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1

Catalytic reaction mechanism of L-lactate dehydrogenase: an ab initio study  

Microsoft Academic Search

Studies on the catalytic reaction mechanism of L-lactate dehydrogenase have been carried out by using quantum chemical ab\\u000a initio calculation at HF\\/6-31G* level. It is found that the interconversion reaction of pyruvate to L-lactate is dominated\\u000a by the hydride ion Hr transfer, and the transfers of the hydride ionH\\u000a r and protonH\\u000a r are a quasi-coupled process, in which the

Ruobing Hou; Zhida Chen; Xianghui Yi; Jiang Bian; Guangxian Xu

2000-01-01

2

Acrosome reaction in bovine spermatozoa: role of reactive oxygen species and lactate dehydrogenase C4.  

PubMed

After capacitation, mammalian spermatozoa accomplish the acrosome reaction (AR), a well-controlled exocytosis process crucial to fertilize mature oocytes that involves several protein kinases such as protein kinase A (PKA), C (PKC), and tyrosine kinase (PTK). Reactive oxygen species (ROS) are involved in both bovine sperm capacitation and AR. Lactate dehydrogenase C4 (LDH-C4) was associated with bovine and mouse sperm capacitation. Our aims were to study the participation of LDH-C4 to contribute with the status redox required for AR and the role of ROS in the regulation of PKA, PKC, and PTK involved in the exocytotic event. Sodium oxamate, an inhibitor of LDH-C4, prevented the AR induced by lysophosphatidylcholine (LPC) or NADH. Hydrogen peroxide promoted and superoxide dismutase (scavenger of superoxide), catalase (scavenger of hydrogen peroxide), diphenyleneiodinum, diphenyliodonium, cibacron blue, and lapachol (inhibitors of NADPH oxidase) prevented the AR, suggesting that ROS and a sperm oxidase are involved in the AR induced by these compounds. Inhibitors of PKA, PKC, and PTK also prevented the AR induced by LPC or NADH, suggesting the involvement of these kinases in the process. These results suggest that LDH-C4 may participate in the regulation of the redox status required to achieve the AR in bovine spermatozoa and that ROS are key elements in the regulation of protein kinases associated with the AR process. PMID:16112812

O'Flaherty, C; Breininger, E; Beorlegui, N; Beconi, M T

2005-10-30

3

The lactate dehydrogenase catalyzed pyruvate adduct reaction: simultaneous general acid-base catalysis involving an enzyme and an external catalyst.  

PubMed

The pH dependence of the reaction catalyzed by lactate dehydrogenase, where pyruvate adds covalently to NAD to yield a NAD-Pyr adduct, together with published data on the pH dependence of parameters in the normal redox reaction suggests similar binding modes for enolpyruvate and lactate in their complexes with E X NAD (where E is one-fourth of the tetramer), for ketopyruvate in its complexes with the protonated species, E X H X NAD and E X H X NADH, and for the NAD--Pyr adduct and NADH plus pyruvate in their complexes with E X H. These similarities, together with previous data, suggest a reaction scheme for the formation of the enzyme-adduct complex that includes the relevant proton-transfer steps. Seven different amine chloride buffers were used in a study of the reverse adduct reaction, i.e., the decomposition of E X H X NAD--Pyr. These act with varying efficiencies as external general acid catalysts; the enzyme apparently acts as a (internal) general base. The involvement of the amine chloride buffers as external general catalysts is supported by the concentration dependence of the buffer effect, by a Brönsted plot, and by solvent deuterium isotope effects. The involvement of the enzyme as an internal general catalyst is inferred from the pH dependence of the reaction and the identities of the nearby groups in the E X H X NAD--Pyr complex (from crystallographic studies). The dependence of the adduct reaction on chloride concentration indicates the presence of dead-end inhibitor complexes of E X H X Cl and E X H X NAD X Cl. Chloride also accelerates the decomposition of the adduct in the complex E X H X NAD--Pyr by binding to this complex. PMID:6477888

Burgner, J W; Ray, W J

1984-07-31

4

Genetics Home Reference: Lactate dehydrogenase deficiency  

MedlinePLUS

... dehydrogenase-B pieces (subunits) of the lactate dehydrogenase enzyme. This enzyme is found throughout the body and is important ... cells. There are five different forms of this enzyme, each made up of four protein subunits. Various ...

5

Quantitative Electrophoretic Determination of Lactate Dehydrogenase Isoenzymes.  

National Technical Information Service (NTIS)

Lactate dehydrogenase isoenzymes of human serum and tissue extracts were separated by agar gel electrophoresis on a microscope slide. The isoenzyme activity was identified by a procedure utilizing p-iodonitrotetrazolium and quantitated by densitometry. Fi...

N. M. Papadopoulos J. A. Kintzios

1966-01-01

6

Convergent Evolution of Trichomonas vaginalis Lactate Dehydrogenase from Malate Dehydrogenase  

Microsoft Academic Search

Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species. The derived amino acid sequence of T. vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two T. vaginalis

Gang Wu; Andras Foser; Benno Ter Kuile; Andrej Sali; Miklos Muller

1999-01-01

7

Structural adaptations of lactate dehydrogenase isozymes.  

PubMed Central

The three-dimensional structures of dogfish M4 (muscle) and pig H4 (heart) lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) have been determined and correlated with the amino acid sequences of the dogfish M4, pig M4, pig H4, chicken M4, and chicken H4 lactate dehydrogenase isozymes. These results have been related to the known differences of physicochemical properties between the M and H lactate dehydrogenase isozymes. By far the largest structural alterations occur in the transition between the "apo" and "ternary complex" conformational states of the enzyme rather than between species or isozymes. The major catalytic difference can be explained by a replacement of alanine (in the M chain) with a glutamine (in the H chain) in the vicinity of the binding site of the coenzyme phosphates. The known immunological differentiation of the M and H isozymes is consistent with the differences in their amino acid sequences.

Eventoff, W; Rossmann, M G; Taylor, S S; Torff, H J; Meyer, H; Keil, W; Kiltz, H H

1977-01-01

8

Serum Lactate Dehydrogenase Levels as a Predictive Marker of Oxaliplatin-Induced Hypersensitivity Reactions in Japanese Patients with Advanced Colorectal Cancer  

PubMed Central

Objective: Clinical laboratory test data obtained prior to treatments were previously analyzed from the standpoint of susceptibility to hypersensitivity reactions in patients treated with the platimun anticancer agent, oxaliplatin (L-OHP). In the present study, the time course from the first to last cycle of the treatment was additionally analyzed to determine a better predictor of these reactions. Methods: A total of 20 laboratory test data were obtained from 108 Japanese patients with advanced colorectal cancer who were treated with the L-OHP-containing regimens, FOLFOX4 and/or mFOLFOX6. The averages and variation coefficients (CV%) of the data until the last cycle of the treatment were compared between patients with hypersensitivity reactions and those without. Results: The average serum lactate dehydrogenase (LDH) level was lower in patients with grade 1/2 reactions (P=0.016), whereas its CV% value was higher in patients with grade 3/4 reactions (P=0.005) than in those without reactions. An increase in serum LDH levels was observed in some patients with grade 3/4 reactions as the cycle number increased, and thereafter hypersensitivity reactions occurred. This phenomenon was not always observed, but was never detected in patients with grade 1/2 reactions. Conclusions: Serum LDH levels may be a predictive marker of hypersensitivity reactions in patients treated with L-OHP. Further extensive examinations with a larger number of patients are needed to establish a patient management strategy.

Seki, Kyoko; Tsuduki, Yasuo; Ioroi, Takeshi; Yamane, Michiko; Yamauchi, Hiroko; Shiraishi, Yukinari; Ogawa, Tadaaki; Nakata, Izumi; Nishiguchi, Kohshi; Matsubayashi, Teruhisa; Takakubo, Yoshihide; Yamamori, Motohiro; Kuwahara, Akiko; Okamura, Noboru; Sakaeda, Toshiyuki

2014-01-01

9

Catecholamine regulation of lactate dehydrogenase in rat brain cell culture  

SciTech Connect

The mechanism of catecholamine induction of the soluble cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27) was studied in the rat glial tumor cell line, C6. Lactate dehydrogenase was partially purified from extracts of (/sup 3/H)leucine-labeled cells by affinity gel chromatography and quantitatively immunoprecipitated with anti-lactate dehydrogenase-5 IgG and with antilactate dehydrogenase-1 IgG. The immunoprecipitates were dissociated and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Using this methodology, the increased enzyme activity of lactate dehydrogenase in norepinephrine-treated C6 cells was observed to be concomitant with the increased synthesis of enzyme molecules. Despite the continued presence of norepinephrine, the specific increase in the rate of synthesis of lactate dehydrogenase was transient. It was first detected at 4 h, was maximum at 9 h, and returned to basal levels by 24 h. The half-life of lactate dehydrogenase enzyme activity was 36 h during the induction and 40 h during deinduction. The half-life for decay of /sup 3/H-labeled lactate dehydrogenase was 41 h. These observations suggest that the increase in lactate dehydrogenase activity in norepinephrine-treated cells does not involve any change in the rate of degradation. Norepinephrine increased the specific rate of synthesis of both lactate dehydrogenase-5 (a tetramer of four M subunits) and lactate dehydrogenase-1 (a tetramer of four H subunits), although to different extents. Since these subunits are coded for by two separate genes on separate chromosomes, it suggests that the regulatory mechanism involves at least two separate sites of action.

Kumar, S.; McGinnis, J.F.; de Vellis, J.

1980-03-25

10

Plant lactate dehydrogenase: NADH kinetics and inhibition by ATP  

Microsoft Academic Search

Lactate dehydrogenase (LDH), isolated from either turnip (Brassica rapa, Cruciferae) or from leek (Allium porrum, Liliaceae) shows normal Michaelis-Menten kinetics with NAD+ in the lactate dehydrogenase direction, but non-hyperbolic kinetics with NADH in the pyruvate reductase direction. These kinetics are not ‘sigmoidal’ and appear consistently as two intersecting straight lines in reciprocal plots, representing a sharp decline in the effectiveness

Padraig O'Carra; Patricia Mulcahy

1997-01-01

11

Structure, Regulation and Evolution of Vertebrate Lactate Dehydrogenase Genes  

Microsoft Academic Search

Steven Shoei-Lung Li (1998) Structure, regulation and evolution of vertebrate lactate dehydrogenase genes. Zoological Studies 37(1): 1-6. In vertebrates, L-lactate dehydrogenase (LDH) isozymes A (muscle) and B (heart) are best suited for pyruvate reduction and lactate oxidation, respectively. In mammals and columbid birds, a 3rd LDH-C isozyme is expressed in testis. In advanced teleost fish a 3rd LDH isozyme is

Steven Shoei-Lung Li

1998-01-01

12

D- and L-lactate dehydrogenases during invertebrate evolution  

Microsoft Academic Search

BACKGROUND: The L-lactate and D-lactate dehydrogenases, which are involved in the reduction of pyruvate to L(-)-lactate and D(+)-lactate, belong to evolutionarily unrelated enzyme families. The genes encoding L-LDH have been used as a model for gene duplication due to the multiple paralogs found in eubacteria, archaebacteria, and eukaryotes. Phylogenetic studies have suggested that several gene duplication events led to the

Melania E Cristescu; David J Innes; Jonathon H Stillman; Teresa J Crease

2008-01-01

13

Transient-kinetic studies of pig muscle lactate dehydrogenase  

PubMed Central

1. The very fast pre-steady-state formation of NADH catalysed by pig M4 lactate dehydrogenase was equivalent to the enzyme-site concentration at pH values greater than 8.0 and to one-half the site concentration at pH6.8. 2. The rate of dissociation of NADH from the enzyme at pH8.0 (450s?1) in the absence of other substrates is faster than the steady-state oxidation of lactate (80s?1). The latter process is therefore controlled by a step before NADH dissociation but subsequent to the hydride transfer. 3. The oxidation of enzyme–NADH by excess of pyruvate was studied as a first-order process at pH9.0. There was no effect of NADD on this reaction and it was concluded that the ternary complex undergoes a rate-limiting change before the hydride-transfer step. 4. Some conclusions about the reactions catalysed by the M4 isoenzyme were drawn from a comparison of these results with those obtained with the H4 isoenzyme and liver alcohol dehydrogenase. ImagesFig. 3.Fig. 4.Fig. 5.

Stinson, R. A.; Gutfreund, H.

1971-01-01

14

Expression cloning of the nox, mdh and ldh genes from Thermus species encoding NADH oxidase, malate dehydrogenase and lactate dehydrogenase  

Microsoft Academic Search

The Thermus thermophilus HB8 mdh and ldh genes and the T. aquaticus EP00276 nox and mdh genes encoding the biotechnologically important enzymes NADH oxidase (EC 1.6.99.3), malate dehydrogenase (EC 1.1.1.37) and lactate dehydrogenase (EC 1.1.1.27) were cloned on the basis of known sequences from related species using the polymerase chain reaction. The nox and mdh genes were directly placed under

Ho-Jin Park; Roland Kreutzer

1994-01-01

15

21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.  

Code of Federal Regulations, 2013 CFR

...biological activity) in serum. Measurements of lactate dehydrogenase isoenzymes are used in the diagnosis and treatment of liver diseases, such as viral hepatitis, and myocardial infarction. (b) Classification. Class...

2013-04-01

16

Purification and substrate kinetics of plant lactate dehydrogenase  

Microsoft Academic Search

Lactate dehydrogenase (LDH) from turnip (Brassica rapa; Cruciferae), purified to electrophoretic homogeneity using affinity chromatography, has a native Mr, of 157 × 1O3 and a subunit Mr, of 38 × 103. The LDH from turnip shows the same relative effectiveness (relative Vmax and Km values) as the mammalian H4 and M4 isoenzymes with pyruvate, lactate and glyoxylate (oxoacetate and dihydroxyacetate)

Patricia Mulcahy; Padraig O'Carra

1997-01-01

17

Genetic Variation and Relative Catalytic Efficiencies: Lactate Dehydrogenase B Allozymes of Fundulus Heteroclitus  

Microsoft Academic Search

In order to evaluate whether functional differences exist between allelic variants of a B type lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) in the teleost fish Fundulus heteroclitus (Linnaeus), the kinetic properties of pyruvate reduction were examined. While the pH dependence and the temperature dependence for maximal catalysis were indistinguishable among the allozymes, reaction velocities at low pyruvate concentrations were

Dennis A. Powers

1979-01-01

18

Properties of lactate dehydrogenase in a psychrophilic marine bacterium.  

PubMed Central

Lactate dehydrogenase (EC 1.1.1.27) from Vibrio marinus MP-1 was purified 15-fold and ammonium activated. The optimum pH for pyruvate reduction was 7.4. Maximum lactate dehydrogenase activity occurred at 10 to 15 degrees C, and none occurred at 40 degrees C. The crude-extract enzyme was stable between 15 and 20 degrees C and lost 50% of its activity after 60 min at 45 degrees C. The partially purified enzyme was stable between 8 and 15 degrees C and lost 50% of its activity after 60 min at 30 degrees C. The thermal stability of lactate dehydrogenase was increased by mercaptoethanol, with 50% remaining activity at 42 degrees C. Images

Mitchell, P; Yen, H C; Mathemeier, P F

1985-01-01

19

Genetic Analysis with Man-Mouse Somatic Cell Hybrids: Linkage between Human Lactate Dehydrogenase B and Peptidase B Genes  

Microsoft Academic Search

Evidence of an active linkage between the human genes that control lactate dehydrogenase B and peptidase B is presented. It is also concluded that there is no link between the genes for lactate dehydrogenase A and lactate dehydrogenase B.

A. Silvana Santachiara; M. Nabholz; V. Miggiano; A. J. Darlington; W. Bodmer

1970-01-01

20

Ontogenic studies of intestinal lactate dehydrogenase isozymes in the hamster.  

PubMed

Our study of the developing small intestine presents a detailed report of the catalytic isozymic and kinetic properties of lactate dehydrogenase. These results allow for a better understanding of the properties of this intestinal enzyme during devlopment as well as relating these properties to the changing metabolic roles of this tissue during development. PMID:7407273

Walden, R; Schiller, C M

1980-01-01

21

Lactate dehydrogenase from the Antarctic eelpout, Lycodichthys dearborni  

Microsoft Academic Search

As part of our studies to examine the molecular basis of cold-adaptation, we have determined the kinetic properties, thermal stability and deduced amino acid sequence of the enzyme lactate dehydrogenase (LDH) from an Antarctic zoarcid fish, Lycodichthys dearborni. Unlike Antarctic notothenioid fish which are endemic to the Southern Ocean, zoarcid fish are cosmopolitan and have a substantially longer evolutionary history

Miriam Sharpe; Christopher Love; Craig Marshall

2001-01-01

22

Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose.  

PubMed

Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40?°C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50?°C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 ?moles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50?°C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates. PMID:22065761

Wang, Qingzhao; Ingram, Lonnie O; Shanmugam, K T

2011-11-22

23

Phylogenetic Analysis of Vertebrate Lactate Dehydrogenase (LDH) Multigene Families  

Microsoft Academic Search

.   In this paper we analyzed 49 lactate dehydrogenase (LDH) sequences, mostly from vertebrates. The amino acid sequence differences\\u000a were found to be larger for a human–killifish pair than a human–lamprey pair. This indicates that some protein sequence convergence\\u000a may occur and reduce the sequence differences in distantly related species. We also examined transitions and transversions\\u000a separately for several species

Yi-Ju Li; Stephen C.-M. Tsoi

2002-01-01

24

Sequence Analysis of Teleost Retina-Specific Lactate Dehydrogenase C: Evolutionary Implications for the Vertebrate Lactate Dehydrogenase Gene Family  

Microsoft Academic Search

At least two gene duplication events have led to the three lactate dehydrogenase (LDH; EC 1.1.1.27) isozymes (LDH-A, LDH-B, and LDH-C) of chordates. The prevailing model for the evolution of the LDH loci involves duplication of a primordial LDH locus near the origin of vertebrates, giving rise to Ldh-A and Ldh-B. A third locus, designated Ldh-C, is expressed in the

J. M. Quattro; H. A. Woods; D. A. Powers

1993-01-01

25

Metabolic engineering of lactate dehydrogenase rescues mice from acidosis.  

PubMed

Acidosis causes millions of deaths each year and strategies for normalizing the blood pH in acidosis patients are greatly needed. The lactate dehydrogenase (LDH) pathway has great potential for treating acidosis due to its ability to convert protons and pyruvate into lactate and thereby raise blood pH, but has been challenging to develop into a therapy because there are no pharmaceutical-based approaches for engineering metabolic pathways in vivo. In this report we demonstrate that the metabolic flux of the LDH pathway can be engineered with the compound 5-amino-2-hydroxymethylphenyl boronic acid (ABA), which binds lactate and accelerates the consumption of protons by converting pyruvate to lactate and increasing the NAD(+)/NADH ratio. We demonstrate here that ABA can rescue mice from metformin induced acidosis, by binding lactate, and increasing the blood pH from 6.7 to 7.2 and the blood NAD(+)/NADH ratio by 5 fold. ABA is the first class of molecule that can metabolically engineer the LDH pathway and has the potential to have a significant impact on medicine, given the large number of patients that suffer from acidosis. PMID:24898534

Acharya, Abhinav P; Rafi, Mohammad; Woods, Elliot C; Gardner, Austin B; Murthy, Niren

2014-01-01

26

Metabolic engineering of lactate dehydrogenase rescues mice from acidosis  

PubMed Central

Acidosis causes millions of deaths each year and strategies for normalizing the blood pH in acidosis patients are greatly needed. The lactate dehydrogenase (LDH) pathway has great potential for treating acidosis due to its ability to convert protons and pyruvate into lactate and thereby raise blood pH, but has been challenging to develop into a therapy because there are no pharmaceutical-based approaches for engineering metabolic pathways in vivo. In this report we demonstrate that the metabolic flux of the LDH pathway can be engineered with the compound 5-amino-2-hydroxymethylphenyl boronic acid (ABA), which binds lactate and accelerates the consumption of protons by converting pyruvate to lactate and increasing the NAD+/NADH ratio. We demonstrate here that ABA can rescue mice from metformin induced acidosis, by binding lactate, and increasing the blood pH from 6.7 to 7.2 and the blood NAD+/NADH ratio by 5 fold. ABA is the first class of molecule that can metabolically engineer the LDH pathway and has the potential to have a significant impact on medicine, given the large number of patients that suffer from acidosis.

Acharya, Abhinav P.; Rafi, Mohammad; Woods, Elliot C.; Gardner, Austin B.; Murthy, Niren

2014-01-01

27

Lactate dehydrogenase C and energy metabolism in mouse sperm.  

PubMed

We demonstrated previously that disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) led to male infertility due to defects in sperm function, including a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD(+) cofactor essential for activity of glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS), or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD:NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the lactate dehydrogenase (LDH) inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported our earlier conclusion that LDHA, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC, but suggested that LDHC has an additional role in the maintenance of energy metabolism in sperm. By coimmunoprecipitation coupled with mass spectrometry, we identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport, and/or sequestration. This led us to hypothesize that in addition to its role in glycolysis, LDHC is part of a complex involved in ATP homeostasis that is disrupted in sperm lacking LDHC. PMID:21565994

Odet, Fanny; Gabel, Scott A; Williams, Jason; London, Robert E; Goldberg, Erwin; Eddy, Edward M

2011-09-01

28

Lactate Dehydrogenase C and Energy Metabolism in Mouse Sperm1  

PubMed Central

We demonstrated previously that disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) led to male infertility due to defects in sperm function, including a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD+ cofactor essential for activity of glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS), or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD:NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the lactate dehydrogenase (LDH) inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported our earlier conclusion that LDHA, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC, but suggested that LDHC has an additional role in the maintenance of energy metabolism in sperm. By coimmunoprecipitation coupled with mass spectrometry, we identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport, and/or sequestration. This led us to hypothesize that in addition to its role in glycolysis, LDHC is part of a complex involved in ATP homeostasis that is disrupted in sperm lacking LDHC.

Odet, Fanny; Gabel, Scott A.; Williams, Jason; London, Robert E.; Goldberg, Erwin; Eddy, Edward M.

2011-01-01

29

Presence of bound substrate in lactate dehydrogenase from carp liver.  

PubMed

While attempting to purify UDP-galactose 4-epimerase from carp liver extract at pH 8.0, it was observed that the preparation even after dialysis could reduce NAD to NADH, interfering epimerase assay. The NAD reduction activity and the epimerase were co-eluted in a series of chromatographic steps. Mass spectrometric analysis of semi-purified fraction revealed that carp liver lactate dehydrogenase (LDH) contained bound lactate which was converted to pyruvate in the presence of NAD. The enzyme-bound lactate and the association with epimerase stabilized LDH from trypsin digestion and thermal inactivation at 45 degrees C by factors of 2.7 and 4.2 respectively, as compared to substrate-free LDH. LDH and epimerase do not belong to any one pathway, but are the rate-limiting enzymes of two different pathways of carbohydrate metabolism. Typically, strongly associated enzymes work in combination, such as two enzymes of the same metabolic pathway. In that background, co-purification of LDH and epimerase as reloaded in this study was an unusual phenomenon. PMID:22803333

Banerjee, Nupur; Bhattacharyya, Debasish

2012-06-01

30

Not only osmoprotectant: betaine increased lactate dehydrogenase activity and L-lactate production in lactobacilli.  

