reduced MG-63 cell proliferation with IC₅₀ values of 6.47, 2.93, and 6.10 µM, respectively, and inhibited hLDHA with EC₅₀ values of 3.03, 0.63, and 3.26 µM, respectively.     

An amperometric lactate biosensor based on lactate dehydrogenase immobilized onto graphene oxide nanoparticles‐modified pencil graphite electrode

A novel amperometric lactate biosensor was developed based on immobilization of lactate dehydrogenase onto graphene oxide nanoparticles‐decorated pencil graphite electrode. The enzyme electrode was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy (FTIR), and cyclic voltammetry at different stages of its construction. The biosensor showed optimum response within 5 s at pH 7.3 (0.1 M sodium phosphate buffer) and 35°C, when operated at 0.7 V. The biosensor exhibited excellent sensitivity (detection limit as low as 0.1 μM), fast response time (5 s), and wider linear range (5–50 mM). Analytical recovery of added lactic acid in serum was between 95.81–97.87% and within‐batch and between‐batch coefficients of variation were 5.04 and 5.40%, respectively. There was a good correlation between serum lactate values obtained by standard colorimetric method and the present biosensor (r = 0.99). The biosensor measured lactate levels in sera of apparently healthy subjects and persons suffering from lactate acidosis and other biological materials (milk, curd, yogurt, beer, white wine, and red wine). The enzyme electrode lost 25% of its initial activity after 60 days of its regular uses, when stored dry at 4°C.     

Determination of lactate dehydrogenase isoenzymes in single lymphocytes from normal and leukemia cell lines

This work demonstrates that our previously developed technique for single-erythrocyte analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) can be applied to study individual lymphocytes, with some modification in the cell lysing procedure. A tesla coil was shown to be capable of lysing the lymphocyte cells inside the capillary. The electromagnetic field induced by the tesla coil was believed to be responsible for breaking the cell membrane. The lactate dehydrogenase (LDH) isoenzyme activities and the relative ratios between different LDH isoenzymes were measured for normal lymphocytes as well as B-type and T-type acute lymphoblastic leukemia cells. Both the LDH activity and the isoenzyme ratios show large variations among individual cells. The former is expected due to variations in cell size. The latter implies that single-cell measurements are less useful than the average values over a cell population as markers for leukemia.     

Identification of substituted 2-thio-6-oxo-1,6-dihydropyrimidines as inhibitors of human lactate dehydrogenase

A novel 2-thio-6-oxo-1,6-dihydropyrimidine-containing inhibitor of human lactate dehydrogenase (LDH) was identified by high-throughput screening (IC₅₀ = 8.1 μM). Biochemical, surface plasmon resonance, and saturation transfer difference NMR experiments indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of the screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC₅₀ = 0.48 μM). A crystal structure of an optimized compound bound to human LDHA was obtained and explained many of the observed structure–activity relationships.     

The Alcaligenes eutrophus ldh structural gene encodes a novel type of lactate dehydrogenase

Abstract The lactate dehydrogenase gene, ldh , of Alcaligenes eutrophus H16 was identified on a 14-kbp Eco RI restriction fragment of a genomic library in the cosmid pHC79 by hybridization with a 50-mer synthetic oligonucleotide which was derived from the N-terminal amino acid sequence of the purified enzyme. Recombinant strains of Escherichia coli JM83, which harboured a 2.0-kbp Pst I subfragment in pUC9-1, expressed LDH at a high level, if ldh was downstream from and colinear to the E. coli lac promoter. The nucleotide sequence of a region of 4245 bp revealed several open reading frames which might represent coding regions. One represented the ldh gene. The amino acid sequence deduced from ldh exhibited 29% and 36% identity to the L-malate dehydrogenase of Methanothermus fervidus and to the putative translation product of an E. coli sequence of unknown function, respectively. The ldh was separated by short intergenic regions from two other open reading frames: ORF5 was located downstream of and colinear to ldh , and its putative translational product revealed 38 to 56% amino acid identity to penicillin-binding proteins. ORF3 was located upstream of and colinear to ldh , and its putative gene translational product represented a hydrophobic protein. A sequence, which resembled the A. eutrophus alcohol dehydrogenase promoter, was detected upstream of ORF3, which most probably represents the first transcribed gene of an operon consisting of ORF3, ldh and ORF5.     

