Characterization Of Lactate Dehydrogenase Research Articles

Cloning, Expression, Purification, and Characterization of Lactate Dehydrogenase from Plasmodium knowlesi: A Zoonotic Malaria Parasite.

Plasmodium knowlesi is the only Plasmodium that causes zoonotic disease among the Plasmodium that cause infection in humans. It is fatal due to its short asexual growth cycle within 24 h. Lactate dehydrogenase (LDH), an enzyme that catalyzes the final step of glycolysis, is a biomarker for diagnosing infection by Plasmodium spp. parasite. Therefore, this study aimed to efficiently produce the soluble form of P. knowlesi LDH (PkLDH) using a bacterial expression system for studying malaria caused by P. knowlesi. Recombinant pET-21a(+)-PkLDH plasmid was constructed by inserting the PkLDH gene into a pET-21a(+) expression vector. Subsequently, the recombinant plasmid was inserted into the protein-expressing Escherichia coli Rosetta(DE3) strain, and the optimal conditions for overexpression of the PkLDH protein were established using this strain. We obtained a yield of 52.0 mg/L PkLDH from the Rosetta(DE3) strain and confirmed an activity of 483.9 U/mg through experiments. This methodology for high-efficiency PkLDH production can be utilized for the development of diagnostic methods and drug candidates for distinguishing malaria caused by P. knowlesi.

Purification and Characterization of Lactate Dehydrogenase in the Foot Muscle and Hepatopancreas of Otala lactea.

Lactate dehydrogenase (LDH) has a crucial role in maintaining ATP production as the terminal enzyme in anaerobic glycolysis. This study will determine the effect of posttranslational modifications (PTMs) on the activity of LDH in the foot muscle and hepatopancreas of an estivating snail, Otala lactea. LDH in foot muscle of O. lactea was purified to homogeneity and partially purified in hepatopancreas in a two-step and three-step process, respectively. The kinetic properties and stability of these isoforms were determined where there was a significant difference in Km and I50 values with pyruvate and urea separately in foot muscle; however, hepatopancreas exhibited significant differences in Km and I50 in salt between control and stress. Interestingly, hepatopancreas has a higher affinity for pyruvate in the control state whereas foot muscle has a higher affinity for its substrate in the estivated state. PTMs of each isoform were identified using immunoblotting and dot blots, which prove to be significantly higher in the control state. Overall, foot muscle LDH enters a low phosphorylation state during estivation allowing more efficiency in consuming pyruvate with higher thermal stability but less structural stability. Hepatopancreas LDH becomes dephosphorylated in the estivating snail that decreases the efficiency of the enzyme in the forward direction; however, the snail has an increased tolerance to the presence of salt when water becomes scarce. Such tissue-specific regulations indicate the organism's ability to reduce energy consumption when undergoing metabolic depression.

Cloning, E. coli Expression, and Characterization of Heart Lactate Dehydrogenase B From River Buffalo (Bubalus bubalis)

Lactate dehydrogenase is an enzyme of glycolytic pathway which catalyzes the interconversion of pyruvate and lactate. The present study describes cDNA cloning, E. coli expression and characterization of lactate dehydrogenase B (LDH-B) from the heart ventricles of river buffalo (Bubalus bubalis). Total RNA was isolated from the heart tissue, a 1005bp cDNA encoding complete polypeptide chain of 334 amino acids was generated by reverse transcriptase reaction and analyzed for nucleotide sequence. The consensus sequence obtained from both strands has shown 84% to 98% homology with that of different mammalian species. The attributed gene was cloned, expressed in BL21 (DE3) RIPL Codon Plus strain of E. coli using pET21a (+) plasmid. The purified recombinant enzyme displayed a KM value of 50 µM for pyruvate, an optimum activity at 35°C and pH 7.0. The enzyme was found as a homotetramer of 140 kDa on FPLC based gel-filtration column. Molecular weight of a subunit of enzyme as determined by mass spectrometric analysis was 36530.21 Da. The present study describes the first ever report about the cDNA sequence and characteristics of recombinant LDH-B from River buffalo.