PubMed

Lactobacilli are commonly used for industrial production of polymer-grade L-lactic acid. The present study tested the Tween 80 alternative betaine in L-lactate production by several industrial lactobacilli. In flask fermentation of Lactobacillus casei, Lactobacillus buchneri, Lactobacillus lactis and Lactobacillus rhamnosus, the betaine addition (2g/l) had similar osmoprotectant effect with Tween 80 but had increased the lactate dehydrogenase activities and L-lactate production than Tween 80 control. In fed-batch fermentation of L. casei, betaine supplementation improved the L-lactic acid titer to 190 g/l, the yield to 95.5% (g L-lactic acid/g glucose), the productivity to 2.6g/lh, and the optical purity to 97.0%. The results demonstrated that supplementation of Tween 80 alternative - betaine in the fermentation medium is feasible for industrial l-lactic acid fermentation by lactobacilli, which will improve the lactate production but will not increase the process costs and modify any process conditions. PMID:24035452

Zou, Huibin; Wu, Zaiqiang; Xian, Mo; Liu, Hui; Cheng, Tao; Cao, Yujin

2013-11-01

31

Human Lactate Dehydrogenase A Inhibitors: A Molecular Dynamics Investigation  

PubMed Central

Lactate dehydrogenase A (LDHA) is an important enzyme in fermentative glycolysis, generating most energy for cancer cells that rely on anaerobic respiration even under normal oxygen concentrations. This renders LDHA a promising molecular target for the treatment of various cancers. Several efforts have been made recently to develop LDHA inhibitors with nanomolar inhibition and cellular activity, some of which have been studied in complex with the enzyme by X-ray crystallography. In this work, we present a molecular dynamics (MD) study of the binding interactions of selected ligands with human LDHA. Conventional MD simulations demonstrate different binding dynamics of inhibitors with similar binding affinities, whereas steered MD simulations yield discrimination of selected LDHA inhibitors with qualitative correlation between the in silico unbinding difficulty and the experimental binding strength. Further, our results have been used to clarify ambiguities in the binding modes of two well-known LDHA inhibitors.

Shi, Yun; Pinto, B. Mario

2014-01-01

32

Conformational heterogeneity within the Michaelis complex of lactate dehydrogenase  

PubMed Central

A series of isotope edited IR measurements, both static as well as temperature jump relaxation spectroscopy, are performed on lactate dehydrogenase (LDH) to determine the ensemble of structures available to its Michaelis complex. There clearly has been a substantial reduction in the number of states available to the pyruvate substrate (as modeled by the substrate mimic, oxamate) and NADH when bound to protein compared to dissolved in solution, as determined by the bandwidths and positions of the critical C2=O band of bound substrate mimic and the C4-H stretch of NADH reduced nicotinamide group. Moreover, it is found that a strong ionic bond (characterized by a signature IR band discovered in this study) is formed between the carboxyl group of bound pyruvate with (presumably) Arg171, forming a strong ‘anchor’ within the protein matrix. However, conformational heterogeneity within the Michaelis complex is found that has an impact on both catalytic efficiency and thermodynamics of the enzyme.

Deng, Hua; Vu, Dung V.; Clinch, Keith; Desamero, Ruel; Dyer, R. Brian; Callender, Robert

2011-01-01

33

Conformational heterogeneity within the Michaelis complex of lactate dehydrogenase.  

PubMed

A series of isotope edited IR measurements, both static as well as temperature jump relaxation spectroscopy, are performed on lactate dehydrogenase (LDH) to determine the ensemble of structures available to its Michaelis complex. There clearly has been a substantial reduction in the number of states available to the pyruvate substrate (as modeled by the substrate mimic, oxamate) and NADH when bound to protein compared to dissolved in solution, as determined by the bandwidths and positions of the critical C(2)?O band of the bound substrate mimic and the C(4)-H stretch of the NADH reduced nicotinamide group. Moreover, it is found that a strong ionic bond (characterized by a signature IR band discovered in this study) is formed between the carboxyl group of bound pyruvate with (presumably) Arg171, forming a strong "anchor" within the protein matrix. However, conformational heterogeneity within the Michaelis complex is found that has an impact on both catalytic efficiency and thermodynamics of the enzyme. PMID:21568287

Deng, Hua; Vu, Dung V; Clinch, Keith; Desamero, Ruel; Dyer, R Brian; Callender, Robert

2011-06-16

34

Pig heart lactate dehydrogenase. Binding of pyruvate and the interconversion of pyruvate-containing ternary complexes.  

PubMed Central

1. Lactate oxidation catalysed by pig heart lactate dehydrogenase was studied in the presence of inhibitory concentrations of pyruvate. Experimental results show the presence of an intermediate which occurs immediately after the hydride transfer step, but before the dissociation of pyruvate and the H+ produced by the reaction. The rate constant for pyruvate dissociation and the dissociation constant for pyruvate from the ternary complex differ from those obtained in pyruvate reduction experiments. 2.In single-turnover pyruvate reduction by pig heart lactate dehydrogenase at pH8.0 pyruvate can bind to the enzyme before a H+ is taken up, and the subsequent uptake of a H+ is governed by a step that is also rate-limiting for single-turnover and steady-state NADH oxidation. 3. Observation of various intermediates in the single-turnover pyruvate reduction experiments has made it possible to determine separately the dissociation constant and Km value for pyruvate at pH8.0, and also the catalytic turnover rate and Km for pyruvate under first-order conditions at different pH values. 4. Further studies on single-turnover pyruvate reduction carried out in 2H2O, or in water at low temperature, show another step which, under these conditions, is slower than that controlling H+ uptake and rate-limiting for NADH oxidation. A scheme is presented which explains these results. Images Fig. 1. Fig. 3. Fig. 5. Fig. 7. Fig. 9. Fig. 11. Fig. 12.

Boland, M J; Gutfreund, H

1975-01-01

35

Cloning, E. coli expression, and characterization of heart lactate dehydrogenase B from river buffalo (Bubalus bubalis).  

PubMed

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. PMID:24299182

Nadeem, Muhammad Shahid; Moran, Jenny; Murtaza, Bibi Nazia; Muhammad, Khushi; Ahmad, Habib

2014-01-01

36

Fabrication of lactate biosensor based on lactate dehydrogenase immobilized on cerium oxide nanoparticles.  

PubMed

An electrochemical biosensor was developed to determine lactate that plays an important role in clinical diagnosis, fermentation and food quality analysis. Abnormal concentration of lactate has been related to diseases such as hypoxia, acute heart disorders, lactic acidosis, muscle fatigue and meningitis. Also, lactate concentration in blood helps to evaluate the athletic performance in sports. The main aim of the work is to fabricate NADH/LDH/Nano-CeO2/GCE bio-electrode for sensing lactate in human blood samples. Toward this, CeO2 nanoparticles were synthesized by a hydroxide mediated approach using cerium nitrate hexahydrate (Ce(NO3)3·6H2O) and NaOH as precursors. X-ray diffraction (XRD) and Field Emission Scanning Electron Microscopy (FE-SEM) studies were carried out to determine the structural and morphological characteristics of CeO2 nanoparticles. XRD pattern indicated the formation of highly crystalline CeO2 nanoparticles with face centered cubic structure. The FE-SEM studies revealed the formation of nanospherical particles of size 29.73±2.59 nm. The working electrode was fabricated by immobilizing nicotinamide adenine dinucleotide (NADH) and lactate dehydrogenase (LDH) on GCE surface with CeO2 nanoparticles as an interface. Electrochemical studies were carried out through cyclic voltammetry using a three electrode system with NADH/LDH/NanoCeO2/GCE as a working electrode, Ag/AgCl saturated with 0.1M KCl as a reference electrode and Pt wire as a counter electrode. From the amperometric study, the linearity was found to be in the range of 0.2-2 mM with the response time of less than 4s. PMID:24034216

Nesakumar, Noel; Sethuraman, Swaminathan; Krishnan, Uma Maheswari; Rayappan, John Bosco Balaguru

2013-11-15

37

NAD-Independent L-Lactate Dehydrogenase Is Required for L-Lactate Utilization in Pseudomonas stutzeri SDM  

PubMed Central

Background Various Pseudomonas strains can use l-lactate as their sole carbon source for growth. However, the l-lactate-utilizing enzymes in Pseudomonas have never been identified and further studied. Methodology/Principal Findings An NAD-independent l-lactate dehydrogenase (l-iLDH) was purified from the membrane fraction of Pseudomonas stutzeri SDM. The enzyme catalyzes the oxidation of l-lactate to pyruvate by using FMN as cofactor. After cloning its encoding gene (lldD), l-iLDH was successfully expressed, purified from a recombinant Escherichia coli strain, and characterized. An lldD mutant of P. stutzeri SDM was constructed by gene knockout technology. This mutant was unable to grow on l-lactate, but retained the ability to grow on pyruvate. Conclusions/Significance It is proposed that l-iLDH plays an indispensable function in Pseudomonas l-lactate utilization by catalyzing the conversion of l-lactate into pyruvate.

Dou, Peipei; Ma, Cuiqing; Li, Lixiang; Kong, Jian; Xu, Ping

2012-01-01

38

Critical role for lactate dehydrogenase A in aerobic glycolysis that sustains pulmonary microvascular endothelial cell proliferation  

PubMed Central

Pulmonary microvascular endothelial cells possess both highly proliferative and angiogenic capacities, yet it is unclear how these cells sustain the metabolic requirements essential for such growth. Rapidly proliferating cells rely on aerobic glycolysis to sustain growth, which is characterized by glucose consumption, glucose fermentation to lactate, and lactic acidosis, all in the presence of sufficient oxygen concentrations. Lactate dehydrogenase A converts pyruvate to lactate necessary to sustain rapid flux through glycolysis. We therefore tested the hypothesis that pulmonary microvascular endothelial cells express lactate dehydrogenase A necessary to utilize aerobic glycolysis and support their growth. Pulmonary microvascular endothelial cell (PMVEC) growth curves were conducted over a 7-day period. PMVECs consumed glucose, converted glucose into lactate, and acidified the media. Restricting extracellular glucose abolished the lactic acidosis and reduced PMVEC growth, as did replacing glucose with galactose. In contrast, slow-growing pulmonary artery endothelial cells (PAECs) minimally consumed glucose and did not develop a lactic acidosis throughout the growth curve. Oxygen consumption was twofold higher in PAECs than in PMVECs, yet total cellular ATP concentrations were twofold higher in PMVECs. Glucose transporter 1, hexokinase-2, and lactate dehydrogenase A were all upregulated in PMVECs compared with their macrovascular counterparts. Inhibiting lactate dehydrogenase A activity and expression prevented lactic acidosis and reduced PMVEC growth. Thus PMVECs utilize aerobic glycolysis to sustain their rapid growth rates, which is dependent on lactate dehydrogenase A.

Parra-Bonilla, Glenda; Alvarez, Diego F.; Al-Mehdi, Abu-Bakr; Alexeyev, Mikhail

2010-01-01

39

Isolation and Partial Characterization of Lactate Dehydrogenase Isozymes from Normal and Variant Rainbow Trout Livers.  

National Technical Information Service (NTIS)

The homotetrameric lactate dehydrogenase (LDH) isozymes, B2 prime and B2 double prime, from normal and variant rainbow trout livers have been purified to homogeneity by affinity chromatography using a Sepharose-linked oxamate ligand. The two isozymes have...

Y. H. J. Kao

1977-01-01

40

Biochemical development of preimplantation mouse embryos: In vivo activities of fructose 1,6-diphosphate aldolase, glucose 6-phosphate dehydrogenase, malate dehydrogenase, and lactate dehydrogenase  

Microsoft Academic Search

The activities of four enzymes were determined during the first four days of mouse embryogenesis. Two enzymes, fructose 1,6-diphosphate aldolase and malate dehydrogenase, increase about 30% in activity, and this increase is attributed to slow but continued enzyme synthesis. The other two enzymes, glucose 6-phosphate dehydrogenase (X-linked) and lactate dehydrogenase, remain constant for the first two days and then decline

Charles J. Epstein; Evelyn A. Wegienka; Charles W. Smith

1969-01-01

41

Relevance of Lactate Dehydrogenase Activity to the Control of Oxidative Glycolysis in Pancreatic Islet B-Cells  

Microsoft Academic Search

The activities of hexokinase isoenzymes, lactate dehydrogenase, cytosolic NAD-linked glycerophosphate dehydrogenase, mitochondrial FAD-linked glycerophosphate dehydrogenase, and glutamate dehydrogenase were measured in homogenates of rat purified pancreatic B and non-B islet cells. In B cell homogenates, the maximal activity of hexokinase and glucokinase was one to two orders of magnitude lower than that of lactate dehydrogenase. The activity of the mitochondrial

Hassan Jijakli; Joanne Rasschaert; Abdellatif Bakkali Nadi; Viviane Leclercq-Meyer; Abdullah Sener; Willy J. Malaisse

1996-01-01

42

Cloning and Polymorphisms of Yak Lactate Dehydrogenase b Gene  

PubMed Central

The main objective of this work was to study the unique polymorphisms of the lactate dehydrogenase-1 (LDH1) gene in yak (Bos grunniens). Native polyacrylamide gel electrophoresis revealed three phenotypes of LDH1 (a tetramer of H subunit) in yak heart and longissimus muscle extracts. The corresponding gene, ldhb, encoding H subunits of three LDH1 phenotypes was obtained by RT-PCR. A total of six nucleotide differences were detected in yak ldhb compared with that of cattle, of which five mutations cause amino acid substitutions. Sequence analysis shows that the G896A and C689A, mutations of ldhb gene, result in alterations of differently charged amino acids, and create the three phenotypes (F, M, and S) of yak LDH1. Molecular modeling of the H subunit of LDH indicates that the substituted amino acids are not located within NAD+ or substrate binding sites. PCR-RFLP examination of G896A mutation demonstrated that most LDH1-F samples are actually heterozygote at this site. These results help to elucidate the molecular basis and genetic characteristic of the three unique LDH1 phenotypes in yak.

Wang, Guosheng; Zhao, Xingbo; Zhong, Juming; Cao, Meng; He, Qinghua; Liu, Zhengxin; Lin, Yaqiu; Xu, Yaou; Zheng, Yucai

2013-01-01

43

Structural Studies of Malate Dehydrogenases (MDHs): MDHs in Brevundimonas Species Are the First Reported MDHs in Proteobacteria Which Resemble Lactate Dehydrogenases in Primary Structure  

Microsoft Academic Search

The N-terminal sequences of malate dehydrogenases from 10 bacterial strains, representing seven genera of Proteobacteria, were determined. Of these, the enzyme sequences of species classified in the genus Brevundi- monas clearly resembled those malate dehydrogenases with greatest similarity to lactate dehydrogenases. Additional evidence from subunit molecular weights, peptide mapping, and enzyme mobilities suggested that malate dehydrogenases from species of the

COLIN CHARNOCK

1997-01-01

44

Enzyme activities in fish spermatozoa with focus on lactate dehydrogenase isoenzymes from herring Clupea harengus  

Microsoft Academic Search

The activities of NAD- and NADP-dependent dehydrogenases and creatine kinase were compared in extracts of spermatozoa from herring (Clupea harengus), carp (Cyprinus carpio) and catfish (Clarias gariepinus). The activity of malic enzyme in herring spermatozoa was approximately 5 and 36 times higher than in carp and catfish spermatozoa. In contrast, lactate dehydrogenase activity in herring spermatozoa was very low. Herring

Jadwiga Gronczewska; Marek S Zi?tara; Anna Biegniewska; Edward F Skorkowski

2003-01-01

45

D-Lactate Dehydrogenase Binding in Escherichia coli dld- Membrane Vesicles Reconstituted for Active Transport*  

PubMed Central

When membrane vesicles prepared from a D-lactate dehydrogenase mutant of E. coli ML 308-225 are treated with a homogeneous preparation of D-lactate dehydrogenase, the enzyme binds to the vesicles and they regain the capacity to catalyze D-lactate oxidation and D-lactate-dependent active transport. Although membranebound enzyme increases linearly with addition of increasing quantities of enzyme, reconstituted transport activity and D-lactate oxidation are saturable functions of the amount of enzyme bound. The maximal specific transport activity obtained in the reconstituted system is similar in magnitude to that of wild type vesicles. Titration studies with 2-(N-dansyl)-aminoethyl-?-D-thiogalactoside demonstrate that there is at least a 7- to 8-fold excess of lac carrier protein relative to D-lactate dehydrogenase. Hydroxybutynoate-inactivated enzyme does not bind to the vesicles, indicating that the coenzyme moiety is critically involved in binding. Conformational changes are also apparently involved since 0.6 M guanidine·HCl is required for optimal binding and reconstitution. The relative unreactivity of reconstituted vesicles towards vinylglycolic acid suggests that D-lactate dehydrogenase is bound to the outer surface of the reconstituted vesicles.

Short, Steven A.; Kaback, H. Ronald; Kohn, Leonard D.

1974-01-01

46

Elevation of serum lactate dehydrogenase in patients with pectus excavatum  

PubMed Central

Introduction Pectus excavatum is the most common congenital chest wall deformity and the depression of the anterior chest wall, which compresses the internal organs. The aim of the present study is to investigate the effects of pectus excavatum on blood laboratory findings. Material and Methods From March 2011 to December 2011, 71 patients with pectus excavatum who visited Seoul Saint Mary Hospital for Nuss procedure were reviewed and analyzed. The blood samples were routinely taken at the day before surgery and pectus bar removal was usually performed in 2 to 3 years after Nuss procedure. To investigate the effects on blood laboratory findings, preoperative routine blood laboratory data and postoperative changes of abnormal laboratory data were analyzed. Results Only lactate dehydrogenase (LDH), one of 26 separate routine laboratory tests, was abnormal and significantly elevated than normal value (age <10, p?=?0.008; age ?10, p?

2014-01-01

47

Energy landscape of the Michaelis complex of lactate dehydrogenase: relationship to catalytic mechanism.  

PubMed

Lactate dehydrogenase (LDH) catalyzes the interconversion between pyruvate and lactate with nicotinamide adenine dinucleotide (NAD) as a cofactor. Using isotope-edited difference Fourier transform infrared spectroscopy on the "live" reaction mixture (LDH·NADH·pyruvate ? LDH·NAD(+)·lactate) for the wild-type protein and a mutant with an impaired catalytic efficiency, a set of interconverting conformational substates within the pyruvate side of the Michaelis complex tied to chemical activity is revealed. The important structural features of these substates include (1) electronic orbital overlap between pyruvate's C2?O bond and the nicotinamide ring of NADH, as shown from the observation of a delocalized vibrational mode involving motions from both moieties, and (2) a characteristic hydrogen bond distance between the pyruvate C2?O group and active site residues, as shown by the observation of at least four C2?O stretch bands indicating varying degrees of C2?O bond polarization. These structural features form a critical part of the expected reaction coordinate along the reaction path, and the ability to quantitatively determine them as well as the substate population ratios in the Michaelis complex provides a unique opportunity to probe the structure-activity relationship in LDH catalysis. The various substates have a strong variance in their propensity toward on enzyme chemistry. Our results suggest a physical mechanism for understanding the LDH-catalyzed chemistry in which the bulk of the rate enhancement can be viewed as arising from a stochastic search through an available phase space that, in the enzyme system, involves a restricted ensemble of more reactive conformational substates as compared to the same chemistry in solution. PMID:24576110

Peng, Huo-Lei; Deng, Hua; Dyer, R Brian; Callender, Robert

2014-03-25

48

Determination of lactate dehydrogenase (LDH) activity in milk by a fluorometric assay.  

PubMed

Indigenous L-lactate dehydrogenase (LDH) in milk originates mainly from somatic cells, leucocytes and invading microorganisms. Its activity may be used for detection of mastitis. However, existing methods to measure LDH activity in milk both need pretreatment of the samples and still suffer from methodological problems. The present paper describes a fast, reliable method for determination of LDH activity, suitable for milk samples. The method is based on fluorometric determination of enzyme kinetics when L-lactate is converted to pyruvate. The assay uses raw milk without pretreatment and the method is easily adjustable to large-scale analyses on micro assay plates. Detection is based on (straight line) linear response within 4-7 min of initiation of the reaction. A substrate concentration of 35 mM in the reaction mixture was considered to be optimal for the assay. Intra plate assay precision was approx. 6% (CV) and the inter plate precision approx. 10%. Known inhibitors of LDH activity (oxidative direction), i.e., oxalic acid, oxamate, and pyruvate, were tested in different concentrations in order to verify the specificity of the response. The detailed kinetics of samples analysed indicated that the isoenzyme composition may have differed between milk samples, and that this composition may have been altered in high activity samples. PMID:15909687

Larsen, Torben

2005-05-01

49

Enzymatic production of D-3-phenyllactic acid by Pediococcus pentosaceus D-lactate dehydrogenase with NADH regeneration by Ogataea parapolymorpha formate dehydrogenase.  

PubMed

3-Phenyllactic acid (PLA) is an antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi. Enzymatic production of PLA can be carried out from phenylpyruvic acid by lactate dehydrogenase (LDH); however, the enzymatic reaction is accompanied by NADH oxidation that inhibits PLA biotransformation. Here, NADH regeneration was achieved using the formate dehydrogenase from Ogataea parapolymorpha and introduced into the D-PLA production process using the D-LDH from Pediococcus pentosaceus. Optimum PLA production by dual enzyme treatment was at pH 6.0 and 50 °C with both enzymes at 0.4 ?M. Using 0.2 mM NADH, D-PLA production by NADH regeneration system reached 5.5 mM, which was significantly higher than that by a single-enzyme reaction. PMID:24249102

Yu, Shuhuai; Zhu, Lanjun; Zhou, Chen; An, Tao; Jiang, Bo; Mu, Wanmeng

2014-03-01

50

Serum lactate dehydrogenase isoenzyme 1 as a marker of testicular germ cell tumor.  