Rat lactate dehydrogenase A and B subunit concentrations are not regulated by mRNA abundance in liver and heart

RNA was isolated from rat liver and heart tissues at various times up to 12 weeks after birth, and probed on slot blots with lactate dehydrogenase A and B cDNA probes. Although the relative abundances of LDH A in liver and LDH B in heart increased substantially in the 12 weeks after birth, mRNAs for both isoenzymes remained remarkably stable in both tissues over the same period. The implications of these observations for the regulation of constitutive gene expression are discussed.     

Identification of substituted 3-hydroxy-2-mercaptocyclohex-2-enones as potent inhibitors of human lactate dehydrogenase

A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC₅₀ = 1.7 μM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC₅₀ = 0.18 μM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure–activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F = 45%).     

The role of an NAD-independent lactate dehydrogenase and acetate in the utilization of lactate byClostridium acetobutylicum strain P262

Clostridium acetobutylicum strain P262 utilized lactate at a rapid rate [600 nmol min^–1 (mg protein)^–1], but lactate could not serve as the sole energy source. When acetate was provided as a co-substrate, the growth rate was 0.05 h^–1. Butyrate, carbon dioxide and hydrogen were the end products of lactate and acetate utilization, and the stoichiometry was 1 lactate + 0.4 acetate → 0.7 butyrate + 0.6 H₂ + 1 CO₂. Lactate-grown cells had twofold lower hydrogenase than glucose-grown cells, and the lactate-grown cells used acetate as an alternative electron acceptor. The cells had a poor affinity for lactate (K_s = 1.1 mM), and there was no evidence for active transport. Lactate utilization was catabolyzed by an inducible NAD-independent lactate dehydrogenase (iLDH) that had a pH optimum of 7.5. The iLDH was fivefold more active with d-lactate than l-lactate, and the K _m for d-lactate was 3.2 mM. Lactate-grown cells had little butyraldehyde dehydrogenase activity, and this defect did not allow the conversion of lactate to butanol. Received: 17 October 1994 / Accepted: 30 January 1995     

Characterization of lactate dehydrogenase enzyme in seminal plasma of Japanese quail (Coturnix coturnix japonica)

Lactate dehydrogenase enzyme present in quail seminal plasma has been characterized. Polyacrylamide gel electrophoresis and subsequently with LDH specific staining of seminal plasma revealed a single isozyme in quail semen. Studies on substrate inhibition, pH for optimum activity and inhibitor (urea) indicated the isozyme present in the quail semen has catalytic properties like LDH-1 viz. H-type. Furthermore, unlike other mammalian species, electrophoretic and kinetic investigations did not support the existence of semen specific LDH-X isozyme in quail semen. The effect of exogenous lactate and pyruvate on sperm metabolic activity was also studied. The addition of 1 mM lactate or pyruvate to quail semen increased sperm metabolic activity. Our results suggested that both pyruvate and lactate could be used by quail spermatozoa to maintain their basic functions. Since the H-type isozyme is important for conversion of lactate to pyruvate under anaerobic conditions it was postulated that exogenous lactate being converted into pyruvate via LDH present in semen may be used by sperm mitochondria to generate ATP. During conversion of lactate to pyruvate NADH is being generated that may be useful for maintaining sperm mitochondrial membrane potential.     

The physiological role of the isoenzymes of lactate dehydrogenase in potatoes

Four of the five isoenzymes of lactate dehydrogenase present in potato tubers have been isolated and their kinetic properties examined. The pyruvate-reductase activity of isoenzyme-4 is greatly reduced at low pH, the affinity for both pyruvate and NADH is reduced and ATP has a stronger inhibitory effect. If the design properties of an enzyme dictate a high affinity for substrates, then the Km values for lactate, glyoxylate and NAD are consistent with an oxidative role for isoenzyme-4. The same considerations do not permit a conclusion about the physiological role of isoenzymes-1 to-3. However, an overview of the kinetic properties of these isoenzymes indicates that isoenzyme-1 is best adapted for the role of pyruvate reductase. Consideration of the relationships between kinetic constants and electrophoretic mobilities of the isoenzymes, leads us to predict that isoenzyme-5 is well adapted for a role in the oxidation of lactate or glyoxylate. The lactate dehydrogenase of potato leaves appears to consist prodominantly of an isoenzyme with the same mobility as isoenzyme-2 of the tubers and the two isoenzymes are probably identical. The kinetic properties of this isoenzyme are consistent with roles in either oxidation or reduction.Abbreviation Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Molecular dynamics simulation of lactate dehydrogenase adsorption onto pristine and carboxylic-functionalized graphene