Purification and Characterization of Lactate Dehydrogenase (LDH) from the Leavening Bacterium Enterobacter cloacae GAO

Purification and Characterization of Lactate Dehydrogenase (LDH) from the Leavening Bacterium Enterobacter cloacae GAO

Expression and characterization of lactate dehydrogenase from Schistosoma japonicum

Some effective antiparasitic drugs for chemotherapy schistosomiasis, such as praziquantel and artemether, are proved to act on lactate dehydrogenase (LDH) in vitro, but detailed molecular action mechanisms of these have not yet been elucidated. To this end, the sequence encoding LDH of Schistosoma japonicum (SjLDH) was amplified from the cDNA library of the adult parasite. Sequence analysis revealed an open reading frame (ORF) of 999 bp, encoding 332 amino acids with a molecular weight of 36.120 kDa. It has three transmembrane regions and may be located in the cytoplasm membrane. The encoding ORF was inserted into pET-28a vector and expressed in Escherichia coli (E. coli) induced by IPTG. A 36-kDa protein with 6xHis-tag was purified by metal affinity column and identified by SDS-PAGE and Western blot, which had high LDH activity of 379 U/mg. Characterization such as Michaelis constant (K (m)) and maximum velocity (V (max)), optimum pH, and temperature of the protein were assayed. It provides a model for drug screening on SjLDH.

Quality, Quantity and Function on the Web

Nathalie Soutthiphong (Imperial College School of Medicine, London, UK) asked how much Pf155/RESA [ring erythrocyte surface antigen] is in a ring stage P. falciparum (in pmole or μg)? Ross Coppel (Monash University, Australia) said little was known about the absolute amounts of any parasite protein, even LDH [lactate dehydrogenase] and the heat-shock protein hsp70, which he believed to be the most abundant. He also thought it extremely difficult to quantitate RESA using current methods of detection as they employ `antisera directed against repetitive epitopes, giving a great amplification of signal. Thus there appears to be much more RESA than is there ... Further, RESA is poorly stained by Coomassie relative to other proteins because of its highly charged nature.' David Bzik (Dartmouth College, NH, USA) agreed about `absolute' figures but said various enzymes, including LDH, can be quantitated in terms of specific activity (D.L. Vander Jagt et al., Partial purification and characterization of lactate dehydrogenase from Plasmodium falciparum. Mol. Biochem. Parasitol. 4, 255–264, 1981). But `regarding the `most abundant malaria protein`, I have to vote for the serine repeat antigen (SERA) ... In SDS-PAGE, SERA appears to be more abundant than LDH ... in cDNA libraries, SERA message (∼2% of cDNA!) is more abundant than LDH (∼0.1%).' However, Mark Wiser (Tulane University, New Orleans, USA) wondered if that was true of P. falciparum specifically, as it appeared not to apply to P. vinckei, P. berghei or P. chabaudi. He had no comparisons with LDH or hsp70, but there seemed to be more MSP-1 [merozoite surface protein 1] than SERA homologues. As to function, Leann Tilley (La Trobe University, Victoria, Australia) said RESA protected spectrin from heat-induced denaturation (E. Da Silva et al., The Plasmodium falciparum protein RESA interacts with the erythrocyte cytoskeleton and modifies erythrocyte thermal stability. Mol. Biochem. Parasitol. 66, 59–69, 1994). She thought its role in protein trafficking had not been studied but `it is the only parasite protein known to be at the RBC membrane in the ring-stage of infection and it disappears later in the life cycle when the bulk of trafficking occurs.' Mark Wiser reacted: `highly antigenic acidic phosphoproteins are associated with the red cell membrane at the ring stage of P. chabaudi, unlike RESA, they remain associated with the membrane throughout the blood stage cycle, and analogous proteins seem to exist in P. falciparum' (M.F. Wiser et al., Plasmodium chabaudi: immunogenicity of a highly antigenic glutamate-rich protein. Exp. Parasitol. 85, 43–54, 1997). RESA is synthesized during the schizont stage and is localized to dense granules of merozoites, especially to finger-like projections of the parasitophorous vacuolar membrane (M. Aikawa and C.T. Atkinson, Immunoelectron-microscopy of parasites. Adv. Parasitol. 29, 151–214, 1990; J.G. Culvenor et al., Plasmodium falciparum ring-infected erythrocyte surface antigen is released from merozoite dense granules after erythrocyte invasion. Infect. Immun. 59, 1183–1187, 1991). `Although the bulk of trafficking might occur later in schizogony, there is substantial trafficking during the early ring stage and throughout the blood stage cycle' (M.F. Wiser et al., A novel alternate secretory pathway for the export of Plasmodium proteins into the host erythrocyte. Proc. Natl. Acad. Sci. U. S. A. 94, 9108–9113, 1997). Leann Tilley apologized for forgetting rodent parasites [often done!] but wondered if `protein trafficking to the RBC membrane in say P. chabaudi occurs by the same mechanism as in P. falciparum?' Mark Wiser accepted the apology; his bias is that all `the Plasmodium species will export proteins into the host erythrocyte by the same mechanism.' Finally Jerry McLaughlin (University of Indiana, USA) recommended caution about conclusions from partial-length homologies: `a prediction of function ... may not follow even from a real homology ... Are there hints by location on chromosome(s) as to function, eg. is RESA near virulence genes, etc.?'