PubMed

Serum lactate dehydrogenase isoenzyme 1 activity was determined repeatedly in 21 men with testicular germ cell tumors in connection with orchiectomy and in 25 without neoplasia who underwent exploration of the testis. The highest level in the men without malignancy was 109 units per 1. and a higher pre-orchiectomy level was found in 15 of the tumor patients: 7 of 11 with seminoma and 8 of 10 with nonseminomatous tumors. In the patients with stage 3 disease serum lactate dehydrogenase isoenzyme 1 was increased more often and the activity was higher than in the stage 1 and 2 cancer patients. Within 1 month after orchiectomy the initially increased level decreased to less than 109 units per 1. in 8 of 10 patients with a stage 1 tumor and it remained higher in 5 with stage 2 or 3 disease. Serum lactate dehydrogenase isoenzyme 1 seems to be a useful marker of testicular germ cell tumor. PMID:2845154

von Eyben, F E; Blaabjerg, O; Petersen, P H; Hørder, M; Nielsen, H V; Kruse-Andersen, S; Parlev, E

1988-11-01

51

Detection and characterization of new genetic mutations in individuals heterozygous for lactate dehydrogenase-B(H) deficiency using DNA conformation polymorphism analysis and silver staining  

Microsoft Academic Search

Human lactate dehydrogenase (LDH) — B(H) mutant genes were analyzed by polymerase chain reaction (PCR) and DNA conformation polymorphism. We used polyacrylamide gradient gel and silver staining procedures for DCP analysis, and observed abnormal migration patterns in individuals heterozygous for the LDH-B deficiency. Subsequent sequence determination of the mutant alleles consistently resulted in detection of three single base substitutions (transversions),

Masato Maekawa; Kayoko Sudo; Masato Kitajima; Yukio Matsuura; Steven S.-L. Li; Takashi Kanno

1993-01-01

52

Pressure inactivation of tetrameric lactate dehydrogenase homologues of confamilial deep-living fishes  

Microsoft Academic Search

The susceptibility to inactivation by hydrostatic pressure of the tetrameric (Fig. 1) muscletype (M4) lactate dehydrogenase homologues (LDH, EC 1.1.1.27;l-lactate: NAD+ oxidoreductase) from six confamilial macrourid fishes was compared at 4 °C. These marine teleost fishes occur over depths of 260 to 4815 m. The pressures necessary to half-inactivate the LDH homologues are related to the pressures which the enzymes

John P. Hennessey; Joseph F. Siebenaller

1985-01-01

53

Population screening of lactate dehydrogenase deficiencies in Fukuoka Prefecture in Japan and molecular characterization of three independent mutations in the lactate dehydrogenase-B(H) gene  

Microsoft Academic Search

Screening for lactate dehydrogenase (LDH) subunit deficiencies was performed on 2880 blood samples from healthy individuals in the Fukuoka Prefecture in Japan by means of electrophoresis. The frequencies of heterozygotes with either LDH-A or LDH-B deficiency were found to be 0.104% at each locus. These estimated frequencies of either LDH-A or LDH-B deficiencies were slightly lower than, but not significantly

Masato Maekawa; Kayoko Sudo; Kiyoko Nagura; Steven S.-L. Li; Takashi Kanno

1994-01-01

54

Double mutation of the PDC1 and ADH1 genes improves lactate production in the yeast Saccharomyces cerevisiae expressing the bovine lactate dehydrogenase gene  

Microsoft Academic Search

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there\\u000a is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase\\u000a the lactate yield,

Kenro Tokuhiro; Nobuhiro Ishida; Eiji Nagamori; Satoshi Saitoh; Toru Onishi; Akihiko Kondo; Haruo Takahashi

2009-01-01

55

Purification and properties of a monomeric lactate dehydrogenase from yak Hypoderma sinense larva.  

PubMed

The objective of the present study was to study the characteristics of lactate dehydrogenase (LDH) from Hypoderma sinense larva. H. sinense larvae were collected from yak (Bos grunniens) and identified by a PCR-RFLP method. Analysis of LDH activity showed that the total LDH activity in H. sinense larva was negatively correlated with the length of larva. Polyacrylamide gel electrophoresis of the extracts of H. sinense larvae revealed one band of LDH, which was then purified by affinity chromatography and gel filtration. This enzyme showed an approximately 36 kDa band on SDS-gel under both reducing and non-reducing conditions, in addition, size exclusion chromatography analysis showed that its molecular weight was smaller than bovine serum albumin (67 kDa), indicating that it contains only one subunit. Michaelis constants (Km) values assay revealed that LDH from H. sinense larva showed significantly lower Km for lactate than other animals. LDH of H. sinense larva was stable at 60 °C for 15 min, and also exhibited high catalytic efficiency in a wide range of pH. HgCl? at the concentration of 0.1mM significantly decreased the activity of LDH from H. sinense larva but not at the concentration of 0.01 mM. The results of the present study demonstrate that LDH from H. sinense larva is a thermal stable and pH insensitive enzyme suitable for catalyzing both forward and reverse reactions. PMID:23474203

Li, Pengfei; Jin, Suyu; Huang, Lin; Liu, Haohao; Huang, Zhihong; Lin, Yaqiu; Zheng, Yucai

2013-06-01

56

Evaluation of the intra- and inter-specific genetic variability of Plasmodium lactate dehydrogenase  

Microsoft Academic Search

BACKGROUND: Malaria diagnosis is vital to efficient control programmes and the recent advent of malaria rapid diagnostic tests (RDTs) provides a reliable and simple diagnostic method. However a characterization of the efficiency of these tests and the proteins they detect is needed to maximize RDT sensitivity. METHODS: Plasmodial lactate dehydrogenase (pLDH) gene of wild isolates of the four human species

Arthur M Talman; Linda Duval; Eric Legrand; Véronique Hubert; Seiha Yen; David Bell; Jacques Le Bras; Frédéric Ariey; Sandrine Houze

2007-01-01

57

Impact of Endosulfan on Lactate Dehydrogenase from the Freshwater Catfish Clarias batrachus  

Microsoft Academic Search

The impact of a sublethal concentration of an organochlorine insecticide endosulfan on the activity, specific activity, electrophoretic patterns and kinetic properties of crude and purified lactate dehydrogenase (LDH) from the liver and the skeletal muscle of the freshwater catfish,Clarias batrachus,was evaluated. The endosulfan significantly reduced the activity and the specific activity of liver and muscle LDH but had no effect

Rajnikant Mishra; S. P. Shukla

1997-01-01

58

Lactate dehydrogenase gene function in the blind cave fish, Anoptichthys jordani , and other characins  

Microsoft Academic Search

The functions of the genes encoded for lactate dehydrogenase (LDH) in six genera of characins (teleost) were examined by electrophoretic and immunochemical analyses of the LDH isozymes. The characins possess the LDH A and B loci present in all vertebrates. The eyeless Mexican cave fish (Anoptichthys jordani) and other characins possessing normal eyes, e.g., Mexican tetra (Astyanax mexicanus, which is

Gregory S. Whitt; Frances S. Maeda

1970-01-01

59

Regularities of Bonding of Chlorin e 6 to the Oligomeric Enzyme Lactate Dehydrogenase  

Microsoft Academic Search

Using differential-spectrophotometry, spectral-luminescence, and polarization methods, we have investigated regularities of complexing of a promising photodynamic sensitizer — chlorin e6 — with a key glycolytic enzyme — lactate dehydrogenase (LDH). The parameters of the dye–enzyme complex have been estimated by the difference between the spectral characteristics of the free dye and the dye bonded to the enzyme. It is shown

V. Yu. Plavskii; V. A. Mostovnikov; G. R. Mostovnikova; A. I. Tret'yakova; L. G. Plavskaya

2003-01-01

60

Formation of an Equilibrium Complex of Lactate Dehydrogenase with Chlorin e 6  

Microsoft Academic Search

Using spectrophotometric, spectral-luminescent, and polarization methods, we have detected the formation of strong equilibrium complexes of a key glycolytic enzyme — lactate dehydrogenase — with a promising photodynamic sensitizer — chlorin e6. It has been established that enzymes serve as the most sensitive targets destroying tumor cells subjected to photodynamic therapy.

V. Yu. Plavskii; V. A. Mostovnikov; G. R. Mostovnikova; A. I. Tret'yakova; A. V. Mikulich

2003-01-01

61

Characterization and comparison of epsilon-crystallin and lactate dehydrogenases in the lenses of vertebrates and invertebrates.  

PubMed

Screening of lens homogenates for the identification of lactate dehydrogenases was undertaken for the representative species from five major classes of vertebrates plus the cephalopod of invertebrates. The duck and caiman lenses appeared to contain the highest enzymatic activity of this glycolytic enzyme among all species examined. Biochemical isolation and characterization of epsilon-crystallins from the duck and caiman lenses revealed differences between these structural crystallins and the authentic lactate dehydrogenase of the avian heart regarding some of the kinetic properties. This is in contrast with the claim that duck epsilon-crystallin is identical to heart-type lactate dehydrogenase. PMID:2787637

Chiou, S H; Chang, W P; Chen, C C

1989-06-01

62

Molecular cloning and characterization of lactate dehydrogenase gene from Eimeria tenella.  

PubMed

Lactate dehydrogenase (LDH) is a key enzyme in the glycolytic pathway and is crucial for parasite survival. In this study, we cloned and expressed the LDH of Eimeria tenella (EtLDH). Real-time polymerase chain reaction and Western blot analysis revealed that the expression of EtLDH was developmentally regulated at the messenger RNA (mRNA) and protein levels. EtLDH mRNA levels were higher in second-generation merozoites than in other developmental stages (unsporulated oocysts, sporulated oocysts, and sporozoites). EtLDH protein expression levels were most prominent in second-generation merozoites, moderately expressed in unsporulated oocysts and sporulated oocysts, and weakly detected in sporozoites. Immunostaining with anti-recombinant EtLDH (rEtLDH) antibody indicated that EtLDH was mainly located in the anterior region in free sporozoites and became concentrated in the anterior region of intracellular sporozoites except for the apex after invasion into DF-1 cells. Specific staining of EtLDH protein was more intense in trophozoites and immature first-generation schizonts, but decreased in mature first-generation schizonts. Inhibition of EtLDH function using specific antibodies cannot efficiently reduce the ability of E. tenella sporozoites to invade host cells. These results suggest that EtLDH may be involved in glycolysis during the first-generation merogony stage in E. tenella and has little role in host invasion. PMID:24906988

Dong, Hui; Wang, Yange; Zhao, Qiping; Han, Hongyu; Zhu, Shunhai; Li, Liujia; Wu, Youling; Huang, Bing

2014-08-01

63

Lactate dehydrogenase from Streptococcus mutans: purification, characterization, and crossed antigenicity with lactate dehydrogenases from Lactobacillus casei, Actinomyces viscosus, and Streptococcus sanguis.  

PubMed Central

A cytoplasmic fructose-1,6-diphosphate-dependent lactate dehydrogenase (LDH; EC 1.1.1.27) from Streptococcus mutans OMZ175 was purified to homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The purification consisted of ammonium sulfate precipitation of the cytoplasmic fraction, DEAE-Sephacel and Blue-Sepharose CL.6B chromatography, and Sephacryl S200 gel permeation. The catalytic activity of the purified enzyme required the presence of fructose-1,6-diphosphate with a broad optimum between pH 5 and 6.2. The concentration of fructose-1,6-diphosphate required for half-maximal velocity was around 0.02 mM and was affected by the pyruvate concentration. The enzyme seemed to have at least two binding sites for the activator which interact in a cooperative manner. Increasing concentrations of fructose-1,6-diphosphate up to 2 mM enhanced the relative affinity of the enzyme for pyruvate and modified the pyruvate saturation curve from sigmoidal to hyperbolic. The enzyme activity showed also a sigmoidal response to NADH, exhibiting two binding sites for the cofactor with a Hill coefficient of about 1.9. The molecular weight of the native enzyme was 150,000 as determined by gel permeation on Sephacryl S200. Monomers (38,000 daltons) and dimers (85,000 daltons) were observed by sodium dodecyl sulfate-gel electrophoresis; the latter form was dissociated after reduction with 2-mercaptoethanol, and the enzyme could be considered a tetramer. Antibodies obtained against the purified S. mutans OMZ175 LDH cross-reacted with the sodium dodecyl sulfate-dissociated forms of LDHs from different S. mutans serotypes, Streptococcus sanguis OMZ9, Lactobacillus casei ATCC 4646, and Actinomyces viscosus NY 1. A competitive enzyme-linked immunosorbent assay allowed us to detect a very close relationship between the native states of L-LDHs from S. mutans serotypes and S. sanguis. Cross-reactions were also observed with the LDHs from A. viscosus and L. casei, the latter being the least related. A very weak immunological relationship was obtained between the L-LDH from S. mutans OMZ175 and the D-LDH from Lactobacillus leichmannii, whereas no cross-reaction could be detected with mammal LDHs. Images

Sommer, P; Klein, J P; Scholler, M; Frank, R M

1985-01-01

64

Regulation of Cyclic GMP, Cyclic amp and Lactate Dehydrogenase by Putative Neutrotransmitters in the C6 Rat Glioma Cell Line.  

National Technical Information Service (NTIS)

In C6 cells norepinephrine and dopamine caused transient increases in cyclic GMP and cyclic AMP, as well as an induction of lactate dehydrogenase. All of these responses were blocked by 1-propranolol, suggesting mediation by a beta -receptor. Phentolamine...

J. E. Bottenstein J. de Vellis

1977-01-01

65

The preparation and kinetics of lactate dehydrogenase attached to water-insoluble particles and sheets  

PubMed Central

1. The preparation of lactate dehydrogenase covalently attached to anion-exchange cellulose particles and sheets by use of a dichloro-sym-triazinyl dyestuff, Procion brilliant orange MGS, is described. 2. The stability and kinetic properties of these preparations were investigated. 3. An equation is derived to describe the change in concentration of a substrate when passed through a uniform bed of a substrate-inhibited enzyme. A number of theoretical curves are shown to illustrate the system. 4. A titrimetric assay for lactate dehydrogenase is described, and shown to be stoicheiometric over the range pH5·0–9·2. 5. The results are discussed in relation to previous work, and the effects of charged groups on the support, and of the diffusion film surrounding any particle in suspension, are treated qualitatively.

Wilson, R. J. H.; Kay, G.; Lilly, M. D.

1968-01-01

66

Alkaline-induced unfolding and salt-induced folding of pig heart lactate dehydrogenase under high pH conditions  

Microsoft Academic Search

The alkaline-induced unfolding and the salt-induced folding of pig heart lactate dehydrogenase under high pH conditions have been followed by fluorescence emission spectra and circular dichroism spectra. The results for alkaline-induced denaturation of lactate dehydrogenase show that at low ionic strength, increasing the pH value increased the extent of unfolding of the enzyme to the maximum ultimate unfolded conformation at

Ji-Hong Bai; Hao-Jing Wang; Hai-Meng Zhou

1998-01-01

67

Electrophoretic patterns of esterases and lactate- and malate-dehydrogenases from Alcaligenes species  

Microsoft Academic Search

Esterases and lactate-(LDH) and malate-(MDH) dehydrogenases of 34 strains ofAlcaligenes faecalis, 16 strains ofA. denitrificans subspxylosoxydans (A. xylosoxydans), 5 strains ofA. piechaudii, and 10 strains ofA. denitrificans subspdenitrificans (A. denitrificans) were analyzed by horizontal polyacrylamide-agarose gel electrophoresis. Four types of esterases were identified inA. faecalis, 3 inA. xylosoxydans, 3 inA. piechaudii, and 14 inA. denitrificans by their spectra of hydrolytic

Chantal Bizet; Bertrand Picard; Fredj Tekaia; Philippe Goullet

1993-01-01

68

Functional and Structural Properties of Lactate Dehydrogenase from Embryos of Different Fishes  

Microsoft Academic Search

Functional and structural properties of L-lactate dehydrogenase (LDH, EC 1.1.1.27) from embryos of various fish species (Atlantic salmon, rainbow trout, autumn cisco, least [Siberian] cisco, Siberian sturgeon, sterlet, loach, carp, goldfish, and zebrafish) were analysed. The minimum Km for pyruvate from embryos is correlated with the optimal temperatures of development in nature. LDH from embryos of fish adapted to low

O. S Klyachko; N. D Ozernyuk

1998-01-01

69

Lactate Dehydrogenase-B cDNA from the Teleost Fundulus heteroclitus: Evolutionary Implications 1  

Microsoft Academic Search

A cDNA that encodes the heart-type lactate dehydrogenase (LDH-B) from the teleost fish Fundulus heteroclitus was cloned and sequenced. The protein encoded by the cDNA was analyzed in relation to 13 LDH proteins from a variety of taxa. One of the deductions from this analysis is that LDH-B proteins have residues in the active site that are unique and that

Douglas L. Crawford; Henry R. Constantino; Dennis A. Powers

70

Characterization of lactate dehydrogenase isozyme pattern and morphology of three marine fish cell lines  

Microsoft Academic Search

Three continuous marine fish cell lines of FG (i.e., Flounder Gill) from flounder (Paralichthys olivaceus) gill, SPH (i.e., Sea Perch Heart) from sea perch (Lateolabrax japonicus) heart and RSBF (i.e., Red Sea Bream Fin) from red sea bream (Pagrosomus major) fin, were characterized by lactate dehydrogenase (LDH) isozyme and morphological analysis. The LDH isozyme patterns of these\\u000a three cell lines

Hua-Rong Guo; Shi-Cui Zhang; Hong-Yan Li; Shang-Liang Tong; Jian-Hai Xiang

2002-01-01

71

Interdemic variation in haematocrit and lactate dehydrogenase in the African cyprinid Barbus neumayeri  

Microsoft Academic Search

This study evaluated whether the African cyprinid Barbus neumayeri from Rwembaita Swamp (low-oxygen) and Njuguta River (high-oxygen) in the Kibale National Park, Uganda differed in traits related to aerobic and anaerobic metabolic potential. Haematocrit was measured as an index of blood oxygen-carrying capacity, and tissue activities and isozyme composition of lactate dehydrogenase (LDH) were measured as indices of tissue anaerobic

M. L. M ARTINEZ; L. J. C HAPMAN; J. M. G RADY; B. B. R EES

2004-01-01

72

Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer  

PubMed Central

DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2:1 protein:aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer:protein complex crystal structure was solved at 2.1-Å resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson–Crick base-paired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics.

Cheung, Yee-Wai; Kwok, Jane; Law, Alan W. L.; Watt, Rory M.; Kotaka, Masayo; Tanner, Julian A.

2013-01-01

73

Purification and partial characterization of a d (?)-lactate dehydrogenase from Desulfovibrio desulfuricans (ATCC 7757)  

Microsoft Academic Search

Summary  A membrane-boundd(–)-lactate dehydrogenase (LDH), an important enzyme in carbon and energy metabolism in sulfate-reducing bacteria of the genusDesulfovibrio, was solubilized from the membrane fraction ofDesulfovibrio desulfuricans (ATCC 7757). The enzyme was purified 84-fold to a final specific activity of 525 nmol DCPIP-reduced\\/min\\/mg protein by ammonium sulfate precipitation, chloroform extraction, gel filtration with Sephadex G-150, and hydrophobic column chromatography withN-octylamine Sepharose

M. H. Czechowski; H. W. Rossmoore

1990-01-01

74

Differential expression of two tomato lactate dehydrogenase genes in response to oxygen deficit  

Microsoft Academic Search

Two different cDNAs encoding lactate dehydrogenase (LDH) were isolated from a library of hypoxically treated tomato roots and sequenced. The use of gene-specific probes on northern blots showed that Ldh2 mRNA was predominant in well-oxygenated roots and levels remained stable upon oxygen deficit; in contrast, Ldh1 mRNA accumulated to high levels within 2 h of hypoxia or anoxia. Immunoblot analyses

Véronique Germain; Philippe Raymond; Bérénice Ricard

1997-01-01

75

Collecting and assessing human lactate dehydrogenase-A conformations for structure-based virtual screening.  

PubMed

Human lactate dehydrogenase-A (LDHA) is emerging as a promising anticancer target. Up to now, structure-based investigations for identifying inhibitors of this enzyme have not explicitly accounted for active site flexibility. In the present study, by combining replica exchange molecular dynamics with network and cluster analyses, we identified reliable LDHA conformations for structure-based ligand design. The selected conformations were challenged and validated by retrospective virtual screening simulations. PMID:24138094

Buonfiglio, Rosa; Ferraro, Mariarosaria; Falchi, Federico; Cavalli, Andrea; Masetti, Matteo; Recanatini, Maurizio

2013-11-25

76

Immobilization of glucose oxidase and lactate dehydrogenase onto magnetic nanoparticles for bioprocess monitoring system  

Microsoft Academic Search

Glucose oxidase (GOD) and lactate dehydrogenase (LDH) were immobilized onto magnetic nanoparticles, viz. Fe3O4, via carbodiimide and glutaraldehyde. The immobilization efficiency was largely dependent upon the immobilization time and\\u000a concentration of glutaraldehyde. The magnetic nanoparticles had a mean diameter of 9.3 nm and were superparamagnetic. The\\u000a immobilization of GOD and LDH on the nanoparticles slightly decreased their saturation magnetization. However,

Ok-Jae Sohn; Chun-Kwang Kim; Jong Il Rhee

2008-01-01

77

Impact of High Pyruvate Concentration on Kinetics of Rabbit Muscle Lactate Dehydrogenase  

Microsoft Academic Search

In order to evaluate the effectiveness of l-lactate dehydrogenase (LDH) from rabbit muscle as a regenerative catalyst of the biologically important cofactor nicotinamide\\u000a adenine dinucleotide (NAD), the kinetics over broad concentrations were studied to develop a suitable kinetic rate expression.\\u000a Despite robust literature describing the intricate complexations, the mammalian rabbit muscle LDH lacks a quantitative kinetic\\u000a rate expression accounting for

Matthew Warren Eggert; Mark E. Byrne; Robert P. Chambers

78

Induction of Lactate Dehydrogenase Isozymes by Oxygen Deficit in Barley Root Tissue 1  

PubMed Central

Lactate dehydrogenase (LDH) activity in attached roots of barley and other cereals increased up to 20-fold during several days of severe hypoxia, reaching a maximum of about 2 micromoles per minute per gram fresh weight. In barley, induction of LDH activity was significant at 2.6% O2 and greatest at 0.06%, the lowest O2 concentration tested. Upon return to aerobic conditions, induced LDH activity declined with an apparent half-life of 2 days. The isozyme profile of barley LDH comprised 5 bands, consistent with a tetrameric enzyme with subunits encoded by two different Ldh genes. Changes in staining intensity of the isozymes as a function of O2 level suggested that one Ldh gene was preferentially expressed in severe hypoxia. When tracer [U-14C]glucose was supplied to induced roots under hypoxic conditions, lactate acquired label, but much less than either ethanol or alanine. Most of the [14C] lactate was secreted into the medium, whereas most other labeled anionic products were retained in the root. Neither hypoxic induction of LDH, nor lactate secretion by induced roots, is predicted from the Davies-Roberts hypothesis, which holds that lactate glycolysis ceases soon after the onset of hypoxia due to acidosis brought about by lactate accumulation in the cytoplasm. These results imply a functional significance for LDH beyond that assigned it in this hypothesis. Images Fig. 5 Fig. 6

Hoffman, Neil E.; Bent, Andrew F.; Hanson, Andrew D.

1986-01-01

79

Physical and functional association of lactate dehydrogenase (LDH) with skeletal muscle mitochondria.  

PubMed

The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO2) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO2. However, subsequent addition of NAD(+) significantly increased JO2, and was abolished by the inhibitor of mitochondrial pyruvate transport, ?-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD(+)-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria. PMID:23873936

Elustondo, Pia A; White, Adrienne E; Hughes, Meghan E; Brebner, Karen; Pavlov, Evgeny; Kane, Daniel A

2013-08-30

80

Design and synthesis of new enzymes based on the lactate dehydrogenase framework.  