ABSTRACT

Lactate dehydrogenase (LDH) is a tetrameric enzyme which is composed of two subunits known as LDHA and LDHB, which are encoded by the LDHA and LDHB genes respectively. LDH catalyses the last step in anaerobic glycolysis through the reversible conversion of pyruvate to lactate via coupled oxidation of NADH cofactor. The LDHA plays an important regulatory role in anaerobic glycolysis, by catalysing the final step of the process. Therefore, it is likely that increases in the expression level of LDHA in cancer cells could facilitate the efficiency of anaerobic glycolysis. Measuring the level of serum LDHA is a key step in the diagnosis of many cancer types. In this study, the adsorption, stability, and dynamics of LDHA on the surface of pristine graphene (PG) and carboxylated graphene (COOH-Graphene) were investigated using its molecular dynamics simulation. Variations in root mean square deviation, root mean square fluctuation, solvent accessible surface area and adsorption energy of the LDHA during the simulation were calculated to analyse the effect of PG and COOH-Graphene on the overall conformation of LDHA. Results showed that the adsorption of LDHA on COOH-Graphene is mostly mediated by electrostatic interactions, whereas on the PG, both Van der Waals and π-π interactions are prominent.     

Interaction of bovine skeletal muscle lactate dehydrogenase with liposomes. Comparison with the data for the heart enzyme

The effects of pH, salt concentration and the presence of oxidized and reduced forms of coenzyme on the interaction of skeletal muscle lactate dehydrogenase with the liposomes derived from the total fraction of bovine erythrocyte lipids were investigated by ultracentrifugation and were compared with those results obtained using the heart-rate isoenzyme which we have previously studied. Liposomes are good adsorptive systems for both types of isoenzyme. In the presence of erythrocyte lipid liposomes, bovine muscle and heart lactate dehydrogenases form two kinds of complex: lactate dehydrogenase adsorbed to liposomes and soluble lactate dehydrogenase-phospholipid complexes. Soluble protein-phospholipid complexes reveal different dependences of their stabilities on pH values and it seems that the nature of the binding site in either isozyme is different. In addition, absorption of the isoenzymes on the liposomes also reveals in difference in the effects of NAD and NADH. While the presence of NAD dissociates LDH-H₄ from the liposomes and NADH does not influence its adsorption, NAD promotes the binding of LDH-M₄, and NADH favors the dissociation.     

A study of lactate dehydrogenase levels and turnover rates during postnatal development in the rat

Specific radioimmunoassays for lactate dehydrogenase A and B subunits have been employed to quantify cellular contents of these proteins more precisely than hitherto possible and to monitor changes during postnatal development. Liver, skeletal muscle, heart muscle and kidney cortex all demonstrated alterations in cellular levels of lactate dehydrogenase subunits over the first 56 days of life, the particular pattern being specific to each tissue. Studies on the turnover of lactate dehydrogenase in vivo and in vitro indicated that the developmental changes in total lactate dehydrogenase content in liver and kidney were regulated at some point(s) during both the biosynthesis and the degradation of the proteins.     

An unusual fructose 1,6-bisphosphate-activated lactate dehydrogenase from the ascidian Pyura stolonifera

Lactate dehydrogenase (LDH) was purified from the siphon muscle of the intertidal ascidian Pyura stolonifera. The enzyme is unique among chordate LDHs but resembles some bacterial and platyhelminth LDHs in being activated by fructose 1,6-bisphosphate (FBP). Concentrations of FBP in the range 5μM to 0.5 mM increase V_max of the pyruvate reductase reaction by 130% to 210%, and decrease K_m pyruvate 5 to 11 fold and K_m NADH 2.5 to 5 fold. The enzyme is also activated by inorganic phosphate, but requires a 50 fold higher concentration to attain the maximum activation achieved by 0.5 mM FBP. Of a range of metabolites tested, including other glycolytic sugar phosphates, only FBP and inorganic phosphate activated the enzyme. FBP activation was not observed with 16 representative vertebrate LDH homotetramers, but did occur to a limited extent with LDH from an echinoderm. LDH was the only pyruvate reductase enzyme detected in P. stolonifera siphon muscle, and its activity was much greater than that of phosphorylase or phosphofructokinase. The LDH reaction is utilized by P. Stolonifera during prolonged siphon closure on exposure to air when lactate, but not succinate, accumulates in the siphon muscle. While the ascidian enzyme provides the first example of a FBP activated LDH from a chordate, it remains to be determined if this unusual property has any role in metabolic regulation.     