Purification and characterization of lactate dehydrogenase from Varanus liver

Abstract Lactate dehydrogenase was purified 21-fold fromliver of Varanus bengalensis using colchicine-sepharose column chromatography. The crudeenzyme showed two isoenzymes (LDH-5 and LDH- 4 )by agarose gel electrophoresis (AGE). The p u r i f i e denzyme showed a single band after SDS-PAGEcorresponding to molecular mass of 35 kDa. Themolecular mass of native enzyme was about 140kDa. The optimum pH for the forward reaction was7.5 while that for the reverse reaction was pH 9.5.The K m values for pyruvate, NADH, lactate andNAD + were 0.17 ± 0.037, 0.02 ± 0.004, 12.4 ± 3.05 and0.38 ± 0.032 mM, respectively. Pre-heating ofenzyme showed that its t 5 0 was 40-50˚C. Oxalateand n-hexanediol were inhibitors for both forwardand reverse reactions. Among divalent ions, Cu + + was shown to be more effective inhibitor for theforward reaction.K e y w o r d s : LDH isoenzyme, liver, properties, purification, Varanus bengalensis Introduction Lactate dehydrogenase ( L -lactate:NAD oxidoreductase,EC 1.1.1.27; LDH) plays an important role in the regu-lation of anaerobic glycolysis through reoxidation ofNADH (Everse and Kaplan, 1973; Javed

Immobilization and Characterization of Lactate Dehydrogenase on TEOS Derived Sol-Gel Films

Physical adsorption and physical entrapment techniques have been utilized for the immobilization of lactate dehydrogenase (LDH) on tetraethylorthosilicate (TEOS) derived sol-gel films. The enzyme (LDH) activity has been assayed as a function of time, temperature, pH and pyruvate concentration. The results of photometric measurements used for monitoring the reaction yield a response time of about 1 min, linearity over a concentration range of 0–1.5 × 10-3 M and detection limit of 5 × 10-5 M. The TEOS sol-gel films containing LDH have been found to be stable for about 30 days at temperatures 4 to 10°C.

Age-dependent variations of lactate dehydrogenase and creatine kinase activities in water buffalo calf serum.

The electrophoretic patterns of the serum enzymes lactate dehydrogenase and creatine kinase from water buffalo calves are described. Differences in total activities as well as their relative distribution were seen at ages ranging from 1 to 10 weeks. While total lactate dehydrogenase activity increased by over 100%, total creatine kinase increased by almost 400%. The relative activities of lactate dehydrogenase 1 and 5 decreased with age. Lactate dehydrogenase 2 and 3 increased and lactate dehydrogenase 4 did not change. In relation to creatine kinase, the prevalent isoenzyme was creatine kinase-MM, but it's relative activity gradually decreased in comparison to the other two isoenzymes (creatine kinase-MB and creatine kinase-BB). Creatine kinase-BB was completely absent until the 3rd week of age. The percentage modifications of creatine kinase isoenzymes were correlated to age. The results suggest that isoenzymatic separation and characterization of lactate dehydrogenase and creatine kinase in relation to the various tissues can significantly contribute to the diagnosis of diseases which are linked to tissue damage.

Purification and characterization of lactate dehydrogenase from Geotrichum candidum

An appreciable quantity of lactate dehydrogenase (EC 1.1.2.3) was present in the crude extract of Geotrichum candidum grown previously in sauerkraut processing waste effluents. The enzyme catalyses the oxidation of lactate to pyruvate with potassium ferricyanide and 2,6-dichlorophenol indophenol as electron acceptors. The enzyme was purified 64-fold by ammonium sulfate fractionation, gel filtration, and ion exchange chromatography with a yield of more than 5%. The specific activity of the purified enzyme was more than 6 units per mg protein. pH and temperature optima were 7·5 and 30°C, respectively. The enzyme was stable at pH values between 7·0 and 8·0, but lost most of its activity at 40°C in 10 min. The enzyme was specific for l(+)-lactate and its K m value with potassium ferricyanide as an electron acceptor was 0·71 mm. The enzyme had a molecular weight of > 200 000 as determined by gel filtration, and its pl was estimated to be 7·5. The enzyme activity was inhibited by heavy metals, and EDTA could be used to prevent heavy metal inhibition.