PubMed

Analysis of the mechanism and structure of lactate dehydrogenases is summarized in a map of the catalytic pathway. Chemical probes, single tryptophan residues inserted at specific sites and a crystal structure reveal slow movements of the protein framework that discriminate between closely related small substrates. Only small and correctly charged substrates allow the protein to engulf the substrate in an internal vacuole that is isolated from solvent protons, in which water is frozen and hydride transfer is rapid. The closed vacuole is very sensitive to the size and charge of the substrate and provides discrimination between small substrates that otherwise have too few functional groups to be distinguished at a solvated protein surface. This model was tested against its ability to successfully predict the design and synthesis of new enzymes such as L-hydroxyisocaproate dehydrogenase and fully active malate dehydrogenase. Solvent friction limits the rate of forming the vacuole and thus the maximum rate of catalysis. PMID:1678537

Dunn, C R; Wilks, H M; Halsall, D J; Atkinson, T; Clarke, A R; Muirhead, H; Holbrook, J J

1991-05-29

81

Lactate and malate dehydrogenase in the fan-shell associated shrimp, Pontonia pinnophylax (Otto): Effects of temperature and urea  

Microsoft Academic Search

In Pontonia pinnophylax (Otto), a crustacean decapod inhabiting the mantle cavity of Pinna nobilis L. (Bivalvia: Pteriomorpha), the lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activity, and their electrophoretic patterns, were compared in relation to heat and urea inactivation. Activity was higher in LDH than in MDH, and the electrophoretic patterns showed a predominance of LDH-A4 and the presence of

G. Laganà; S. Giacobbe; E. Bellocco; C. Mannucci; A. Galtieri; S. Ficarra; A. Kotyk; U. Leuzzi

2007-01-01

82

On the pathway of forming enzymatically productive ligand-protein complexes in lactate dehydrogenase.  

PubMed

We have carried out a series of studies on the binding of a substrate mimic to the enzyme lactate dehydrogenase (LDH) using advanced kinetic approaches, which begin to provide a molecular picture of the dynamics of ligand binding for this protein. Binding proceeds via a binding-competent subpopulation of the nonligated form of the protein (the LDH/NADH binary complex) to form a protein-ligand encounter complex. The work here describes the collapse of the encounter complex to form the catalytically competent Michaelis complex. Isotope-edited static Fourier transform infrared studies on the bound oxamate protein complex reveal two kinds of oxamate environments: 1), a major populated structure wherein all significant hydrogen-bonding patterns are formed at the active site between protein and bound ligand necessary for the catalytically productive Michaelis complex and 2), a minor structure in a configuration of the active site that is unfavorable to carry out catalyzed chemistry. This latter structure likely simulates a dead-end complex in the reaction mixture. Temperature jump isotope-edited transient infrared studies on the binding of oxamate with LDH/NADH suggest that the evolution of the encounter complex between LDH/NADH and oxamate collapses via a branched reaction pathway to form the major and minor bound species. The production of the catalytically competent protein-substrate complex has strong similarities to kinetic pathways found in two-state protein folding processes. Once the encounter complex is formed between LDH/NADH and substrate, the ternary protein-ligand complex appears to "fold" to form a compact productive complex in an all or nothing like fashion with all the important molecular interactions coming together at the same time. PMID:18390601

Deng, Hua; Brewer, Scott; Vu, Dung M; Clinch, Keith; Callender, Robert; Dyer, R Brian

2008-07-01

83

Multichannel Simultaneous Determination of Activities of Lactate Dehydrogenase  

SciTech Connect

It is very important to find the best conditions for some enzymes to do the best catalysis in current pharmaceutical industries. Based on the results above, we could say that this set-up could be widely used in finding the optimal condition for best enzyme activity of a certain enzyme. Instead of looking for the best condition for enzyme activity by doing many similar reactions repeatedly, we can complete this assignment with just one run if we could apply enough conditions.

Ma, L.

2000-09-12

84

Binding ligands and cofactor to L-lactate dehydrogenase from human skeletal and heart muscles.  

PubMed

Binding affinities of cofactor and ligands to the active site of two different isoforms of lactate dehydrogenase (LDH), heart and skeletal muscles (H4 and M4, respectively), can be used for medical and biological applications. Herein, a hybrid QM/MM computational approach based on free energy perturbation methods has been carried out to estimate binding affinities and binding isotope effects (BIEs) for NADH/NAD(+) and oxamate, pyruvate, L-lactate, and D-lactate ligands to the M4 and H4 isoforms of L-LDH. Here, we show that determining how cofactor and ligands interact with the active site of LDH isoforms advanced the still open discussion on the intracellular lactate shuttle hypothesis. In our discussion we deny the key concept of this hypothesis showing, based on interaction energy values, that there is no evidence that the M4 type of LDH in the skeletal muscles cells served as a catalyst of the conversion of lactate to pyruvate. Additionally, theoretical determination of BIEs for H4 and M4 types of LDH shows that there is a way of using the BIEs as a tool capable to distinguish these isoforms, and for this purpose D-lactate labeled with deuterium in positions 11 or 7, 8, 9 ([11-2H]-BIE and [7,8,9-2H3]-BIE) or L-lactate labeled only in position 11 ([11-2H]-BIE) could be used. We propose the BIEs as a useful tool which can be applied in order to experimentally determine the types of LDH. PMID:21526780

?widerek, Katarzyna; Paneth, Piotr

2011-05-19

85

Factors Affecting the Activity of the Lactate Dehydrogenase of Streptococcus cremoris  

PubMed Central

Studies with partially purified extracts of the nicotinamide adenine dinucleotide-linked l(+)-lactate dehydrogenase of Streptococcus cremoris US3 showed that fructose-1,6-diphosphate (FDP) was essential for the catalytic reduction of pyruvate in the pH range 5.0 to 7.0, outside of which the organism does not grow. In the absence of FDP, enzyme activity was observed only in the region of pH 8.0. The optimal pH for the oxidation of lactate was approximately 8.0 in the presence and absence of FDP. The FDP-activated enzyme was markedly inhibited by inorganic phosphate. The enzyme lost activity on standing at 5 C in alkaline triethanolamine, was quite stable at pH 6.0 to 6.5, and underwent irreversible denaturation below pH 5.0. Inorganic phosphate or FDP increased the stability of the enzyme in alkaline buffers. Some distinguishing properties of individual lactate dehydrogenases, activated by FDP, are discussed.

Jonas, H. A.; Anders, R. F.; Jago, G. R.

1972-01-01

86

The specific interaction of Cibacron and related dyes with cyclic nucleotide phosphodiesterase and lactate dehydrogenase  

PubMed Central

1. Reactive Blue 2 (Cibacron Blue 3G-A) is a competitive inhibitor of bovine heart cyclic nucleotide phosphodiesterase (Ki 0.3?m). The Ki increases with increasing temperature, suggesting that hydrophobic interactions are not largely responsible for the binding of the dye. Another 25 sulphonated aromatic dyes are also competitive inhibitors of the cyclic nucleotide phosphodiesterase (Ki values in the range of 0.06–13.6?m). 2. These dyes (covalently linked to Dextran 40) inhibit bovine heart cyclic nucleotide phosphodiesterase. Reactive Blue 2 (covalently linked to Dextran 40) is a competitive inhibitor of the phosphodiesterase (Ki 0.4?m). 3. Bovine heart cyclic nucleotide phosphodiesterase is retained on a column of Reactive Blue 2-Sephacryl S-200 and can be eluted from the column by 3?:5?-cyclic AMP. 4. A variety of the dyes (either free or covalently linked to Dextran 40) are competitive inhibitors of rabbit muscle lactate dehydrogenase. 5. The effectiveness of a wide range of structurally dissimilar dyes as competitive inhibitors of lactate dehydrogenase and cyclic nucleotide phosphodiesterase compromises proposals for the use of Reactive Blue 2 as a specific probe for the dinucleotide-binding structural domain present in many dehydrogenases and kinases. Detailed information of the various dyes used has been deposited as Supplementary Publication SUP 50089 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

Ashton, Anthony R.; Polya, Gideon M.

1978-01-01

87

Changes in antibody specificities and cytokine release after infection with lactate dehydrogenase-elevating virus.  

PubMed

Lactate dehydrogenase-elevating virus (LDV) is an apparently innocuous and persistent virus that can modify mouse immune reactions. We have shown that LDV-infected mice immunized with human growth hormone (hGH) showed a deep modification of the specificity of the anti-hGH antibodies (Ab) in CBA/Ht mice but not BALB/c animals. The aim of this work was to extend the previous observations to another mouse strain, C57BL/6, as well as to an antigen unrelated to hGH, ovalbumin (OVA), and to explore at the same time the production of various cytokines at serum and cellular levels. The amount of Ab directed to hGH or OVA native antigenic determinants versus the concentration of Ab to cryptic epitopes was evaluated by ELISA competition experiments. Results indicated that LDV infection affected Ab specificity solely in CBA/Ht mice. In CBA/Ht the virus infection was associated with a reduction of the Ab titers to hGH native epitopes and with a decrease of IL-13 and IL-17 serum levels, but Ab to native OVA epitopes were increased with a simultaneous increase of IL-17. Accordingly, only lymph node cells from infected CBA/Ht mice immunized with OVA were found to produce INF-?, IL-13 and IL-17. Thus, a correlation of cytokine production with a change in Ab specificity after a viral infection was found, although this phenomenon was restricted to a given antigen and to the genetic background of immunized animals. These observations suggest that an apparent harmless virus can affect some immunological mechanisms, which could lead, for example, to inflammatory or autoimmune disorders. PMID:23391715

Aparicio, José L; Saxena, Anubha; Coutelier, Jean-Paul; Van Snick, Jacques; Retegui, Lilia A

2013-03-01

88

Simultaneous purification of L-malate dehydrogenase and L-lactate dehydrogenase from bovine heart by biomimetic-dye affinity chromatography  

Microsoft Academic Search

Two commercially important enzymes, L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (MDH) were purified simultaneously from bovine heart, on an agarose affinity adsorbent. This adsorbent bears a dye-ligand composed of an anthraquinone chlorotriazine chromophore linked to a biomimetic terminal 4-aminophenyloxanylic acid moiety. The purification protocol exploited the biomimetic affinity adsorbent, in combination with a cross-linked agarose DEAE anion-exchanger. The procedure comprised

N. E. Labrou; Y. D. Clonis

1997-01-01

89

Glucose6-phosphate dehydrogenase and lactate dehydrogenase in the green-lipped mussel ( Perna viridis): Possible biomarkers for hypoxia in the marine environment  

Microsoft Academic Search

Green-lipped mussels (Perna viridis) were collected from a well-oxygenated site in Hong Kong and maintained in situ at this and three other sites with different dissolved oxygen (DO) regimes. The transplanted mussels were retrieved after a 4-week field exposure. An estimation of the activities of glucose-6-phosphate dehydrogenase (G6PDH) and lactate dehydrogenase (LDH) in the adductor muscles of the mussels showed

Rudolf S. S. Wu; Paul K. S. Lam

1997-01-01

90

An electrochemical biosensor with nanointerface for lactate detection based on lactate dehydrogenase immobilized on zinc oxide nanorods.  

PubMed

Hepatic immaturity is observed particularly in children whose age is under three, when the lactate concentration is greater than the normal level in blood. An electrochemical lactate biosensor was developed by immobilizing lactate dehydrogenase (LDH) on to ZnO nanorods at pH 7.4 via chitosan. Growth of polycrystalline ZnO nanorods towards (101) plane was confirmed using XRD. The FE-SEM study revealed the formation of ZnO nanorods with an aspect ratio of 3.24. Immobilization of LDH on ZnO nanorods was confirmed using FTIR spectra and surface coverage. Electrochemical studies were carried out through cyclic voltammetry and amperometry using three electrode system with Au/NanoZnO/LDH as working electrode, Ag/AgCl in 0.1 M KCl as reference electrode and Pt wire as counter electrode. The sensitivity of the biosensor was found to be 1.832 ?A ?mol(-1) L exhibiting linearity 0.2-0.8 ?mol L(-1) with the detection and quantification limits of 4.73 and 15.75 nmol L(-1) respectively. The response time of Au/NanoZnO/LDH bioelectrode was found to be <1 s. Prediction band for net current was framed to enhance specificity. Michaelis-Menten constant (KM(app)) and maximum rate (Imax) values for immobilized LDH were found to be 0.38 ?mol L(-1) and 2.798 ?A respectively. Repeatability and reproducibility of LDH biosensor were also reported. PMID:24231089

Nesakumar, Noel; Thandavan, Kavitha; Sethuraman, Swaminathan; Krishnan, Uma Maheswari; Rayappan, John Bosco Balaguru

2014-01-15

91

Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth of Corynebacterium glutamicum on D-lactate  

PubMed Central

Background Corynebacterium glutamicum is able to grow with lactate as sole or combined carbon and energy source. Quinone-dependent L-lactate dehydrogenase LldD is known to be essential for utilization of L-lactate by C. glutamicum. D-lactate also serves as sole carbon source for C. glutamicum ATCC 13032. Results Here, the gene cg1027 was shown to encode the quinone-dependent D-lactate dehydrogenase (Dld) by enzymatic analysis of the protein purified from recombinant E. coli. The absorption spectrum of purified Dld indicated the presence of FAD as bound cofactor. Inactivation of dld resulted in the loss of the ability to grow with D-lactate, which could be restored by plasmid-borne expression of dld. Heterologous expression of dld from C. glutamicum ATCC 13032 in C. efficiens enabled this species to grow with D-lactate as sole carbon source. Homologs of dld of C. glutamicum ATCC 13032 are not encoded in the sequenced genomes of other corynebacteria and mycobacteria. However, the dld locus of C. glutamicum ATCC 13032 shares 2367 bp of 2372 bp identical nucleotides with the dld locus of Propionibacterium freudenreichii subsp. shermanii, a bacterium used in Swiss-type cheese making. Both loci are flanked by insertion sequences of the same family suggesting a possible event of horizontal gene transfer. Conclusions Cg1067 encodes quinone-dependent D-lactate dehydrogenase Dld of Corynebacterium glutamicum. Dld is essential for growth with D-lactate as sole carbon source. The genomic region of dld likely has been acquired by horizontal gene transfer.

2010-01-01

92

In vivo regulation of alcohol dehydrogenase and lactate dehydrogenase in Rhizopus oryzae to improve L-lactic acid fermentation.  

PubMed

Rhizopus oryzae is becoming more important due to its ability to produce an optically pure L: -lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4'-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways. PMID:21416338

Thitiprasert, Sitanan; Sooksai, Sarintip; Thongchul, Nuttha

2011-08-01

93

The influence of oxygen on radiation-induced structural and functional changes in glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase  

NASA Astrophysics Data System (ADS)

Proteins are major targets for oxidative damage due to their abundance in cells and high reactivity with free radicals. In the present study we examined the influence of oxygen on radiation-induced inactivation and structural changes of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH). We chose these two enzymes because they occur at high concentrations and participate in the most important processes in organisms; furthermore, they show considerable similarity in their structure. Protein solutions were irradiated with X-rays in doses ranging from 0.1 to 0.7 kGy, in air and N2O. The much higher radiation inactivation of GAPDH as compared to LDH is correlated with substantially greater structural changes in this protein, mainly involving the loss of free thiol groups (-SH). Of lesser importance in the differentiation of the radiosensitivity of the studied enzymes are tryptophan residues. Molecular oxygen, present during irradiation, increased to a significantly greater extent the inactivation and structural changes of GAPDH than that of LDH. The results suggest that the greater effect of oxygen on GAPDH is due to the higher efficiency of the superoxide radical, the higher amount of hydroperoxides generated, and the higher degree of unfolding of this protein.

Rodacka, Aleksandra; Serafin, Eligiusz; Bubinski, Michal; Krokosz, Anita; Puchala, Mieczyslaw

2012-07-01

94

Rapid Detection of Lactate Dehydrogenase and Genotyping of Plasmodium falciparum in Saliva of Children with Acute Uncomplicated Malaria  

PubMed Central

The diagnosis of malaria in biological fluids other than blood using non-invasive, rapid diagnostic techniques provides a valuable approach in case management and epidemiological studies of malaria. Rapid detection of Plasmodium falciparum lactate dehydrogenase (pLDH) in saliva samples from 130 of 144 children with microscopically confirmed P. falciparum infection was evaluated using Optimal-IT dipsticks. Genotyping of parasites was also performed in saliva and blood samples from a cohort of patients by polymerase chain reaction (PCR). The sensitivity of the dipstick in whole-blood, whole-saliva, or supernatant of spun saliva samples was 97.2%, 77.9%, and 48.4%, respectively. The sensitivity of the dipstick in whole-saliva samples was significantly higher than in supernatant of spun saliva samples (P < 0.0005). Mutant T76 allele was detectable in 60% and 57% of blood and saliva samples, respectively. This finding shows rapid detection of pLDH in patient saliva.

Gbotosho, Grace O.; Happi, Christian T.; Folarin, Onikepe; Keyamo, Ochuko; Sowunmi, Akintunde; Oduola, Ayoade M. J.

2010-01-01

95

Cationic Surfactant-Based Colorimetric Detection of Plasmodium Lactate Dehydrogenase, a Biomarker for Malaria, Using the Specific DNA Aptamer  

PubMed Central

A simple, sensitive, and selective colorimetric biosensor for the detection of the malarial biomarkers Plasmodium vivax lactate dehydrogenase (PvLDH) and Plasmodium falciparum LDH (PfLDH) was demonstrated using the pL1 aptamer as the recognition element and gold nanoparticles (AuNPs) as probes. The proposed method is based on the aggregation of AuNPs using hexadecyltrimethylammonium bromide (CTAB). The AuNPs exhibited a sensitive color change from red to blue, which could be seen directly with the naked eye and was monitored using UV-visible absorption spectroscopy and transmission electron microscopy (TEM). The reaction conditions were optimized to obtain the maximum color intensity. PvLDH and PfLDH were discernible with a detection limit of 1.25 pM and 2.94 pM, respectively. The applicability of the proposed biosensor was also examined in commercially available human serum.

Lee, Seonghwan; Manjunatha, D H; Jeon, Weejeong; Ban, Changill

2014-01-01

96

Cationic surfactant-based colorimetric detection of Plasmodium lactate dehydrogenase, a biomarker for malaria, using the specific DNA aptamer.  

PubMed

A simple, sensitive, and selective colorimetric biosensor for the detection of the malarial biomarkers Plasmodium vivax lactate dehydrogenase (PvLDH) and Plasmodium falciparum LDH (PfLDH) was demonstrated using the pL1 aptamer as the recognition element and gold nanoparticles (AuNPs) as probes. The proposed method is based on the aggregation of AuNPs using hexadecyltrimethylammonium bromide (CTAB). The AuNPs exhibited a sensitive color change from red to blue, which could be seen directly with the naked eye and was monitored using UV-visible absorption spectroscopy and transmission electron microscopy (TEM). The reaction conditions were optimized to obtain the maximum color intensity. PvLDH and PfLDH were discernible with a detection limit of 1.25 pM and 2.94 pM, respectively. The applicability of the proposed biosensor was also examined in commercially available human serum. PMID:24992632

Lee, Seonghwan; Manjunatha, D H; Jeon, Weejeong; Ban, Changill

2014-01-01

97

Human lactate dehydrogenase A (LDHA) rescues mouse Ldhc-null sperm function.  

PubMed

By targeted disruption of the lactate dehydrogenase c (Ldhc) gene, we demonstrated that spermatozoa require Ldhc for capacitation, motility, and fertilizing capacity. Ldhc expression is restricted to the developing germ cells that, however, are apparently not compromised by the lack of the LDHC isozyme. Because LDHC is abundant in spermatozoa that utilize aerobic glycolysis for energy requirements, its main function was presumed to be the interconversion of pyruvate to lactate with the concomitant oxidation/reduction of NADH to NAD(+). We found that sperm without LDHC were still able to convert lactate to pyruvate as mediated by LDHA that is tightly bound to the fibrous sheath. It was assumed that the level of glycolysis was insufficient to power motility and the subsequent fertilizing capacity of the mutated sperm. To investigate whether LDHC possesses certain unique characteristics essential for fertility, human LDHA was introduced as a transgene to Ldhc-null mice. We report here that the exogenous LDHA rescued the phenotype of the Ldhc-null males. Sperm from the LDHA transgenic males with the Ldhc deletion (LDHA(+)/Ldhc(-/-)) are motile, capable of protein tyrosine phosphorylation, and able to fertilize, thus restoring these properties to LDHC-null sperm. However, the lactate and ATP levels in the rescued sperm did not differ significantly from sperm lacking LDHC. We suggest that it is the localization of the transgene to the sperm cytosol that is mainly responsible for restoration of sperm function and fertility. PMID:23467744

Tang, Huanghui; Duan, Chongwen; Bleher, Reiner; Goldberg, Erwin

2013-04-01

98

Human Lactate Dehydrogenase A (LDHA) Rescues Mouse Ldhc-Null Sperm Function1  

PubMed Central

ABSTRACT By targeted disruption of the lactate dehydrogenase c (Ldhc) gene, we demonstrated that spermatozoa require Ldhc for capacitation, motility, and fertilizing capacity. Ldhc expression is restricted to the developing germ cells that, however, are apparently not compromised by the lack of the LDHC isozyme. Because LDHC is abundant in spermatozoa that utilize aerobic glycolysis for energy requirements, its main function was presumed to be the interconversion of pyruvate to lactate with the concomitant oxidation/reduction of NADH to NAD+. We found that sperm without LDHC were still able to convert lactate to pyruvate as mediated by LDHA that is tightly bound to the fibrous sheath. It was assumed that the level of glycolysis was insufficient to power motility and the subsequent fertilizing capacity of the mutated sperm. To investigate whether LDHC possesses certain unique characteristics essential for fertility, human LDHA was introduced as a transgene to Ldhc-null mice. We report here that the exogenous LDHA rescued the phenotype of the Ldhc-null males. Sperm from the LDHA transgenic males with the Ldhc deletion (LDHA+/Ldhc?/?) are motile, capable of protein tyrosine phosphorylation, and able to fertilize, thus restoring these properties to LDHC-null sperm. However, the lactate and ATP levels in the rescued sperm did not differ significantly from sperm lacking LDHC. We suggest that it is the localization of the transgene to the sperm cytosol that is mainly responsible for restoration of sperm function and fertility.

Tang, Huanghui; Duan, Chongwen; Bleher, Reiner; Goldberg, Erwin

2013-01-01

99

Cardioprotective Activity of A Novel and Potent Competitive Inhibitor of Lactate Dehydrogenase  

PubMed Central

Alkaline incubation of NADH results in the formation of a very potent inhibitor of lactate dehydrogenase. High resolution mass spectroscopy along with NMR characterization clearly showed that the inhibitor is derived from attachment of a glycolic acid moiety to the 4-position of the dihydronicotinamide ring of NADH. The very potent inhibitor is competitive with respect to NADH. The inhibitor added in submicromolar concentrations to cardiomyocytes protects them from damage caused by hypoxia/reoxygenation stress. In isolated mouse hearts, addition of the inhibitor results in a substantial reduction of myocardial infarct size caused by global ischemia/reperfusion injury.