Semiempirical calculations of the oxygen equilibrium isotope effect on binding of oxamate to lactate dehydrogenase

Semiempirical methods have been used in an attempt to predict theoretically the experimentally observed value of 0.9840 for the oxygen isotope effect on binding of oxamate to lactate dehydrogenase. The overall strategy involved vibrational analysis of oxamate in two different environments; that of the active site residues and in aqueous solution. The comparison of calculated values with the experimentally determined isotope effect proved the AM1 Hamiltonian to be superior to the PM3 Hamiltonian in this modelling. While most tested methods of accounting for solvent effects on the vibrational frequencies of the solute yielded similar results it turned out that what was crucial for the purpose of determination of the isotope effect was the model of oxamate in the active site of the enzyme. In particular, the major factor responsible for the inverse value of this isotope effect can be ascribed to the formation of an ordered, bifurcated hydrogen bond between the oxamate carboxylate and the guanidinium group of the active site histidine. Correspondence to: P. Paneth     

Morpholino-mediated in vivo silencing of Cryptosporidium parvum lactate dehydrogenase decreases oocyst shedding and infectivity

Cryptosporidium is a highly prevalent protozoan parasite that is the second leading cause of childhood morbidity and mortality due to diarrhoea in developing countries, and causes a serious diarrheal syndrome in calves, lambs and goat kids worldwide. Development of fully effective drugs against Cryptosporidium has mainly been hindered by the lack of genetic tools for functional characterization and validation of potential molecular drug targets in the parasite. Herein, we report the development of a morpholino-based in vivo approach for Cryptosporidium parvum gene knockdown to facilitate determination of the physiological roles of the parasite’s genes in a murine model. We show that, when administered intraperitoneally at non-toxic doses, morpholinos targeting C. parvum lactate dehydrogenase (CpLDH) and sporozoite 60K protein (Cp15/60) were able to specifically and sustainably down-regulate the expression of CpLDH and Cp15/60 proteins, respectively, in C. parvum-infected interferon-γ knockout mice. Over a period of 6?days of daily administration of target morpholinos, CpLDH and Cp15/60 proteins were down-regulated by 20- to 50-fold, and 10- to 20-fold, respectively. Knockdown of CpLDH resulted in approximately 80% reduction in oocyst load in the feces of mice, and approximately 70% decrease in infectivity of the sporozoites excysted from the shed oocysts. Cp15/60 knockdown did not affect oocyst shedding nor infectivity but, nevertheless, provided a proof-of-principle for the resilience of the morpholino-mediated C. parvum gene knockdown system in vivo. Together, our findings provide a genetic tool for deciphering the physiological roles of C. parvum genes in vivo, and validate CpLDH as an essential gene for the growth and viability of C. parvum in vivo.     

The influence of polystyrene nanoparticles on the fractal kinetics of lactate dehydrogenase

Plastics are ubiquitous in the aquatic environment and their degradation of fragments down to the nanoscale level have raised concerns given their ability to pervade cells. The accumulation of nanoparticles could lead to molecular crowding which can alter the normal functioning of enzymes. The purpose of this study was to examine the influence of polystyrene nanoparticles (NPs) on the fractal kinetics of the lactate dehydrogenase reaction: pyruvate + NADH ? lactate + NAD⁺. The influence of NPs on LDH activity was examined first in vitro to highlight specific effects and secondly in mussels exposed to NPs in vivo for 24h at 15 °C. The reaction rates of LDH were determined with increasing concentrations of pyruvate to reach saturation at circa 1 mM pyruvate. The addition of F-actin, a known binding template for LDH, revealed a characteristic change in reaction rates associated with fractal organization. The addition of 50 and 100 nm transparent NPs also produced these changes. The fractal dimension was determined and revealed that both F-actin and NPs reduced the fractal dimension of the LDH reaction. The addition of viscosity sensor probe in the reaction media revealed viscosity waves during the reaction at low substrate concentrations thought to be associated to synchronized switching between the relaxed and tensed states of LDH. The amplitude and the frequency of viscosity waves were increased by both NPs and F-actin which were associated with increased reaction rates. In mussels exposed to NPs, the isolation of digestive gland subcellular fraction revealed that LDH activity was significantly influenced by the fractal dimension of the LDH reaction where a loss of affinity (high fractal K_M) was detected in mussels exposed to the high concentrations of NPs. It is concluded that polystyrene NPs could change the biophysical properties of the cytoplasm such as the fractal organization of the intracellular environment during the LDH reaction.