The partial purification and characterization of lactate dehydrogenase

This work highlights the effect of drug solubility and polarity and solid layer stacking on the release rate and mechanism of layered double hydroxides-drug (LDH-D) hybrids. With such a purpose, LDH-D hybrids containing three structural related non-steroidal anti-inflammatory drugs (ibuprofen, naproxen or ketoprofen) were synthesized by a simple co-precipitation method. LDH matrixes exhibited a high drug loading capacity, even exceeding the anion exchange capacity of the solid especially with the more apolar drugs. The structure and interfacial properties of the particulate LDH-D hybrids were also dependent on the polarity of the loaded drug. Finally, the release mechanisms in neutral and acidic media were studied with compressed LDH-D hybrids. The hybrids compression leaded to highly stacked platelets that caused a slower and steadier drug release rate than particulate LDH-D hybrids in all cases. In neutral medium, the drugs were exclusively released by anion exchange with HPO42− ions and the release rate was determined by the drug polarity. In acidic medium, weathering was the main release mechanism. However, additional processes (anion exchange, drug solubilization) were concurrent in the latter media, the overall mechanism and release rate being dependent on the drug solubility.

Characterization of lactate dehydrogenase, malate dehydrogenase, pyruvate kinase, and phosphoenolpyruvate carboxykinase in Romanomermis culicivorax postparasitic larvae and adults

1. 1. Romanomermis culicivorax postparasites 48 hr after emergence had high malate dehydrogenase (MDH) and phosphoenolpyruvate carboxykinase (PEPCK) but relatively low pyruvate kinase (PK) and very little lactate dehydrogenase (LDH) activities. 2. 2. The pH optima, coenzyme, cofactor, and ionic requirements of the enzymes were similar to those of other helminths. 3. 3. MDH had a tenfold greater affinity for oxaloacetate (OAA) than malate and was 9 times more active for OAA reduction than malate oxidation. 4. 4. PK and PEPCK did not differ in their apparent K m s for phosphoenolpyruvate ( 0.9 × 10 −3 M and 1.1 × 10- 3 M , respectively), but PEPCK had a greater affinity for IDP ( K m = 1.2 × 10 −4 M ) than PK for ADP ( K m = 5.8 × 10 −4 M . 5. 5. The low activities of LDH and PK but high activities of MDH and PEPCK suggested that anaerobic carbohydrate catabolism followed the fumarate reductase pathway. 6. 6. PEPCK had a lower pH optimum (5.25) than PK (7.25) suggesting that pH may be an important factor in regulating the PK/PEPCK branchpoint.

Partial purification and characterization of lactate dehydrogenase from Plasmodium falciparum

Lactate dehydrogenase (LDH) from Plasmodium falciparum was partially purified by two different procedures. In the first procedure, parasitized erythrocytes (80% parasitemia) were lysed, and the soluble fraction was purified on DEAE-Sephadex to separate the parasite LDH (LDH-P) from the LDH isozymes present in the human erythrocytes. LDH-P was then purified by high-performance liquid chromatography (HPLC) on a TSK-G-3000 SW protein column. This two-step procedure gave LDH-P with specific activity 85 μmol/min/mg protein; this represented a 700-fold increase in specific activity relative to the starting lysate. Alternatively, parasites of P. falciparum were isolated by mechanical rupture of infected erythrocytes followed by differential centrifugation. The 100 000 × g supernatant obtained after lysis of these parasites showed LDH-P specific activity 3.6 μmol/min/mg protein. This activity was free of contaminating erythrocyte LDH as determined by electrophoresis and specific staining for LDH. Further purification of LDH-P by HPLC, as before, gave material with specific activity 98 μmol/min/mg protein. Recoveries of activity on HPLC were 90%, demonstrating the usefulness of this procedure for the partial purification of small quantities of parasite protein. The kinetic properties of LDH-P were compared with those of two of the human isozymes, LDH-H 4 and LDH-M 4. LDH-P resembles LDH-H 4 in its kinetic properties: K M (NADH) is 7, 8.3 and 1.3 μM for LDH-P, LDH-H 4 and LDH-M 4, respectively; K M (pyruvate) is 30, 60 and 180 μM for LDH-P, LDH-H 4 and LDH-M 4. LDH-P differs significantly from LDH-H 4 and LDH-M 4 in that LDH-P is not sensitive to inhibition by high pyruvate nor sensitive to inhibition by the complex between NAD + and pyruvate. LDH-P is inactivated within seconds by sodium deoxycholate at concentrations that do not affect LDH-H 4 and only slowly inactivate LDH-M 4.