Kotlyar, Alexander B.; Randazzo, Antonio; Honbo, Norman; Jin, Zhu-Qui; Karliner, Joel S.; Cecchini, Gary

2009-01-01

100

Ability of Cytosolic Malate Dehydrogenase and Lactate Dehydrogenase to Increase the Ratio of NADPH to NADH Oxidation by Cytosolic Glycerol3-phosphate Dehydrogenase  

Microsoft Academic Search

At the normal pH of the cytosol (7.0 to 7.1) and in the presence of physiological (1.0 mM) levels of free Mg2+, theVmaxof the NADPH oxidation is only slightly lower than theVmaxof NADH oxidation in the cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) reaction. Under these conditions physiological (30 ?M) levels of cytosolic malate dehydrogenase (E.C. 1.1.1.37) inhibited oxidation of 20 ?M

Leonard A. Fahien; José I. Laboy; Zafeer Z. Din; Prakash Prabhakar; Tatyana Budker; Michael Chobanian

1999-01-01

101

Mitochondrial lactate dehydrogenase is involved in oxidative-energy metabolism in human astrocytoma cells (CCF-STTG1).  

PubMed

Lactate has long been regarded as an end product of anaerobic energy production and its fate in cerebral metabolism has not been precisely delineated. In this report, we demonstrate, for the first time, the ability of a human astrocytic cell line (CCF-STTG1) to consume lactate and to generate ATP via oxidative phosphorylation. (13)C-NMR and HPLC analyses aided in the identification of tricarboxylic acid (TCA) cyle metabolites and ATP in the astrocytic mitochondria incubated with lactate. Oxamate, an inhibitor of lactate dehydrogenase (LDH), abolished mitochondrial lactate consumption. Electrophoretic and fluorescence microscopic analyses helped localize LDH in the mitochondria. Taken together, this study implicates lactate as an important contributor to ATP metabolism in the brain, a finding that may significantly change our notion of how this important organ manipulates its energy budget. PMID:18253497

Lemire, Joseph; Mailloux, Ryan J; Appanna, Vasu D

2008-01-01

102

Conformation of NAD+ bound to allosteric L-lactate dehydrogenase activated by chemical modification.  

PubMed

On modification of arginine residues with 2,3-butanedione, the Thermus caldophilus L-lactate dehydrogenase is converted to an activated form that is independent of an allosteric effector, fructose 1,6-bisphosphate (Fru-1,6-P2). The conformation of NAD+ bound to the modified enzyme in the absence of Fru-1,6-P2 was investigated by means of proton NMR, analyzing the time dependence of the transferred nuclear Overhauser effect (TRNOE) and TRNOE action spectra. The inter-proton distances determined on TRNOE analysis indicated that both the nicotinamide riboside moiety and the adenosine moiety of NAD+ were in the anti conformation, the ribose rings being in the C3'-endo form. This conformation was almost the same as that of NAD+ bound to the native enzyme-Fru-1,6-P2 complex, rather than that of NAD+ bound to the free native enzyme. These results suggest that the C3'-endo-anti form of the enzyme-bound NAD+ is essential for the activation of the T. caldophilus L-lactate dehydrogenase. PMID:2722793

Koide, S; Yokoyama, S; Matsuzawa, H; Miyazawa, T; Ohta, T

1989-05-25

103

Crystal structure and thermodynamic properties of d-lactate dehydrogenase from Lactobacillus jensenii.  

PubMed

The thermostable d-lactate dehydrogenase from Lactobacillus jensenii (Ljd-LDH) is a key enzyme in the production of the d-form of lactic acid from pyruvate concomitant with the oxidation of NADH to NAD(+). The polymers of d-lactic acid are used as biodegradable bioplastics. The crystal structures of Ljd-LDH and in complex with NAD(+) were determined at 2.13 and 2.60? resolutions, respectively. The Ljd-LDH monomer consists of the N-terminal substrate-binding domain and the C-terminal NAD-binding domain. The Ljd-LDH forms a homodimeric structure, and the C-terminal NAD-binding domain mostly enables the dimerization of the enzyme. The NAD cofactor is bound to the GxGxxG NAD-binding motif located between the two domains. Structural comparisons of Ljd-LDH with other d-LDHs reveal that Ljd-LDH has unique amino acid residues at the linker region, which indicates that the open-close dynamics of Ljd-LDH might be different from that of other d-LDHs. Moreover, thermostability experiments showed that the T50(10) value of Ljd-LDH (54.5°C) was much higher than the commercially available d-lactate dehydrogenase (42.7°C). In addition, Ljd-LDH has at least a 7°C higher denaturation temperature compared to commercially available d-LDHs. PMID:24794195

Kim, Sangwoo; Gu, Sol-A; Kim, Yong Hwan; Kim, Kyung-Jin

2014-07-01

104

Novel control of lactate dehydrogenase from the freeze tolerant wood frog: role of posttranslational modifications.  

PubMed

Lactate dehydrogenase (LDH), the terminal enzyme of anaerobic glycolysis, plays a crucial role both in sustaining glycolytic ATP production under oxygen-limiting conditions and in facilitating the catabolism of accumulated lactate when stress conditions are relieved. In this study, the effects on LDH of in vivo freezing and dehydration stresses (both of which impose hypoxia/anoxia stress on tissues) were examined in skeletal muscle of the freeze-tolerant wood frog, Rana sylvatica. LDH from muscle of control, frozen and dehydrated wood frogs was purified to homogeneity in a two-step process. The kinetic properties and stability of purified LDH were analyzed, revealing no significant differences in V max, K m and I 50 values between control and frozen LDH. However, control and dehydrated LDH differed significantly in K m values for pyruvate, lactate, and NAD, I 50 urea, and in temperature, glucose, and urea effects on these parameters. The possibility that posttranslational modification of LDH was responsible for the stable differences in enzyme behavior between control and dehydrated states was assessed using ProQ diamond staining to detect phosphorylation and immunoblotting to detect acetylation, methylation, ubiquitination, SUMOylation and nitrosylation of the enzyme. LDH from muscle of dehydrated wood frogs showed significantly lower levels of acetylation, providing one of the first demonstrations of a potential role for protein acetylation in the stress-responsive control of a metabolic enzyme. PMID:23638346

Abboud, Jean; Storey, Kenneth B

2013-01-01

105

Probing Lactate Dehydrogenase Activity in Tumors by Measuring Hydrogen/Deuterium Exchange in Hyperpolarized l-[1-13C,U-2H]Lactate  

PubMed Central

13C magnetic resonance spectroscopy and spectroscopic imaging measurements of hyperpolarized 13C label exchange between exogenously administered [1-13C]pyruvate and endogenous lactate, catalyzed by lactate dehydrogenase (LDH), has proved to be a powerful approach for probing tissue metabolism in vivo. This experiment has clinical potential, particularly in oncology, where it could be used to assess tumor grade and response to treatment. A limitation of the method is that pyruvate must be administered in vivo at supra-physiological concentrations. This problem can be avoided by using hyperpolarized [1-13C]lactate, which can be used at physiological concentrations. However, sensitivity is limited in this case by the relatively small pyruvate pool size, which would result in only low levels of labeled pyruvate being observed even if there was complete label equilibration between the lactate and pyruvate pools. We demonstrate here a more sensitive method in which a doubly labeled lactate species can be used to measure LDH-catalyzed exchange in vivo. In this experiment exchange of the C2 deuterium label between injected hyperpolarized l-[1-13C,U-2H]lactate and endogenous unlabeled lactate is observed indirectly by monitoring phase modulation of the spin-coupled hyperpolarized 13C signal in a heteronuclear 1H/13C spin–echo experiment.

2012-01-01

106

Lactate dehydrogenase isozymes in the trunk and cardiac muscles of an antarctic teleost fish, Notothenia neglecta Nybelin  

Microsoft Academic Search

The distribution and kinetics of lactate dehydrogenase (LDH) isozymes in the red and white trunk muscles, and cardiac muscle of an antarctic teleost fish (Notothenia neglecta Nybelin) have been studied. Pyruvate inhibition of LDH in all three muscle types is very low, being less than 50% even at a concentration of 60mM pyruvate. Activity versus pyruvate concentration profiles are not

Neil A. Fitch

1989-01-01

107

The effect of pressure on muscle lactate dehydrogenase activity of some deep-sea and shallow-water fishes  

Microsoft Academic Search

The activity of crude muscle lactate dehydrogenase (LDH) of several species of bathypelagic and shallow-water fishes has been measured at pressures between 1 and 578 atm and at temperatures of 15° and 25°C. No relationship has been found between the effect of pressure on enzyme activity and the hydrostatic pressure of the organism's environment. Applied hydrostatic pressure reduced activity at

R. G. Gillen

1971-01-01

108

The ontogenic characteristics of lactate dehydrogenase isozymes in mammaliam pre-implantation ova.  

PubMed

The onotgenic characteristics of lactate dehydrogenase (LDH) isozymes in mammalian pre-implantation ova have been reviewed. Evidence has been provided that the ova of mice and other mammalian species contain enzyme activity in a masked form, and display turnover processes which possess distinctive characteristics by comparison with those in adult tissues. Also, the extraordinary high levels of LDH in the extracellular secretion of the mammaliam oviduct have been commented on, along with the influence of reproductive hormones on the activity and type of this enzyme. In addition, attention has been drawn to the unique characteristics of the oval micro-evironment, and the influence which such factors may exert on the realization of enzyme phenotype during early mammalian development. PMID:353393

Masters, C J

1978-06-01

109

Evaluation of the intra- and inter-specific genetic variability of Plasmodium lactate dehydrogenase  

PubMed Central

Background Malaria diagnosis is vital to efficient control programmes and the recent advent of malaria rapid diagnostic tests (RDTs) provides a reliable and simple diagnostic method. However a characterization of the efficiency of these tests and the proteins they detect is needed to maximize RDT sensitivity. Methods Plasmodial lactate dehydrogenase (pLDH) gene of wild isolates of the four human species of Plasmodium from a variety of malaria endemic settings were sequenced and analysed. Results No variation in nucleotide was found within Plasmodium falciparum, synonymous mutations were found for Plasmodium malariae and Plasmodium. vivax; and three different types of amino acid sequence were found for Plasmodium ovale. Conserved and variable regions were identified within each species. Conclusion The results indicate that antigen variability is unlikely to explain variability in performance of RDTs detecting pLDH from cases of P. falciparum, P. vivax or P. malariae malaria, but may contribute to poor detection of P. ovale.

Talman, Arthur M; Duval, Linda; Legrand, Eric; Hubert, Veronique; Yen, Seiha; Bell, David; Le Bras, Jacques; Ariey, Frederic; Houze, Sandrine

2007-01-01

110

Synthesis, cytotoxicity for mimics of catalase: Inhibitors of lactate dehydrogenase and hypoxia inducible factor.  

PubMed

Lactate dehydrogenase A (LDH-A) is a potentially important metabolic target for the inhibition of the highly activated glycolysis pathway in cancer cells. Two Mn(II) complexes with ligand containing di(pyridylmethyl) amine and pyrrol-ketone were used to attenuate the activity of LDH-A. The inhibition of the manganese(II) complexes on the proliferation of HepG-2 cells is related to their ability to disproportionate H2O2. Importantly, the synthesized mimic of catalase can decrease the expression of hypoxia inducible factor (HIF-1?) in HepG-2 cells. So we envision that the multifunctional mimics of catalase could attenuate the activity of LDH-A signaling the cancer cells to death through HIF-1? involved path. PMID:24763359

Xue, Juan-Juan; Chen, Qiu-Yun; Kong, Meng-Yun; Zhu, Chun-Yin; Gen, Zhi-Rong; Wang, Zhi-Lin

2014-06-10

111

Complete Nucleotide Sequence of the Mouse Lactate Dehydrogenase-a Functional Gene: Comparison of the Exon-Intron Organization of Dehydrogenase Genes  

PubMed Central

The complete sequence of 12,851 nucleotides of the mouse lactate dehydrogenase-A (LDH-A) gene has been determined. It includes eight exons, seven introns, promoter and regulatory regions. The B1 repetitive elements present in intron III and VI are oriented in opposite orientation, and they share 72% sequence homology. The exon-intron organization of mouse LDH-A gene is compared with the organizations of other dehydrogenase genes, and the molecular evolution of the nicotinamide adenine dinucleotide binding domains is discussed.

Fukasawa, Kayoko M.; Li, Steven S.-L.

1987-01-01

112

Active and inactive state structures of unliganded Lactobacillus casei allosteric L-lactate dehydrogenase.  

PubMed

Lactobacillus casei L-lactate dehydrogenase (LCLDH) is activated through the homotropic and heterotropic activation effects of pyruvate and fructose 1,6-bisphosphate (FBP), respectively, and exhibits unusually high pH-dependence in the allosteric effects of these ligands. The active (R) and inactive (T) state structures of unliganded LCLDH were determined at 2.5 and 2.6 A resolution, respectively. In the catalytic site, the structural rearrangements are concerned mostly in switching of the orientation of Arg171 through the flexible intersubunit contact at the Q-axis subunit interface. The distorted orientation of Arg171 in the T state is stabilized by a unique intra-helix salt bridge between Arg171 and Glu178, which is in striking contrast to the multiple intersubunit salt bridges in Lactobacillus pentosus nonallosteric L-lactate dehydrogenase. In the backbone structure, major structural rearrangements of LCLDH are focused in two mobile regions of the catalytic domain. The two regions form an intersubunit linkage through contact at the P-axis subunit interface involving Arg185, replacement of which with Gln severely decreases the homotropic and hetertropic activation effects on the enzyme. These two regions form another intersubunit linkage in the Q-axis related dimer through the rigid NAD-binding domain, and thus constitute a pivotal frame of the intersubunit linkage for the allosteric motion, which is coupled with the concerted structural change of the four subunits in a tetramer, and of the binding sites for pyruvate and FBP. The unique intersubunit salt bridges, which are observed only in the R state structure, are likely involved in the pH-dependent allosteric equilibrium. PMID:19787773

Arai, Kazuhito; Ishimitsu, Toshihiro; Fushinobu, Shinya; Uchikoba, Hiroyuki; Matsuzawa, Hiroshi; Taguchi, Hayao

2010-02-15

113

Lactate Dehydrogenase A is a potential prognostic marker in clear cell renal cell carcinoma  

PubMed Central

Background Over 90% of cancer-related deaths in clear cell renal cell carcinoma (RCC) are caused by tumor relapse and metastasis. Thus, there is an urgent need for new molecular markers that can potentiate the efficacy of the current clinical-based models of prognosis assessment. The objective of this study is to evaluate the potential significance of lactate dehydrogenase A (LDHA), assessed by immunohistochemical staining, as a prognostic marker in clear cell renal cell carcinoma in relation to clinicopathological features and clinical outcome. Methods We assessed the expression of LDHA at the protein level, by immunohistochemistry, and correlated its expression with multiple clinicopathological features including tumor size, clinical stage, histological grade, disease-free and overall survival in 385 patients with primary clear cell renal cell carcinoma. We also correlated the LDHA expression with overall survival, at mRNA level, in an independent data set of 170 clear cell renal cell carcinoma cases from The Cancer Genome Atlas databases. Cox proportional hazards models adjusted for the potential clinicopathological factors were used to test for associations between the LDHA expression and both disease-free survival and overall survival. Results There is statistically significant positive correlation between LDHA level of expression and tumor size, clinical stage and histological grade. Moreover, LDHA expression shows significantly inverse correlation with both disease-free survival and overall survival in patients with clear cell renal cell carcinoma. Our results are validated by examining LDHA expression, at the mRNA level, in the independent data set of clear cell renal cell carcinoma cases from The Cancer Genome Atlas databases which also shows that higher lactate dehydrogenase A expression is associated with significantly shorter overall survival. Conclusion Our results indicate that LDHA up-regulation can be a predictor of poor prognosis in clear cell renal cell carcinoma. Thus, it represents a potential prognostic biomarker that can boost the accuracy of other prognostic models in patients with clear cell renal cell carcinoma.

2014-01-01

114

The approach to the Michaelis complex in lactate dehydrogenase: the substrate binding pathway.  

PubMed

We examine here the dynamics of forming the Michaelis complex of the enzyme lactate dehydrogenase by characterizing the binding kinetics and thermodynamics of oxamate (a substrate mimic) to the binary lactate dehydrogenase/NADH complex over multiple timescales, from nanoseconds to tens of milliseconds. To access such a wide time range, we employ standard stopped-flow kinetic approaches (slower than 1 ms) and laser-induced temperature-jump relaxation spectroscopy (10 ns-10 ms). The emission from the nicotinamide ring of NADH is used as a marker of structural transformations. The results are well explained by a kinetic model that has binding taking place via a sequence of steps: the formation of an encounter complex in a bimolecular step followed by two unimolecular transformations on the microsecond/millisecond timescales. All steps are well described by single exponential kinetics. It appears that the various key components of the catalytically competent architecture are brought together as separate events, with the formation of strong hydrogen bonding between active site His(195) and substrate early in binding and the closure of the catalytically necessary protein surface loop over the bound substrate as the final event of the binding process. This loop remains closed during the entire period that chemistry takes place for native substrates; however, motions of other key molecular groups bringing the complex in and out of catalytic competence appear to occur on faster timescales. The on-enzyme K(d) values (the ratios of the microscopic rate constants for each unimolecular step) are not far from one. Either substantial, approximately 10-15%, transient melting of the protein or rearrangements of hydrogen bonding and solvent interactions of a number of water molecules or both appear to take place to permit substrate access to the protein binding site. The nature of activating the various steps in the binding process seems to be one overall involving substantial entropic changes. PMID:15980172

McClendon, Sebastian; Zhadin, Nick; Callender, Robert

2005-09-01

115

Molecular cloning and characterization of lactate dehydrogenase gene 1 in the silkworm, Bombyx mori.  

PubMed

Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate into lactate and constitutes a major checkpoint of anaerobic glycolysis. Recently, LDH draws a great deal of attention for its potential to be used as a novel diagnostic and therapeutic target for various diseases, including cancer and malaria. Insect LDHs have been mainly identified from fruit fly and mosquitoes, but not from silkworm. In this study, a novel LDH homologue, designated as BmLDH1, was firstly identified and characterized from the silkworm, Bombyx mori. The BmLDH1 cDNA contains an open reading frame of 996 bp, and encodes a protein of 331 amino acid residues with calculated molecular mass of 36 kDa. Sequence comparison showed BmLDH1 is a highly conserved protein. RT-PCR revealed BmLDH1 is transcripted in all tissues and in all developmental stages tested, indicating its essential roles for silkworm physiology and development. The BmLDH1 gene was subcloned and expressed in E. coli, and was further characterized by Western blot and Mass Spectrometry. The expressed protein contained the LDH activity, and could be inhibited by reduced glutathione in vitro. Immunofluoresence showed that the BmLDH1 was located in the cytoplasm. The cloned BmLDH1 sequence was deposited in the GenBank (accession number EU334850). PMID:20852941

Xia, Hengchuan; Wu, Chao; Xu, Qinggang; Shi, Jing; Feng, Fan; Chen, Keping; Yao, Qin; Wang, Yong; Wang, Lin

2011-03-01

116

Testis-specific lactate dehydrogenase is expressed in somatic tissues of plateau pikas?  

PubMed Central

LDH-C4 is a lactate dehydrogenase that catalyzes the interconversion of pyruvate with lactate. In mammals the, Ldh-c gene was originally thought to be expressed only in testis and spermatozoa. Plateau pika (Ochotona curzoniae), belonging to the genus Ochotona of the Ochotonidea family, is a hypoxia tolerant mammal living at 3000–5000 m above sea levelon the Qinghai-Tibet Plateau. We found that the expression pattern of six LDH isoenzymes in the somatic tissues of female and male plateau pikas to be the same as those in testis and sperm, suggesting that LDH-C4 was expressed in somatic tissues of plateau pika. Here we report the detection of LDHC in the somatic tissues of plateau pika using RT-PCR, Western blotting and immunohistochemistry. Our results indicate that Ldh-c mRNA is transcribed in the heart, liver, lung, kidney, brain, skeletal muscle and testis. In somatic tissues LDHC was translated in the cytoplasm, while in testis it was expressed in both cytoplasm and mitochondria. The third band from cathode to anode in LDH isoenzymes was identified as LDH-C4. The finding that Ldh-c is expressed in both somatic tissues and testis of plateau pika provides important implications for more in-depth research into the Ldh-c function in mammals.

Wang, Duowei; Wei, Lian; Wei, Dengbang; Rao, Xinfeng; Qi, Xinzhang; Wang, Xiaojun; Ma, Benyuan

2013-01-01

117

Structural characterization of the apo form and NADH binary complex of human lactate dehydrogenase.  

PubMed

Lactate dehydrogenase A (LDH-A) is a key enzyme in anaerobic respiration that is predominantly found in skeletal muscle and catalyses the reversible conversion of pyruvate to lactate in the presence of NADH. LDH-A is overexpressed in many tumours and has therefore emerged as an attractive target for anticancer drug discovery. Crystal structures of human LDH-A in the presence of inhibitors have been described, but currently no structures of the apo or binary NADH-bound forms are available for any mammalian LDH-A. Here, the apo structure of human LDH-A was solved at a resolution of 2.1 Å in space group P4122. The active-site loop adopts an open conformation and the packing and crystallization conditions suggest that the crystal form is suitable for soaking experiments. The soaking potential was assessed with the cofactor NADH, which yielded a ligand-bound crystal structure in the absence of any inhibitors. The structures show that NADH binding induces small conformational changes in the active-site loop and an adjacent helix. A comparison with other eukaryotic apo LDH structures reveals the conservation of intra-loop interactions. The structures provide novel insight into cofactor binding and provide the foundation for soaking experiments with fragments and inhibitors. PMID:24816116

Dempster, Sally; Harper, Stephen; Moses, John E; Dreveny, Ingrid

2014-05-01

118

Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression.  

PubMed

As the result of genetic alterations and tumor hypoxia, many cancer cells avidly take up glucose and generate lactate through lactate dehydrogenase A (LDHA), which is encoded by a target gene of c-Myc and hypoxia-inducible factor (HIF-1). Previous studies with reduction of LDHA expression indicate that LDHA is involved in tumor initiation, but its role in tumor maintenance and progression has not been established. Furthermore, how reduction of LDHA expression by interference or antisense RNA inhibits tumorigenesis is not well understood. Here, we report that reduction of LDHA by siRNA or its inhibition by a small-molecule inhibitor (FX11 [3-dihydroxy-6-methyl-7-(phenylmethyl)-4-propylnaphthalene-1-carboxylic acid]) reduced ATP levels and induced significant oxidative stress and cell death that could be partially reversed by the antioxidant N-acetylcysteine. Furthermore, we document that FX11 inhibited the progression of sizable human lymphoma and pancreatic cancer xenografts. When used in combination with the NAD(+) synthesis inhibitor FK866, FX11 induced lymphoma regression. Hence, inhibition of LDHA with FX11 is an achievable and tolerable treatment for LDHA-dependent tumors. Our studies document a therapeutical approach to the Warburg effect and demonstrate that oxidative stress and metabolic phenotyping of cancers are critical aspects of cancer biology to consider for the therapeutical targeting of cancer energy metabolism. PMID:20133848

Le, Anne; Cooper, Charles R; Gouw, Arvin M; Dinavahi, Ramani; Maitra, Anirban; Deck, Lorraine M; Royer, Robert E; Vander Jagt, David L; Semenza, Gregg L; Dang, Chi V

2010-02-01

119

Structural characterization of the apo form and NADH binary complex of human lactate dehydrogenase  

PubMed Central

Lactate dehydrogenase A (LDH-A) is a key enzyme in anaerobic respiration that is predominantly found in skeletal muscle and catalyses the reversible conversion of pyruvate to lactate in the presence of NADH. LDH-A is overexpressed in many tumours and has therefore emerged as an attractive target for anticancer drug discovery. Crystal structures of human LDH-A in the presence of inhibitors have been described, but currently no structures of the apo or binary NADH-bound forms are available for any mammalian LDH-A. Here, the apo structure of human LDH-A was solved at a resolution of 2.1?Å in space group P4122. The active-site loop adopts an open conformation and the packing and crystallization conditions suggest that the crystal form is suitable for soaking experiments. The soaking potential was assessed with the cofactor NADH, which yielded a ligand-bound crystal structure in the absence of any inhibitors. The structures show that NADH binding induces small conformational changes in the active-site loop and an adjacent helix. A comparison with other eukaryotic apo LDH structures reveals the conservation of intra-loop interactions. The structures provide novel insight into cofactor binding and provide the foundation for soaking experiments with fragments and inhibitors.

Dempster, Sally; Harper, Stephen; Moses, John E.; Dreveny, Ingrid

2014-01-01

120

A molecular design that stabilizes active state in bacterial allosteric L-lactate dehydrogenases.  

PubMed

l-Lactate dehydrogenase (l-LDH) of Lactobacillus casei (LCLDH) is a typical bacterial allosteric l-LDH that requires fructose 1,6-bisphosphate (FBP) for its enzyme activity. A mutant LCLDH was designed to introduce an inter-subunit salt bridge network at the Q-axis subunit interface, mimicking Lactobacillus pentosus non-allosteric l-LDH (LPLDH). The mutant LCLDH exhibited high catalytic activity with hyperbolic pyruvate saturation curves independently of FBP, and virtually the equivalent K(m) and V(m) values at pH 5.0 to those of the fully activated wild-type enzyme with FBP, although the K(m) value was slightly improved with FBP or Mn(2+) at pH 7.0. The mutant enzyme exhibited a markedly higher apparent denaturating temperature (T(1/2)) than the wild-type enzyme in the presence of FBP, but showed an even lower T(1/2) without FBP, where it exhibited higher activation enthalpy of inactivation (?H(‡)). This result is consistent with the fact that the active state is more unstable than the inactive state in allosteric equilibrium of LCLDH. The LPLDH-like network appears to be conserved in many bacterial non-allosteric l-LDHs and dimeric l-malate dehydrogenases, and thus to be a key for the functional divergence of bacterial l-LDHs during evolution. PMID:21828088

Arai, Kazuhito; Ichikawa, Jun; Nonaka, Shinta; Miyanaga, Akimasa; Uchikoba, Hiroyuki; Fushinobu, Shinya; Taguchi, Hayao

2011-11-01

121

Difference spectroscopic and kinetic studies on the interaction of lactate dehydrogenase with structurally related triazine dyes.  

PubMed

Difference spectroscopy and enzyme kinetics were employed to study the interaction of lactate dehydrogenase (LDH) from rabbit muscle with the azo-dye Procion Red HE-3B and two of its structural variants in order to follow the significance of the sulphonated terminal rings for the strength and specificity of binding. Procion Red HE-3B possesses a significantly higher affinity to LDH compared to the dye Cibacron Blue F3G-A, a well characterized pseudo-biospecific ligand of dehydrogenases. Moreover, Procion Red HE-3B showed competition towards the cofactor NAD+/NADH. The enzyme-dye complex is mainly stabilized by hydrophobic interactions, but other binding forces cannot be excluded. LDH possesses one dye-binding site per subunit. As a binding region the active center of LDH, preferentially the hydrophobic nicotinamide pocket is involved. Removal of the negatively charged sulphonic acid group from the terminal rings of Procion Red HE-3B decreases the affinity to LDH significantly but does not change the type of binding. Addition of an anilino group to the terminal rings of Procion Red HE-3B does not affect the affinity to the active site significantly but enables the binding on other sites with lower affinity in dependence on the dye concentration. PMID:1824535

Cadelis, F; Kirchberger, J; Vijayalakshmi, M A; Kopperschläger, G

1991-01-01

122

Analysis of lactate and malate dehydrogenase enzyme profiles of selected major carps of wetland of Calcutta.  

PubMed

The East Calcutta Wetland (ECW), a Ramsar site in India, acts as the only sink for both city sewages as well as effluents from the surrounding small-scale industries and is alarmingly polluted with heavy metals. The three best edible major carp species rohu (Labeo rohita,), catla (Catla catla,) and mrigala (Cirrhinus mrigala) were undertaken to monitor lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) by cellulose acetate electrophoresis (CAE) to assess the effects of pollutants, if any. Crude tissue extracts were prepared from brain, eye, heart, skeletal muscle and kidney tissue respectively from each type of fish. No differences were not found in MDH of catla from both sites for all tissues analyzed in this study. Rohu also showed similar mobility for all tissues except for heart tissue which was distinctly different in fishes from ECW site than that of its counterpart from non ECW site. On the other hand, MDH of two tissues of mrigala, eye and muscle respectively showed different migration patterns. LDH profiles for all tissues of three fish species from both the sites were consistently similar, only the expression levels of muscle LDH of mrigala and kidney LDH of rohu varied little. PMID:23360005

Manna, Madhumita; Chakraborty, Priyanka

2012-07-01

123

Design, synthesis, and biological evaluation of Plasmodium falciparum lactate dehydrogenase inhibitors.  

PubMed

Plasmodium falciparum lactate dehydrogenase (pfLDH) is a key enzyme for energy generation of malarial parasites and is a potential antimalarial chemotherapeutic target. It is known that the oxamate moiety, a pyruvate analog, alone shows higher inhibition against pfLDH than human LDHs, suggesting that it can be used for the development of selective inhibitors. Oxamic acid derivatives were designed and synthesized. Derivatives 5 and 7 demonstrated activities against pfLDH with IC50 values of 3.13 and 1.75 muM, respectively, and have 59- and 7-fold selectivity over mammalian LDH, respectively. They also have micromolar range activities against Plasmodium falciparum malate dehydrogenase (pfMDH), which may fill the role of pfLDH when the activity of pfLDH is reduced. Thus, certain members of these oxamic acid derivatives may have dual inhibitory activities against both pfLDH and pfMDH. It is presumed that dual LDH/MDH inhibitors would have enhanced potential as antimalarial drugs. PMID:17636950

Choi, Seoung-ryoung; Pradhan, Anupam; Hammond, Nicholas L; Chittiboyina, Amar G; Tekwani, Babu L; Avery, Mitchell A

2007-08-01

124

Reactions upstream of glycerate-1,3-bisphosphate drive Corynebacterium glutamicum (D)-lactate productivity under oxygen deprivation.  

PubMed

We previously demonstrated the simplicity of oxygen-deprived Corynebacterium glutamicum to produce D-lactate, a primary building block of next-generation biodegradable plastics, at very high optical purity by introducing heterologous D-ldhA gene from Lactobacillus delbrueckii. Here, we independently evaluated the effects of overexpressing each of genes encoding the ten glycolytic enzymes on D-lactate production in C. glutamicum. We consequently show that while the reactions catalyzed by glucokinase (GLK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase had positive effects on D-lactate productivity by increasing 98, 39, 15, 13, and 10 %, respectively, in 10 h reactions in minimal salts medium, the reaction catalyzed by pyruvate kinase had large negative effect by decreasing 70 %. The other glycolytic enzymes did not affect D-lactate productivity when each of encoding genes was overexpressed. It is noteworthy that all reactions associated with positive effects are located upstream of glycerate-1,3-bisphosphate in the glycolytic pathway. The D-lactate yield also increased by especially overexpressing TPI encoding gene up to 94.5 %. Interestingly, overexpression of PFK encoding gene reduced the yield of succinate, one of the main by-products of D-lactate production, by 52 %, whereas overexpression of GAPDH encoding gene increased succinate yield by 26 %. Overexpression of GLK encoding gene markedly increased the yield of dihydroxyacetone and glycerol by 10- and 5.8-fold in exchange with decreasing the D-lactate yield. The effect of overexpressing glycolytic genes was also evaluated in 80 h long-term reactions. The variety of effects of overexpressing each of genes encoding the ten glycolytic enzymes on D-lactate production is discussed. PMID:23712891

Tsuge, Yota; Yamamoto, Shougo; Suda, Masako; Inui, Masayuki; Yukawa, Hideaki

2013-08-01

125

Physiological and fermentation properties of Bacillus coagulans and a mutant lacking fermentative lactate dehydrogenase activity.  

PubMed

Bacillus coagulans, a sporogenic lactic acid bacterium, grows optimally at 50-55 °C and produces lactic acid as the primary fermentation product from both hexoses and pentoses. The amount of fungal cellulases required for simultaneous saccharification and fermentation (SSF) at 55 °C was previously reported to be three to four times lower than for SSF at the optimum growth temperature for Saccharomyces cerevisiae of 35 °C. An ethanologenic B. coagulans is expected to lower the cellulase loading and production cost of cellulosic ethanol due to SSF at 55 °C. As a first step towards developing B. coagulans as an ethanologenic microbial biocatalyst, activity of the primary fermentation enzyme L-lactate dehydrogenase was removed by mutation (strain Suy27). Strain Suy27 produced ethanol as the main fermentation product from glucose during growth at pH 7.0 (0.33 g ethanol per g glucose fermented). Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH) acting in series contributed to about 55% of the ethanol produced by this mutant while pyruvate formate lyase and ADH were responsible for the remainder. Due to the absence of PDH activity in B. coagulans during fermentative growth at pH 5.0, the l-ldh mutant failed to grow anaerobically at pH 5.0. Strain Suy27-13, a derivative of the l-ldh mutant strain Suy27, that produced PDH activity during anaerobic growth at pH 5.0 grew at this pH and also produced ethanol as the fermentation product (0.39 g per g glucose). These results show that construction of an ethanologenic B. coagulans requires optimal expression of PDH activity in addition to the removal of the LDH activity to support growth and ethanol production. PMID:20677017

Su, Yue; Rhee, Mun Su; Ingram, Lonnie O; Shanmugam, K T

2011-03-01

126

Molecular basis of evolutionary adaptation at the lactate dehydrogenase-B locus in the fish Fundulus heteroclitus.  

PubMed

At the extremes of its natural distribution, populations of the common killifish Fundulus heteroclitus experience a difference of more than 15 degrees C in mean annual temperature. These populations are virtually fixed for two different codominant alleles at the heart-type lactate dehydrogenase locus (Ldh-B) which code for allozymes with different and adaptive kinetic responses to temperature. Two populations near the extremes of the species range (i.e., Maine and Georgia) were further studied for thermal adaptation at this locus. In the absence of any kinetic differences one would predict that to maintain a constant reaction velocity, 2 to 3 times as much enzyme would be required for each 10 degrees C decrease in environmental temperature. Consistent with this adaptive strategy and in addition to the adaptive kinetic characteristics, the LDH-B4 enzyme (EC 1.1.1.27) concentration and its mRNA concentration were approximately twice as great in the northern population as in the southern population. Acclimation experiments allow us to conclude that these differences are due to a combination of fixed genetic traits (evolutionary adaptation) and plastic responses to temperature (physiological acclimation). Furthermore, our calculations show that the LDH-B4 reaction velocities are essentially equivalent for these two populations, even though they live in significantly different thermal environments. PMID:2594773

Crawford, D L; Powers, D A

1989-12-01

127

Genetic and physiological analysis of the lethal effect of L-(+)-lactate dehydrogenase deficiency in Streptococcus mutans: complementation by alcohol dehydrogenase from Zymomonas mobilis.  

PubMed Central

CH4ts is a previously isolated recombinant mutant of Streptococcus mutans NG8 which produces a thermolabile L-(+)-lactate dehydrogenase (LDH) activity. It does not grow at 42 degrees C under a variety of cultivation conditions. In this study, we show that a batch culture of CH4ts shifted from 30 to 42 degrees C underwent rapid cessation of growth and accelerated cell death. The mutant grew at 42 degrees C in continuous culture under glucose-limiting conditions. Under these conditions, lactate production was replaced by production of ethanol and, to a smaller extent, acetoin. The cloned Zymomonas mobilis gene for alcohol dehydrogenase II, placed under the control of the S. mutans spaP regulatory signals, complemented LDH deficiency. The alcohol dehydrogenase-complemented mutant grew as well or better than NG8 on a variety of carbon sources at 42 degrees C and produced significant amounts of ethanol in place of lactic acid. These results are in accord with other approaches indicating that S. mutans has other enzymatic activities, including pyruvate formate-lyase and pyruvate dehydrogenase, for pyruvate metabolism. However, at high glucose concentrations, the levels of activity of these enzymes are apparently insufficient to compensate for the absence of LDH.

Hillman, J D; Chen, A; Snoep, J L

1996-01-01

128

Oxidation of D-lactate and L-lactate by Neisseria meningitidis: purification and cloning of meningococcal D-lactate dehydrogenase.  

PubMed Central

Neisseria meningitidis was found to contain at least two lactate-oxidizing enzymes. One of these was purified 460-fold from spheroplast membranes and found to be specific primarily for D-lactate, with low-affinity activity for L-lactate. The gene for this enzyme (dld) was cloned, and a dld mutant was constructed by insertional inactivation of the gene. The mutant was unable to grow on D-lactate but retained the ability to grow on L-lactate, providing evidence for a second lactate-oxidizing enzyme with specificity for L-lactate. High-affinity L-lactate-oxidizing activity was detected in intact bacteria of both the dld+ and dld mutant strains. This L-lactate-oxidizing activity was also seen in sonicated bacteria but was reduced substantially on detergent solubilization or on preparation of spheroplast membranes. Images

Erwin, A L; Gotschlich, E C

1993-01-01

129

Mechanism of reaction of allylamine with the quinoprotein methylamine dehydrogenase.  

PubMed Central

Allylamine did not serve as an efficient substrate for methylamine dehydrogenase (EC 1.4.99.3) in a steady-state assay of activity and appeared to act as a competitive inhibitor of methylamine oxidation by methylamine dehydrogenase. Transient kinetic studies, however, revealed that allylamine rapidly reduced the tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase. The rate of TTQ reduction by allylamine was 322 s-1, slightly faster than the rate of reduction by methylamine. These data were explained by a kinetic mechanism in which allylamine and methylamine are alternative substrates for methylamine dehydrogenase. The apparent competitive inhibition by allylamine is due to a very slow rate of release of the aldehyde product, 0.28 s-1, relative to a rate of 18.6 s-1 for the release of the aldehyde product of methylamine oxidation. A reaction mechanism is proposed for the oxidative deamination of allylamine by methylamine dehydrogenase. This mechanism is discussed in relation to the reaction mechanisms of topa-bearing quinoprotein amine oxidases, the flavoprotein monoamine oxidase and the mammalian semicarbazide-sensitive amine oxidase.

Davidson, V L; Graichen, M E; Jones, L H

1995-01-01

130

A review of the use of serum lactate dehydrogenase measurement in patients presenting to the paediatric emergency department  

Microsoft Academic Search

AimThe aim of our investigation was to review the use of serum lactate dehydrogenase (LDH) testing in the Paediatric Emergency Department and to develop an appropriate algorithm for its use.MethodsWe obtained case records of patients that had serum LDH measured in the Department over a 3 year period and examined the justification and result of each test and any subsequent

A D Ross; E Abrahamson

2011-01-01

131

Inhibitor screening of lactate dehydrogenase C4 from black-lipped pika in the Western Sichuan Plateau.  

PubMed

Studies indicated that lactate dehydrogenase C4 (LDH-C4) was a good target protein for development of contraceptive drugs. Virtual screening and in vitro enzyme assay using pika LDH-C4 as target protein revealed NSC61610, NSC215718, and NSC345647 with Ki of 7.8, 27, and 41??M separately. This study might be helpful for development of pika contraceptive drugs. PMID:25036963

Zhang, Qinglian; He, Qinghua; Xue, Fulai; Ma, Jinhu

2014-04-01

132

Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes  

Microsoft Academic Search

To elucidate mechanisms of enzymatic ad- aptation to extreme cold, we determined kinetic properties, thermal stabilities, and deduced amino acid sequences of lactate dehydrogenase A4 (A4-LDH) from nine Antarctic (21.86 to 1°C) and three South American (4 to 10°C) noto- thenioid teleosts. Higher Michaelis-Menten constants (Km) and catalytic rate constants (kcat) distinguish orthologs of Antarctic from those of South American

PETER A. FIELDS; GEORGE N. SOMERO

1998-01-01

133

Expression of LDH-C Isozyme among Lizard Taxa: Evolutionary Implications for the Vertebrate Lactate Dehydrogenase Gene Family  

Microsoft Academic Search

Chien-Hsien Kuo, San Kao, Ching-Feng Weng and Sin-Che Lee (1999) Expression of LDH-C isozyme among lizard taxa: evolutionary implications for the vertebrate lactate dehydrogenase gene family. Zoological Studies 38(3): 344-349. In order to more completely understand the complete basis for the multiple LDH isozymes in lizards, seven species of Taiwanese lizards belonging to 4 families and 7 genera were sampled.

Chien-Hsien Kuo; San Kao; Ching-Feng Weng; Sin-Che Lee

1999-01-01

134

Analysis of a genetic mutation in an electrophoretic variant of the human lactate dehydrogenase-B(H) subunit  

Microsoft Academic Search

An electrophoretic variant of the lactate dehydrogenase (LDH)-B(H) subunit was discovered in a patient with diabetes mellitus. His LDH activity in serum was slightly lower than normal and the LDH isozyme pattern showed an abnormal migration indicating an LDH-B subunit variant of the fast type. The LDH containing the variant subunit revealed a decreased heat stability. DNA analysis of the

Masato Maekawa; Kayoko Sudo; Masato Kitajima; Yukio Matsuura; Steven S.-L. Li; Takashi Kanno

1993-01-01

135

Observation and identification of lactate dehydrogenase anomaly in a postburn patient  

PubMed Central

Objective: Lactate dehydrogenase (LDH) anomaly is one of the macroenzymes. Macroenzymes are enzymes in serum that have formed high molecular mass complexes, either by self polymerisation or by association with other serum components. The aim of this study was to identify the properties of LDH anomaly and observe the changes from admission to discharge in a postburn patient with LDH anomaly in his serum. Methods: LDH isoenzymes of the serum were electrophoretically fractionated with terylene cellulose acetate supporting media; LDH anomaly was identified by counter immunoelectrophoresis. Results: An abnormal LDH-4 band and an extra band on the cathode of LDH-5 were observed in the serum of this patient and were found to be part of an LDH-IgG complex. As his symptoms improved, the patient's LDH anomaly gradually disappeared. The appearance and disappearance of the anomaly seemed to be related to the progression of the patient's burns. Conclusion: In clinical practice, it is important to keep in mind the possibility of an LDH anomaly in patients when the LDH level is abnormally high or does not seem to be related to the clinical state. Early discovery of an LDH anomaly in a patient's serum may be useful for diagnosis and treatment.

Liu, Z; Zhang, Y; Zhang, X; Yang, X

2004-01-01

136

Glucose levels and succinate and lactate dehydrogenase activity in EMT6/Ro tumor spheroids.  

PubMed

To evaluate the effects of glucose on the development of cell heterogeneity and the occurrence of necrotic areas in the center of tumor spheroids, a procedure (combining microdissection of small tissue samples from frozen-dried cryosections and microchemical analysis) was developed to measure glucose in distinct, concentrically arranged, microregions of tumor spheroids: the outermost area of proliferating cells, the area of nonproliferating cells and 2 central "necrotic" areas, with either abundant or little intercellular space. Since glucose levels, for analytical reasons, had to be expressed on a dry weight basis, and because of the morphological heterogeneity of the microregions of tumor spheroids, it was necessary to measure and take into account the regional differences in cell density (water content), in order to obtain adequate estimates of the glucose levels in the various microregions. At glucose concentrations of 5.5 and 3.6 mM in the culture medium, the glucose levels varied between 3.5 and 1.4 mmoles/kg wet weight and were lowest in those central areas where the cell density was lowest. Histochemical demonstration of the distribution of lactate and succinate dehydrogenase activity indicates a considerably higher capacity of tumor cells for anaerobic than for aerobic energy production. PMID:7774614

Teutsch, H F; Goellner, A; Mueller-Klieser, W

1995-03-01

137

Molecular Nature of Spontaneous Mutations in Mouse Lactate Dehydrogenase-a Processed Pseudogenes  

PubMed Central

The presence of at least ten mouse LDH-A pseudogenes was demonstrated in the genomic blot analysis, and four different processed pseudogenes have thus far been isolated and characterized. In this report, the nucleotide sequences of two different mouse lactate dehydrogenase-A processed pseudogenes, M11 and M14, were determined and compared with the protein-coding sequences of the mouse and rat LDH-A functional genes. In the pseudogene M11, the sequence of 64 nucleotides from codon no. 257 to 278 was tandemly duplicated. In the pseudogene M14, the sequence of 22 nucleotides from codon no. 68 to 75 was replaced by an inserted repetitive sequence of 242 nucleotides homologous to a mouse truncated R element. The pattern of nucleotide substitutions accumulated in mouse LDH-A pseudogenes M11 and M14, as well as that of pseudogene M10 identified previously, was analyzed, and the substitution frequencies of the C or G at the CG dinucleotide were found to be high.

Fukasawa, Kayoko M.; Tanimura, Masako; Sakai, Ikuya; Sharief, Farida S.; Chung, Fu-Zon; Li, Steven S.-L.

1987-01-01

138

Serum lactate dehydrogenase isoenzyme 1 in patients with seminoma stage I followed with surveillance.  

PubMed

Serum lactate dehydrogenase isoenzyme I catalytic concentration (S-LD-1) was measured in patients with testicular seminoma clinical stage I followed with surveillance after orchiectomy. The serum samples were obtained before orchiectomy in 110 patients (group A) and soon after orchiectomy in 55 patients (group B). In group A, 60 patients (55%) had elevated S-LD-1 and 10 patients (9%) had elevated serum human chorionic gonadotropin concentrations (S-hCG). In group B, median S-LD-1 was lower than that of group A and decreased with increasing time after orchiectomv (p = 0.001, Jonckheere-Terpstra test, one-sided). After a median follow-up of 5.1 years, 23 patients (21%) in group A had relapses. The patients with elevated S-LD-1 and those with normal S-LD-1 had a similar relapse-free survival (p = 0.79, log-rank test). Thus patients with seminoma stage I had elevated S-LD-1 more often than elevated S-hCG but an elevation in S-LD-1 did not predict a relapse during follow-up with surveillance. Further studies are required to elucidate the value of S-LD-1 in monitoring the surveillance of patients with seminoma stage I. PMID:11990523

von Eyben, Finn Edler; Madsen, Ebbe Lindegaard; Blaabjerg, Ole; Petersen, Per Hyltoft; Jacobsen, Grete Krag; Specht, Lena; Pedersen, Bent Nørgaard; von der Maase, Hans

2002-01-01

139

Interaction of lactate dehydrogenase with structurally related triazine dyes using affinity partitioning and affinity chromatography.  