Enzymes of coccidid: Purification and properties of l-lactate dehydrogenase from Eimeria stiedae

The initial extensive purification and characterization of lactate dehydrogenase (EC 1.1.1.27) from sporozoa was accomplished. Fractional precipitation with ammonium sulfate, followed by chromatography on DEAE-Sephadex ® yielded a 500–1000-fold purification of the enzyme from unsporulated Eimeria stiedae oocysts. One isoenzyme was demonstrated by electrophoresis in polyacrylamide gel. The subunits of this isoenzyme were dissociated and recombined to form another active isoenzyme. The rate of conversion of pyruvate to lactate by this enzyme was strongly influenced by specific anions. In Γ/2 0.1 glycine-sodium glycinate buffer at 40 C, the K m for lactate was 3.03 × 10 −2 M and the K m for NAD was 3.34 × 10 −4 M. The enzyme accepted NAD and certain of its analogs as coenzymes. The 3-acetylpyridine analog of NADP served as coenzyme, whereas NADP did not. The enzyme was peculiar because it accepted α-NAD as coenzyme. A pyruvate-NAD complex apparently did not compete with NADH for the same binding site on the enzyme molecule. The rate of conversion of pyruvate to lactate was maximal at 40 C. The effect of hydrogen ion concentration on the rate of interconversion of pyruvate and lactate varied according to the buffer system in which the reaction occurred.

A comparative physico-chemical characterization of lactate dehydrogenase: Isozymes in Brook trout, Lake trout and their hybrid Splake trout.

AbstractPeptide maps and amino acid composition of LDH crystallized from skeletal muscle extracts of brook trout (Salvelinus fontinalis) and lake trout (S. namaycush) are similar to those of the hybrid splake trout. The molecular weights are identical to mammalian LDH on the basis of sucrose density gradient centrifugation. The five A′–A isozymes are restricted to LDH from trout muscle and migrate very slowly toward the anode. The B′–B subunit series of isozymes are ubiquitous in trout tissues, although cell specific proportions of isozymes occur. While the A′–A subunit genes must be present in all trout cells, they are active only in the muscle where their products are responsible for essentially all of the LDH activity in this tissue.Isozyme patterns reveal that B subunit‐containing tetramers (less anodal) predominate in LDH from liver, whereas B′ subunit molecules (more anodal) prevail in LDH from trout heart. Therefore, extracts from heart, muscle, and liver tissues were utilized to characterize and compare the duplicated subunit tetramers of LDH in terms of several physico‐chemical and physiological parameters.Substrate kinetics show that trout B′ subunit tetramers are very sensitive to increases in pyruvate and lactate. These isozymes attain maximum initial velocity at about 0.8 μmole of pyruvate, while substrate inhibition begins at 7 μmoles. The B and the A′–A tetramers reach maximum initial velocity at 1.3 and 3.5 μmoles of pyruvate, respectively, and inhibition does not begin until substrate concentrations of about 20 μmoles are reached. Results are similar with lactate as substrate. Only the B′ tetramers exhibit substrate inhibition with lactate.An analysis of competitive pyruvate and oxamate inhibition reveals that the LDH active site(s) binds one molecule of inhibitor (or substrate).The inactivating temperature for trout B′ subunit tetramers is 70–75°C; for B subunit tetramers, 65–70°C; and for A′–A tetramers, 46–54°. The A′–A and the B subunit‐containing tetramers possess the generally lower thermal stability characteristic associated with LDH‐5 from most other animals, but thermolability is much more pronounced in the A′–A tetramers. Kinetics of the thermal inactivation process show that it is a first‐order, unimolecular event consistent with protein denaturation.Homologies of trout LDH isozymes with those of higher vertebrates are discussed in relation to these data.

Kinetic characterization of lactate dehydrogenase of normal and virus-infected chorioallantoic membrane of the chick embryo

Kinetic characterization of lactate dehydrogenase of normal and virus-infected chorioallantoic membrane of the chick embryo