PubMed

Affinity partitioning in aqueous two-phase systems consisting of dextran and dye-liganded polyethylene glycol was employed to study the interaction of lactate dehydrogenase (LDH) from rabbit muscle (E.C. 1.1.1.27) with Procion Red HE-3B and four structurally related derivatives of this dye in order to follow the significance of the terminal rings of Procion Red HE-3B for the strength of interaction. The study revealed that the arrangement of the two 1-amino-8-naphthol-3,6-disulphonic acid rings seems to be a prerequisite for the interaction of azonaphthol dyes with LDH. The negatively charged sulfonic acid group at the terminal rings of Procion Red HE-3B enhances the affinity of the ligand for LDH significantly. The removal of this sulphonic acid group or splitting off the complete terminal rings decreases the affinity to LDH and improves the competitive effect of NAD+. The results of affinity partitioning are compared with those of affinity chromatography and kinetic data. The usefulness and the choice of parameters of affinity partitioning as an analytical tool to predict the chromatographic behaviour of dye ligands are discussed. PMID:2625437

Kirchberger, J; Cadelis, F; Kopperschläger, G; Vijayalakshmi, M A

1989-12-01

140

Muscular cholinesterase and lactate dehydrogenase activities in deep-sea fish from the NW Mediterranean.  

PubMed

Organisms inhabiting submarine canyons can be potentially exposed to higher inputs of anthropogenic chemicals than their counterparts from the adjacent areas. To find out to what extend this observation applies to a NW Mediterranean canyon (i.e. Blanes canyon) off the Catalan coast, four deep-sea fish species were collected from inside the canyon (BC) and the adjacent open slope (OS). The selected species were: Alepocephalus rostratus, Lepidion lepidion, Coelorinchus mediterraneus and Bathypterois mediterraneus. Prior to the choice of an adequate sentinel species, the natural variation of the selected parameters (biomarkers) in relation to factors such as size, sex, sampling depth and seasonality need to be characterised. In this study, the activities of cholinesterases (ChEs) and lactate dehydrogenase (LDH) enzymes were determined in the muscle of the four deep-sea fish. Of all ChEs, acetylcholinesterase (AChE) activity was dominant and selected for further monitoring. Overall, AChE activity exhibited a significant relationship with fish size whereas LDH activity was mostly dependent on the sex and gonadal development status, although in a species-dependent manner. The seasonal variability of LDH activity was more marked than for AChE activity, and inside-outside canyon (BC-OS) differences were not consistent in all contrasted fish species, and in fact they were more dependent on biological traits. Thus, they did not suggest a differential stress condition between sites inside and outside the canyon. PMID:24296242

Koenig, Samuel; Solé, Montserrat

2014-03-01

141

Characterization of lactate dehydrogenase isozyme pattern and morphology of three marine fish cell lines  

NASA Astrophysics Data System (ADS)

Three continuous marine fish cell lines of FG (i.e., Flounder Gill) from flounder ( Paralichthys olivaceus) gill, SPH (i.e., Sea Perch Heart) from sea perch ( Lateolabrax japonicus) heart and RSBF (i.e., Red Sea Bream Fin) from red sea bream ( Pagrosomus major) fin, were characterized by lactate dehydrogenase (LDH) isozyme and morphological analysis. The LDH isozyme patterns of these three cell lines and their corresponding tissues of origin were investigated and compared. The results showed: (1) No difference was found in the LDH isozyme patterns of FG and flounder gill tissue. However, the LDH isozyme patterns of SPH and RSBF were significantly different from their corresponding tissues of origin; (2) LDH isozyme patterns of FG, SPH and RSBF were markedly different from each other and could serve as genetic markers for species identification and detection of cross contamination. Morphological change analysis of these three cell lines in comparison to their original tissues indicated that FG cells still appeared epithelioid without morphological transformation. However, morphological changes were found in SPH and RSBF compared to their original tissues. Therefore, the cellular morphology was still plastic in the relatively stable culture conditions, and it was possible that change of LDH patterns was related to morphological changes of fish cells in vitro.

Guo, Hua-Rong; Zhang, Shi-Cui; Li, Hong-Yan; Tong, Shang-Liang; Xiang, Jian-Hai

2002-09-01

142

Identification of proteins interacting with lactate dehydrogenase in claw muscle of the porcelain crab Petrolisthes cinctipes  

PubMed Central

Biochemical adaptation of enzymes involves conservation of activity, stability and affinity across a wide range of intracellular and environmental conditions. Enzyme adaptation by alteration of primary structure is well known, but the roles of protein-protein interactions in enzyme adaptation are less well understood. Interspecific differences in thermal stability of lactate dehydrogenase (LDH) in porcelain crabs (genus Petrolisthes) are related to intrinsic differences among LDH molecules and by interactions with other stabilizing proteins. Here, we identified proteins that interact with LDH in porcelain crab claw muscle tissue using co-immunoprecipitation, and showed LDH exists in high molecular weight complexes using size exclusion chromatography and Western blot analyses. Co-immunoprecipitated proteins were separated using 2D SDS PAGE and analyzed by LC/ESI using peptide MS/MS. Peptide MS/MS ions were compared to an EST database for Petrolisthes cinctipes to identify proteins. Identified proteins included cytoskeletal elements, glycolytic enzymes, a phosphagen kinase, and the respiratory protein hemocyanin. Our results support the hypothesis that LDH interacts with glycolytic enzymes in a metabolon structured by cytoskeletal elements that may also include the enzyme for transfer of the adenylate charge in glycolytically produced ATP. Those interactions may play specific roles in biochemical adaptation of glycolytic enzymes.

Cayenne, Andrea P.; Gabert, Beverly; Stillman, Jonathon H.

2011-01-01

143

Role of lactate dehydrogenase in metmyoglobin reduction and color stability of different bovine muscles.  

PubMed

The role of lactate dehydrogenase (LDH) in metmyoglobin reducing activity (MRA) and color stability of different bovine muscles was studied in two consecutive experiments. In experiment 1, three different bovine muscles -M. longissimus lumborum (LL), M. semimembranosus (SM), and M. psoas major (PM) - were obtained (n=7, respectively), cut into steaks, PVC packaged, and then displayed for 7days at 1°C. The LL was the most red over display time and had more (P<0.05) LDH-B activity (catalyzing toward NADH generation), LDH1 isoform expression, NADH, and higher (P<0.05) MRA than the other two muscles studied. The PM had the least color stability and lowest MRA. In experiment 2, LL steaks (n=8) were cut in half, one side syringe-injected with oxamate, and the other injected with distilled water. Inclusion of oxamate decreased (P<0.05) LDH-B activity, NADH, and a* values after 10days display at 1°C. These results suggest that variation in color stability of physiologically different muscles is regulated by different replenishment rates of NADH via different LDH isozymes. PMID:20416707

Kim, Y H; Keeton, J T; Smith, S B; Berghman, L R; Savell, J W

2009-11-01

144

Generation of oxamic acid libraries: antimalarials and inhibitors of Plasmodium falciparum lactate dehydrogenase.  

PubMed

Lactate dehydrogenase (LDH) is a key enzyme in the glycolytic pathway of Plasmodium falciparum (pf) and has several unique amino acids, related to other LDHs, at the active site, making it an attractive target for antimalarial agents. Oxamate, a competitive inhibitor, shows high substrate affinity for pfLDH. This class of compounds has been viewed as potential antimalarial agents. Thus, we have developed an effective automated synthetic strategy for the rapid synthesis of oxamic acid and ester libraries to screen for potential lead inhibitors. One hundred sixty-seven oxamic acids were synthesized using a "catch and release" method with overall yields of 20-70%. Most of the compounds synthesized had some inhibitory effects, but compounds 5 and 6 were the most active against both chloroquine- and mefloquine-resistant strains with IC50 values of 15.4 and 9.41 microM and 20.4 and 8.40 microM, respectively. Some oxamic acids showed activities against pfLDH and mammalian LDH (mLDH) at the micromolar range. These oxamic acids selectively inhibited pfLDH 2-5 fold over mLDH. Oxamic acid 21 was the most active against pfLDH at IC50 = 14 and mLDH at IC50 = 25 microM, suggesting that oxamic acid derivatives are potential inhibitors of pfLDH and that further study is required to develop selective inhibitors of pfLDH over mLDH. PMID:17316052

Choi, Seoung-ryoung; Beeler, Aaron B; Pradhan, Anupam; Watkins, E Blake; Rimoldi, John M; Tekwani, Babu; Avery, Mitchell A

2007-01-01

145

The promoting vibration in human heart lactate dehydrogenase is a preferred vibrational channel  

PubMed Central

We examine if the rate promoting vibration of lactate dehydrogenase is a preferred axis of thermal energy transfer. While it seems plausible that such a mechanistically important motion is also a favored direction of energy transfer, none of the previous studies of rate promoting vibrations in enzymatic catalysis have addressed this question. It is equally likely that the promoting vibration, though catalytically important, has no different properties than any other axis in the protein. Resolution of this issue is important for two reasons: First, if energy is transferred along this axis in a preferred fashion, it shows that the protein is engineered in a way that transfers thermal energy into a motion that is coupled to the chemical step. Second, the discovery of a preferred direction of thermal transfer provides a potential route to experimental verification of the promoting vibration concept. Our computational experiments are specifically designed to mimic potential laser experiment with the deposition of thermal energy in an active site chromophore with subsequent measurement of temperature at various points in the protein. Our results indicate that the promoting vibration is indeed a preferred channel of energy transfer. In addition, we study the vibrational structure of the protein via the dynamical structure factor to show preferred vibrational motion along the promoting vibration axis is an inherent property of the protein structure via thermal fluctuations.

Davarifar, Ardy; Antoniou, Dimitri; Schwartz, Steven D.

2011-01-01

146

Analytical quality specifications for serum lactate dehydrogenase isoenzyme 1 based on clinical goals.  

PubMed

The aim of the study was to deduce analytical quality specifications for the determination of catalytic concentration of serum lactate dehydrogenase isoenzyme 1 (S-LD-1) according to clinical goals (the clinical utility model). We defined clinical goals for false positive and false negative S-LD-1 measurements in the monitoring of patients with testicular germ cell tumors (TGCT), clinical stage I, on a surveillance only program. The absolute S-LD-1 catalytic concentrations were routinely corrected for contamination from preanalytical hemolysis. A reference group of 37 men had a near In-Gaussian distribution for the absolute S-LD-1 catalytic concentration. The geometric mean was 76 U/l and an S-LD-1 >128 U/l (99.72 percentile, the decision limit) indicated a high risk of a relapse of TGCT. We have previously shown that an S-LD-1 >160 U/l (treatment limit) was associated with a suboptimal outcome from the treatment of metastatic TGCT. The maximum allowable analytical positive bias was 5 U/l, and the maximum allowable analytical negative bias was -32 U/l. The maximum allowable analytical coefficient of variation, CV(A), was 11% (approximately 14 U/l) at a bias = -5 U/l. For S-LD-1 measurements not corrected for hemolysis, the decision limit was 145 U/l, the maximum allowable negative bias -19 U/l, and CV(A) 8%(approximately 12 U/l). A routine correction for hemolysis had a large impact on the analytical quality specifications. PMID:10418747

von Eyben, F E; Petersen, P H; Blaabjerg, O; Madsen, E L

1999-05-01

147

Evolutionary factors affecting Lactate dehydrogenase A and B variation in the Daphnia pulex species complex  

PubMed Central

Background Evidence for historical, demographic and selective factors affecting enzyme evolution can be obtained by examining nucleotide sequence variation in candidate genes such as Lactate dehydrogenase (Ldh). Two closely related Daphnia species can be distinguished by their electrophoretic Ldh genotype and habitat. Daphnia pulex populations are fixed for the S allele and inhabit temporary ponds, while D. pulicaria populations are fixed for the F allele and inhabit large stratified lakes. One locus is detected in most allozyme surveys, but genome sequencing has revealed two genes, LdhA and LdhB. Results We sequenced both Ldh genes from 70 isolates of these two species from North America to determine if the association between Ldh genotype and habitat shows evidence for selection, and to elucidate the evolutionary history of the two genes. We found that alleles in the pond-dwelling D. pulex and in the lake-dwelling D. pulicaria form distinct groups at both loci, and the substitution of Glutamine (S) for Glutamic acid (F) at amino acid 229 likely causes the electrophoretic mobility shift in the LDHA protein. Nucleotide diversity in both Ldh genes is much lower in D. pulicaria than in D. pulex. Moreover, the lack of spatial structuring of the variation in both genes over a wide geographic area is consistent with a recent demographic expansion of lake populations. Neutrality tests indicate that both genes are under purifying selection, but the intensity is much stronger on LdhA. Conclusions Although lake-dwelling D. pulicaria hybridizes with the other lineages in the pulex species complex, it remains distinct ecologically and genetically. This ecological divergence, coupled with the intensity of purifying selection on LdhA and the strong association between its genotype and habitat, suggests that experimental studies would be useful to determine if variation in molecular function provides evidence that LDHA variants are adaptive.

2011-01-01

148

Antimalarial Activity of Potential Inhibitors of Plasmodium falciparum Lactate Dehydrogenase Enzyme Selected by Docking Studies  

PubMed Central

The Plasmodium falciparum lactate dehydrogenase enzyme (PfLDH) has been considered as a potential molecular target for antimalarials due to this parasite's dependence on glycolysis for energy production. Because the LDH enzymes found in P. vivax, P. malariae and P. ovale (pLDH) all exhibit ?90% identity to PfLDH, it would be desirable to have new anti-pLDH drugs, particularly ones that are effective against P. falciparum, the most virulent species of human malaria. Our present work used docking studies to select potential inhibitors of pLDH, which were then tested for antimalarial activity against P. falciparum in vitro and P. berghei malaria in mice. A virtual screening in DrugBank for analogs of NADH (an essential cofactor to pLDH) and computational studies were undertaken, and the potential binding of the selected compounds to the PfLDH active site was analyzed using Molegro Virtual Docker software. Fifty compounds were selected based on their similarity to NADH. The compounds with the best binding energies (itraconazole, atorvastatin and posaconazole) were tested against P. falciparum chloroquine-resistant blood parasites. All three compounds proved to be active in two immunoenzymatic assays performed in parallel using monoclonals specific to PfLDH or a histidine rich protein (HRP2). The IC50 values for each drug in both tests were similar, were lowest for posaconazole (<5 µM) and were 40- and 100-fold less active than chloroquine. The compounds reduced P. berghei parasitemia in treated mice, in comparison to untreated controls; itraconazole was the least active compound. The results of these activity trials confirmed that molecular docking studies are an important strategy for discovering new antimalarial drugs. This approach is more practical and less expensive than discovering novel compounds that require studies on human toxicology, since these compounds are already commercially available and thus approved for human use.

Penna-Coutinho, Julia; Cortopassi, Wilian Augusto; Oliveira, Aline Alves; Franca, Tanos Celmar Costa; Krettli, Antoniana Ursine

2011-01-01

149

Lactate dehydrogenase undergoes a substantial structural change to bind its substrate.  

PubMed

Employing temperature-jump relaxation spectroscopy, we investigate the kinetics and thermodynamics of the formation of a very early ternary binding intermediate formed when lactate dehydrogenase (LDH) binds a substrate mimic on its way to forming the productive LDH/NADH.substrate Michaelis complex. Temperature-jump scans show two distinct submillisecond processes are involved in the formation of this ternary binding intermediate, called the encounter complex here. The on-rate of the formation of the encounter complex from LDH/NADH with oxamate (a substrate mimic) is determined as a function of temperature and in the presence of small concentrations of a protein destabilizer (urea) and protein stabilizer (TMAO). It shows a strong temperature dependence with inverse Arrhenius behavior and a temperature-dependent enthalpy (heat capacity of 610 +/- 84 cal/Mol K), is slowed in the presence of TMAO and speeded up in the presence of urea. These results suggest that LDH/NADH occupies a range of conformations, some competent to bind substrate (open structure; a minority population) and others noncompetent (closed), in fast equilibrium with each other in accord with a select fit model of binding. From the thermodynamic results, the two species differ in the rearrangement of low energy hydrogen bonds as would arise from changes in internal hydrogen bonding and/or increases in the solvation of the protein structure. The binding-competent species can bind ligand at or very near diffusion-limited speeds, suggesting that the binding pocket is substantially exposed to solvent in these species. This would be in contrast to the putative closed structure where the binding pocket resides deep within the protein interior. PMID:17483169

Qiu, Linlin; Gulotta, Miriam; Callender, Robert

2007-09-01

150

Regulation of liver lactate dehydrogenase by reversible phosphorylation in response to anoxia in a freshwater turtle.  

PubMed

Lactate dehydrogenase (LDH) is the terminal enzyme of anaerobic glycolysis and key to hypoxia/anoxia survival by most animals. In this study, the effects of anoxic submergence (20 h at 7°C in nitrogen-bubbled water) were assessed on LDH from liver of an anoxia-tolerant freshwater turtle, the red-eared slider (Trachemys scripta elegans). Liver LDH from aerobic and anoxic turtles was purified to homogeneity in two steps. The kinetic properties and thermal stability of purified LDH were analyzed, revealing significant differences between the two enzyme forms in V(max), K(m) pyruvate, and I(50) pyruvate as well as melting temperature determined by differential scanning fluorimetry. The phosphorylation state of aerobic and anoxic forms of LDH was visualized by ProQ Diamond phosphoprotein staining, the results indicating that the anoxic form had a higher phosphorylation state. Incubation studies that promoted protein kinase versus protein phosphatase actions showed that changes in the phosphorylation state of aerobic and anoxic forms mimicked the anoxia-responsive changes in K(m) pyruvate and I(50) pyruvate. The high phosphate form of liver LDH that occurs in anoxic turtles appears to be a less active form. Turtle liver LDH was also subject to another form of posttranslational modification, protein acetylation, with a 70% higher content of acetylated lysine residues on anoxic versus aerobic LDH. This is the first study to show that LDH function in an anoxia-tolerant animal can be differentially modified between aerobic and anoxic states via the mechanism of posttranslational modification. PMID:22735190

Xiong, Zi Jian; Storey, Kenneth B

2012-10-01

151

Polymorphism of the parasite lactate dehydrogenase gene from Plasmodium vivax Korean isolates  

PubMed Central

Background Assaying for the parasitic lactate dehydrogenase (pLDH) is widely used as a rapid diagnostic test (RDT), but the efficacy of its serological effectiveness in diagnosis, that is antibody detection ability, is not known. The genetic variation of Korean isolates was analysed, and recombinant protein pLDH was evaluated as a serodiagnostic antigen for the detection of Plasmodium vivax malaria. Methods Genomic DNA was purified, and the pLDH gene of P. vivax was amplified from blood samples from 20 patients. The samples came from five epidemic areas: Bucheon-si, Gimpo-si, and Paju-si of Gyeonggi Province, Gangwha-gun of Incheon metropolitan city, and Cheorwon-gun of Gangwon Province, South Korea, from 2010 to 2011. The antigenicity of the recombinant protein pLDH was tested by western blot and enzyme-linked immunosorbent assay (ELISA). Results Sequence analysis of 20 Korean isolates of P. vivax showed that the open reading frame (ORF) of 951 nucleotides encoded a deduced protein of 316 amino acids (aa). This ORF showed 100% identity with the P. vivax Belem strain (DQ060151) and P. vivax Hainan strain (FJ527750), 89.6% homology with Plasmodium falciparum FCC1_HN (DQ825436), 90.2% homology with Plasmodium berghei (AY437808), 96.8% homology with Plasmodium knowlesi (JF958130), and 90.2% homology with Plasmodium reichenowi (AB122147). A single-nucleotide polymorphism (SNP) at nucleotide 456 (T to C) was also observed in the isolate from Bucheon, but it did not change in the amino acid sequence. The expressed recombinant protein had a molecular weight of approximately 32 kDa, as analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Of the 40 P. vivax patients, 34 (85.0%) were positive by ELISA. Conclusions The pLDH genes of 19 isolates of P. vivax were identical, except one for SNP at nucleotide 456. This observation indicates that this gene is relatively stable. Based on these results, the relationship between antibody production against pLDH and the pattern of disease onset should be investigated further before using pLDH for serodiagnosis.

2013-01-01

152

The use of ternary complexes to study ionizations and isomerizations during catalysis by lactate dehydrogenase*  

PubMed Central

1. The binding of oxamate to pig heart and pig muscle isoenzymes of lactate dehydrogenase in the presence of NADH was studied by fluorescence titration. The dissociation constant of oxamate from the heart enzyme complex is 3?m and from the muscle isoenzyme 25?m at pH5. These values quantitatively increase with pH as predicted if oxamate can bind only to the enzyme–NADH complex if a group with pK6.9 is protonated. There are four non-interacting oxamate-binding sites per tetramer. 2. o-Nitrophenylpyruvate is a poor substrate for both isoenzymes but has a reasonable affinity to the heart isoenzyme. Initially, it forms an enzyme–NADH–substrate complex, which can be detected either by protein-fluorescence quenching or by NADH-fluorescence quenching. The pH-dependence of the dissociation constant of nitrophenylpyruvate also shows that this ternary complex can only form if a group with pK6.8 is protonated. Taken with the results of chemical-modification experiments, these results allow the pK of 6.8 to be assigned to a system probably involving the imidazole side chain of histidine-195. Formation of a ternary complex from a binary one at pH8 is predicted to result in a proton being taken up from solution. 3. Isotope-effect studies with NADH and its deuterium analogue show that the rapidly formed ternary complex with o-nitrophenylpyruvate slowly isomerizes to give an active ternary complex, which then rapidly decomposes to NAD+. The isomerization is pH-independent, and it is suggested that histidine-195 is still protonated in the activated ternary complex, which is present before hydride transfer. 4. All four subunits of the enzyme are kinetically equivalent with respect to the oxidation of bound NADH by o-nitrophenylpyruvate. 5. A partial mechanism for the enzyme is described which emphasizes the isomerizations and ionizations involved in forming the reduced ternary complex at pH6 and 8.

Holbrook, J. John; Stinson, Robert A.

1973-01-01

153

Creatine kinase and lactate dehydrogenase responses after upper-body resistance exercise with different rest intervals.  

PubMed

The purpose of the current study was to compare serum creatine kinase (CK) and lactate dehydrogenase (LDH) concentrations at multiple time points after resistance exercise sessions that incorporated different rest intervals between sets and exercises. Twenty untrained men (18.65+/-0.49 years, 68.30+/-7.98 kg, and 174.4+/-4.80 cm) performed 2 resistance exercise sessions (i.e., 3 sets with 80% 1 repetition maximum for 5 upper-body exercises) with either 1-minute (SEQ1) or 3-minute (SEQ3) rest between sets and exercises. For each session, CK and LDH concentrations were measured before exercise (PRE) and 24, 48, and 72 hours after exercise (24P, 48P, and 72P). Subjects lifted a 24% greater (p<0.05) volume load during SEQ3 than during SEQ1. Within SEQ1, significant differences in CK concentrations were demonstrated between most time points, except between 24P and 72P. Similarly, within SEQ3, significant differences in CK concentrations were demonstrated between most time points, except between 24P and 72P and between 48P and 72P. The CK concentrations were highest at 48P for both sessions. When the CK concentrations were compared between SEQ1 and SEQ3, no significant differences were demonstrated at any time point. Within SEQ1, a significant difference in LDH concentration was demonstrated between 48P and 72P. Within SEQ3, significant differences in LDH concentrations were demonstrated between PRE and 24P and between PRE and 48P. The LDH concentrations were highest at 72P for SEQ1 and at 24P for SEQ3. When the LDH concentrations were compared between SEQ1 and SEQ3, no significant differences were demonstrated at any time point. These results suggest that muscle damage was similar between rest intervals; however, the volume load completed to induce the muscle damage was significantly greater when 3-minute rest intervals were employed. Therefore, when considered relative to the volume load completed, 1-minute rest intervals during resistance exercise may invoke greater muscle damage. PMID:20508471

Rodrigues, Bernardo M; Dantas, Estélio; de Salles, Belmiro Freitas; Miranda, Humberto; Koch, Alexander J; Willardson, Jeffrey M; Simão, Roberto

2010-06-01

154

Circulating cell-free methylated DNA and lactate dehydrogenase release in colorectal cancer  

PubMed Central

Background Hypermethylation of DNA is an epigenetic alteration commonly found in colorectal cancer (CRC) and can also be detected in blood samples of cancer patients. Methylation of the genes helicase-like transcription factor (HLTF) and hyperplastic polyposis 1 (HPP1) have been proposed as prognostic, and neurogenin 1 (NEUROG1) as diagnostic biomarker. However the underlying mechanisms leading to the release of these genes are unclear. This study aimed at examining the possible correlation of the presence of methylated genes NEUROG1, HLTF and HPP1 in serum with tissue breakdown as a possible mechanism using serum lactate dehydrogenase (LDH) as a surrogate marker. Additionally the prognostic impact of these markers was examined. Methods Pretherapeutic serum samples from 259 patients from all cancer stages were analyzed. Presence of hypermethylation of the genes HLTF, HPP1, and NEUROG1 was examined using methylation-specific quantitative PCR (MethyLight). LDH was determined using an UV kinetic test. Results Hypermethylation of HLTF and HPP1 was detected significantly more often in patients with elevated LDH levels (32% vs. 12% [p = 0.0005], and 68% vs. 11% [p < 0.0001], respectively). Also, higher LDH values correlated with a higher percentage of a fully methylated reference in a linear fashion (Spearman correlation coefficient 0.18 for HLTF [p = 0.004]; 0.49 [p < .0001] for HPP1). No correlation between methylation of NEUROG1 and LDH was found in this study. Concerning the clinical characteristics, high levels of LDH as well as methylation of HLTF and HPP1 were significantly associated with larger and more advanced stages of CRC. Accordingly, these three markers were correlated with significantly shorter survival in the overall population. Moreover, all three identified patients with a worse prognosis in the subgroup of stage IV patients. Conclusions We were able to provide evidence that methylation of HLTF and especially HPP1 detected in serum is strongly correlated with cell death in CRC using LDH as surrogate marker. Additionally, we found that prognostic information is given by both HLTF and HPP1 as well as LDH. In sum, determining the methylation of HLTF and HPP1 in serum might be useful in order to identify patients with more aggressive tumors.

2014-01-01

155

Polymorphism of the parasite lactate dehydrogenase gene from Plasmodium vivax Korean isolates.  

PubMed

BACKGROUND: Assaying for the parasitic lactate dehydrogenase (pLDH) is widely used as a rapid diagnostic test (RDT), but the efficacy of its serological effectiveness in diagnosis, that is antibody detection ability, is not known. The genetic variation of Korean isolates was analysed, and recombinant protein pLDH was evaluated as a serodiagnostic antigen for the detection of Plasmodium vivax malaria. METHODS: Genomic DNA was purified, and the pLDH gene of P. vivax was amplified from blood samples from 20 patients. The samples came from five epidemic areas: Bucheon-si, Gimpo-si, and Paju-si of Gyeonggi Province, Gangwha-gun of Incheon metropolitan city, and Cheorwon-gun of Gangwon Province, South Korea, from 2010 to 2011. The antigenicity of the recombinant protein pLDH was tested by western blot and enzyme-linked immunosorbent assay (ELISA). RESULTS: Sequence analysis of 20 Korean isolates of P. vivax showed that the open reading frame (ORF) of 951 nucleotides encoded a deduced protein of 316 amino acids (aa). This ORF showed 100% identity with the P. vivax Belem strain (DQ060151) and P. vivax Hainan strain (FJ527750), 89.6% homology with Plasmodium falciparum FCC1_HN (DQ825436), 90.2% homology with Plasmodium berghei (AY437808), 96.8% homology with Plasmodium knowlesi (JF958130), and 90.2% homology with Plasmodium reichenowi (AB122147). A single-nucleotide polymorphism (SNP) at nucleotide 456 (T to C) was also observed in the isolate from Bucheon, but it did not change in the amino acid sequence. The expressed recombinant protein had a molecular weight of approximately 32 kDa, as analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Of the 40 P. vivax patients, 34 (85.0%) were positive by ELISA. CONCLUSIONS: The pLDH genes of 19 isolates of P. vivax were identical, except one for SNP at nucleotide 456. This observation indicates that this gene is relatively stable. Based on these results, the relationship between antibody production against pLDH and the pattern of disease onset should be investigated further before using pLDH for serodiagnosis. PMID:23688062

Shin, Hyun-Il; Kim, Jung-Yeon; Lee, Won-Ja; Sohn, Youngjoo; Lee, Sang-Wook; Kang, Yoon-Joong; Lee, Hyeong-Woo

2013-05-21

156

Comparative performance of aldolase and lactate dehydrogenase rapid diagnostic tests in Plasmodium vivax detection  

PubMed Central

Background Misdiagnosis of malaria by commercial rapid diagnostic tests (RDTs) is a major cause of concern in the diagnosis of malaria. This retrospective study was aimed at assessing the relative performance of four RDTs with emphasis on the detection of two Plasmodium vivax antigens: aldolase and lactate dehydrogenase (LDH). Methods Three commercially available Plasmodium LDH or aldolase antigen detection kits (One Step Malaria P.f/P.v, ParaHit Total ver. 1.0, SD Bioline Malaria) and an anti-P. vivax aldolase-specific monoclonal antibody (mAb) pair 1C3-12 F10 were evaluated with P. vivax positive as well as non-P. vivax samples and healthy samples using blood smear examination as standard. Each test was read according to the manufacturer’s instructions. Results MAb 1C3-12 F10 pair targeting P. vivax-specific aldolase exhibited very good specificity and sensitivity of 100 and 97.4%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) of 100 and 99.5%, respectively, were also observed. The anti-P. vivax LDH in the One-Step Malaria P.f/P.v test showed sensitivity, specificity, PPV and NPV of 93.5, 98.0, 88.9 and 98.8%, respectively. ParaHit Total ver. 1.0 targeting the pan-aldolase antigen showed sensitivity, specificity of 97.4 and 99.6%, respectively. PPV and NPV were both 99.5%. SD Bioline had sensitivity, specificity, PPV and NPV of 93.5, 100, 100 and 98.8%, respectively. The overall sensitivity and specificity of all four RDTs were acceptable, especially for the aldolase detection tests. Five (6.5%) of the P. vivax-positive samples (n?=?77) that were confirmed by microscopic examination as well as the two aldolase detection RDTs (mAb 1C3-12 F10 and ParaHit Total ver.1.0) were undetected by the two LDH detection RDTs (One Step Malaria P.f/P.v and SD Bioline). Similarly, two positive samples (2.6%) that were positively confirmed by the LDH detection RDTs were also undetected by the aldolase detection test kits. Conclusion Aldolase and LDH antigens perform differently in different P. vivax samples; hence there is a high risk of misdiagnosis when monoclonal antibodies are used against only one particular antigen in the test. A combination of both aldolase and LDH in RDTs for the rapid diagnosis of P. vivax will enhance the sensitivity of the assay and reduce misdiagnosis.

2014-01-01

157

Macromolecular Crowding Effect upon in Vitro Enzyme Kinetics: Mixed Activation-Diffusion Control of the Oxidation of NADH by Pyruvate Catalyzed by Lactate Dehydrogenase.  

PubMed

Enzyme kinetics studies have been usually designed as dilute solution experiments, which differ substantially from in vivo conditions. However, cell cytosol is crowded with a high concentration of molecules having different shapes and sizes. The consequences of such crowding in enzymatic reactions remain unclear. The aim of the present study is to understand the effect of macromolecular crowding produced by dextran of different sizes and at diverse concentrations in the well-known reaction of oxidation of NADH by pyruvate catalyzed by l-lactate dehydrogenase (LDH). Our results indicate that the reaction rate is determined by both the occupied volume and the relative size of dextran obstacles with respect to the enzyme present in the reaction. Moreover, we analyzed the influence of macromolecular crowding on the Michaelis-Menten constants, vmax and Km. The obtained results show that only high concentrations and large sizes of dextran reduce both constants suggesting a mixed activation-diffusion control of this enzymatic reaction due to the dextran crowding action. From our knowledge, this is the first experimental study that depicts mixed activation-diffusion control in an enzymatic reaction due to the effect of crowding. PMID:24660904

Balcells, Cristina; Pastor, Isabel; Vilaseca, Eudald; Madurga, Sergio; Cascante, Marta; Mas, Francesc

2014-04-17

158

Tyrosine Phosphorylation of Lactate Dehydrogenase A Is Important for NADH/NAD+ Redox Homeostasis in Cancer Cells ?  

PubMed Central

The Warburg effect describes an increase in aerobic glycolysis and enhanced lactate production in cancer cells. Lactate dehydrogenase A (LDH-A) regulates the last step of glycolysis that generates lactate and permits the regeneration of NAD+. LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. We found that the oncogenic receptor tyrosine kinase FGFR1 directly phosphorylates LDH-A. Phosphorylation at Y10 and Y83 enhances LDH-A activity by enhancing the formation of active, tetrameric LDH-A and the binding of LDH-A substrate NADH, respectively. Moreover, Y10 phosphorylation of LDH-A is common in diverse human cancer cells, which correlates with activation of multiple oncogenic tyrosine kinases. Interestingly, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, demonstrate increased respiration through mitochondrial complex I to sustain glycolysis by providing NAD+. However, such a compensatory increase in mitochondrial respiration in Y10F cells is insufficient to fully sustain glycolysis. Y10 rescue cells show decreased cell proliferation and ATP levels under hypoxia and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation enhances LDH-A enzyme activity to promote the Warburg effect and tumor growth by regulating the NADH/NAD+ redox homeostasis, representing an acute molecular mechanism underlying the enhanced lactate production in cancer cells.

Fan, Jun; Hitosugi, Taro; Chung, Tae-Wook; Xie, Jianxin; Ge, Qingyuan; Gu, Ting-Lei; Polakiewicz, Roberto D.; Chen, Georgia Z.; Boggon, Titus J.; Lonial, Sagar; Khuri, Fadlo R.; Kang, Sumin; Chen, Jing

2011-01-01

159

Localization of D-lactate dehydrogenase in membrane vesicles prepared by using a french press or ethylenediaminetetraacetate-lysozyme from Escherichia coli.  

PubMed Central

The localization of D-lactate dehydrogenase in membrane vesicles prepared from Escherichia coli was studied using antibody against the purified enzyme. The activity of D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by using a French press were completely inhibited by this antibody, suggesting that the enzyme is localized on the outside of these vesicles. This and previous results (Futai, 1974) strongly indicate the inversion of these vesicles. The D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by treatment with ethylenediaminetetraacetate-lysozyme were inhibited about 15% by the antibody, whereas proline transport of the vesicles was insensitive to antibody. These results suggest that most of the membrane vesicles have D-lactate dehydrogenase on the inside of the membrane and that such vesicles transport amino acids. This essentially confirms the results of Short, Kaback, and Kohn (1975). However, unlike them we observed that a small but significant portion of activity was sensitive to the antibody as shown above. This portion may represent the completely inverted vesicles in the preparation. Ferricyanide reductase activity cannot be detected in spheroplasts, but about 30 to 50% of the total was detected in membrane vesicles prepared by treatment with ethylenediaminetetraacetate. This confirms our previous findings with membrane prepared by a slightly different procedure. It is concluded that in these vesicles about half the reactive sites for ferricyanide are moved from inside to outside the membrane, whereas 85% of the D-lactate dehydrogenase remains inside the membrane. Images

Futai, M; Tanaka, Y

1975-01-01

160

Lactate-Dehydrogenase 5 is overexpressed in non-small cell lung cancer and correlates with the expression of the transketolase-like protein 1  

Microsoft Academic Search

AIMS: As one of the five Lactate dehydrogenase (LDH) isoenzymes, LDH5 has the highest efficiency to catalyze pyruvate transformation to lactate. LDH5 overexpression in cancer cells induces an upregulated glycolytic metabolism and reduced dependence on the presence of oxygen. Here we analyzed LDH5 protein expression in a well characterized large cohort of primary lung cancers in correlation to clinico-pathological data

Gian Kayser; Ahmad Kassem; Wulf Sienel; Luzie Schulte-Uentrop; Dominik Mattern; Konrad Aumann; Elmar Stickeler; Martin Werner; Bernward Passlick; Axel Hausen

2010-01-01

161

Lactate dehydrogenase-5 (LDH-5) overexpression in non-small-cell lung cancer tissues is linked to tumour hypoxia, angiogenic factor production and poor prognosis  

Microsoft Academic Search

Lactate dehydrogenase-5 (LDH-5) catalyses the reversible transformation of pyruvate to lactate, having a principal position in the anaerobic cellular metabolism. Induction of LDH-5 occurs during hypoxia and LDH-5 transcription is directly regulated by the hypoxia-inducible factor 1 (HIF1). Serum LDH levels have been correlated with poor prognosis and resistance to chemotherapy and radiotherapy in various neoplastic diseases. The expression, however,

M I Koukourakis; A Giatromanolaki; E Sivridis; G Bougioukas; V Didilis; K C Gatter; A L Harris

2003-01-01

162

Pressure-adaptive differences in the binding and catalytic properties of muscle-type (M 4 ) lactate dehydrogenases of shallow- and deep-living marine fishes  

Microsoft Academic Search

The pressure sensitivities of substrate (pyruvate) and cofactor (NADH) binding and catalytic rate of purified muscle-type (M4) lactate dehydrogenases (LDH, EC 1.1.1.27; NAD+: lactate oxidoreductase) from shallow- and deep-living teleost fishes were compared. The LDH's of the shallow species are significantly more pressure-sensitive than the LDH's of the deep-living fishes. The apparent Michaelis constant (Km)1 of pyruvate of the deep-living

Joseph F. Siebenaller; George N. Somero

1979-01-01

163

An apparent progressive and recurrent evolutionary restriction in tissue expression of a gene, the lactate dehydrogenase-C gene, within a family of bony fish (Salmoniformes: Umbridae)  

Microsoft Academic Search

Summary Unexpectedly large differences in the tissue patterns of lactate dehydrogenase-C (Ldh-C) gene regulation were observed among species of fish within the family Umbridae (Salmoniformes). Normally, all the species within a family or order of advanced fishes exhibit the same, tissue-restricted pattern ofl-latate dehydrogenase C4 isozyme synthesis—either eye- or liver-restricted expression, but not both. However, within the Umbridae the more

M. K. Kettler; G. S. Whitt

1986-01-01

164

Purification and characterization of a urea sensitive lactate dehydrogenase from the liver of the African clawed frog, Xenopus laevis.  

PubMed

The African clawed frog, Xenopus laevis, is able to withstand extremely arid conditions by estivating, in conjunction with dehydration tolerance and urea accumulation. Estivating X. laevis reduce their metabolic rate and recruit anaerobic glycolysis, driven by lactate dehydrogenase (LDH; E.C. 1.1.1.27) enzymes that reversibly convert pyruvate and NADH to lactate and NAD(+), to meet newly established ATP demands. The present study investigated purified LDH from the liver of dehydrated and control X. laevis. LDH from dehydrated liver showed a significantly higher K m for L-lactate (1.74 fold), NAD(+) (2.41 fold), and pyruvate (1.78 fold) in comparison to LDH from the liver of control frogs. In the presence of physiological levels of urea found in dehydrated animals, the K m values obtained for dehydrated LDH all returned to control LDH K m values. Dot blot analysis showed post-translational modifications may be responsible for the kinetic modification as the dehydrated form of LDH showed more phosphorylated serine residues (1.54 fold), less methylated lysine residues (0.43 fold), and a higher level of ubiquitination (1.90 fold) in comparison to control LDH. The physiological consequence of dehydration-induced LDH modification appears to adjust LDH function in conjunction with urea levels in dehydrated frogs. When urea levels are high during dehydration, LDH retains its normal function. Yet, as urea levels drop during rehydration, LDH function is reduced, possibly shunting pyruvate to the TCA cycle. PMID:24651940

Katzenback, Barbara A; Dawson, Neal J; Storey, Kenneth B

2014-07-01

165

COMPARISON OF A PARASITE LACTATE DEHYDROGENASE-BASED IMMUNOCHROMATOGRAPHIC ANTIGEN DETECTION ASSAY (OPTIMAL t) WITH MICROSCOPY FOR THE DETECTION OF MALARIA PARASITES IN HUMAN BLOOD SAMPLES  

Microsoft Academic Search

Microscopic examination of blood smears remains the gold standard for malaria diagnosis, but is labor- intensive and requires skilled operators. Rapid dipstick technology provides a potential alternative. A study was conducted in The Gambia to compare the performance of OptiMALt, an immunochromatographic antigen detection assay for the diagnosis of malaria using parasite lactate dehydrogenase, against standard microscopy in patients with

ANGELA HUNT COOKE; PETER L. CHIODINI; TOM DOHERTY; ANTHONY H. MOODY; JUNITA RIES; MARGARET PINDER

166

[The function of lymphocyte populations based on aberrations in the lactate dehydrogenase isoenzyme spectra in systemic lupus erythematosus and rheumatoid arthritis].  

PubMed

The data were obtained on the presence of considerable aberrations of the isoenzymic spectrum of lactate dehydrogenase in different lymphocyte populations and subpopulations. The isoenzymic shifts reflect the changes occurring in the metabolic status of the cells and are sensitive tests detecting functional deficiency of lymphocytes. PMID:2144061

Matve?kov, G P; Kaliia, E S; Levin, V I; San'ko, N M

1990-01-01

167

Lactation  

PubMed Central

Lactation is the most energy-efficient way to provide for the dietary needs of young mammals, their mother's milk being actively protective, immunomodulatory, and ideal for their needs. Intrauterine mammary gland development in the human female is already apparent by the end of the sixth week of gestation. During puberty and adolescence secretions of the anterior pituitary stimulate the maturation of the graafian follicles in the ovaries and stimulate the secretion of follicular estrogens, which stimulate development of the mammary ducts. Pregnancy has the most dramatic effect on the breast, but development of the glandular breast tissue and deposition of fat and connective tissue continue under the influence of cyclic sex-hormone stimulation. Many changes occur in the nipple and breast during pregnancy and at delivery as a prelude to lactation. Preparation of the breasts is so effective that lactation could commence even if pregnancy were discontinued at 16 weeks. Following birth, placental inhibition of milk synthesis is removed, and a woman's progesterone blood levels decline rapidly. The breasts fill with milk, which is a high-density, low-volume feed called colostrum until about 30 hours after birth. Because it is not the level of maternal hormones, but the efficiency of infant suckling and/or milk removal that governs the volume of milk produced in each breast, mothers who permit their infants to feed ad libitum commonly observe that they have large volumes of milk 24-48 hours after birth. The two maternal reflexes involved in lactation are the milk-production and milk-ejection reflex. A number of complementary reflexes are involved when the infant feeds: the rooting reflex (which programmes the infant to search for the nipple), the sucking reflex (rhythmic jaw action creating negative pressure and a peristaltic action of the tongue), and the swallowing reflex. The infant's instinctive actions need to be consolidated into learned behaviour in the postpartum period when the use of artificial teats and dummies (pacifiers) may condition the infant to different oral actions that are inappropriate for breast-feeding. Comparisons of breast milk and cow's milk fail to describe the many important differences between them, e.g., the structural and qualitative differences in proteins and fats, and the bioavailability of minerals. The protection against infection and allergies conferred on the infant, which is impossible to attain through any other feeding regimen, is one of breast milk's most outstanding qualities. The maximum birth-spacing effect of lactation is achieved when an infant is fully, or nearly fully, breast-fed and the mother consequently remains amenorrhoeic.

1989-01-01

168

Effect of sublethal concentration of Solanum nigrum on transaminases and lactate dehydrogenase of Biomphalria arabica, in Saudi Arabia.  

PubMed

Schistosomes have a complex lifecycle with freshwater intermediate host snails. The snail host represents the weakest point in the lifecycle of parasite. Biomphalaria arabica is intermediate host for Schistosoma mansoni in Saudi Arabia. In this work, aspartate and alanine aminotransferases (AST and ALT) and lactate dehydrogenase (LDH) were measured in the tissue homogenate and haemolymph of B. arabica. Besides, the effect of sublethal concentrations (LC25) of dry powdered Solanum nigrum leaf was tested as plant molluscicide against B. arabica. The studied enzymes were altered in molluscicide-treated snails compared to control. AST and ALT were slightly affected but LDH was the most significantly altered enzyme. The role of the biochemical manipulation in affecting host-parasite relationship was discussed. PMID:17580567

El-Ansary, Afaf K; Al Daihan, Soad K

2007-04-01

169

The use of sperm-specific lactate dehydrogenase isoenzyme for the identification of semen in dried stains.  

PubMed

The sperm-specific lactate dehydrogenase (LDH) isoenzyme (Blanco and Zinkham, 1963; Goldberg, 1963) separated from other LDH isoenzymes of semen by polyacrylamide gel electrophoresis has been found to be suitable for specific differentiation of human semen from other human body fluids and semen of commonly encountered animals. Seminal isoenzymes were found to be stable even 4 weeks after storage in tropical conditions. The method gave substantially more positive results than microscopic identification of spermatozoa when applied to a large number of relatively old stains on actual crime articles. It is therefore valuable in a large number of cases involving normal males and also in interpreting results of immunological and enzymological individualisation of semen stains. PMID:1033891

Mokashi, R H; Madiwale, M S

1976-01-01

170

Serum lactate dehydrogenase isoenzyme 1. An early indicator of relapse in patients with testicular germ cell tumors.  

PubMed

The present study aimed to evaluate serum lactate dehydrogenase isoenzyme 1 (S-LD-1) as an indicator of relapse for patients with testicular germ cell tumors. Twenty-seven patients were investigated with repeated determinations of S-LD-1 from diagnosis to relapse; 9 had seminoma and 18 nonseminomatous tumors. Eleven of 21 had raised S-LD-1 at relapse (4 with seminoma). Seven of the 27 patients had a raised S-LD-1 for median 2 months (1.4-4.5 months) before relapse was detected. Thus, S-LD-1 is of use, complementary to clinical examinations, determinations of serum alpha fetoprotein and human chorionic gonadotropin, and CT scans, in monitoring patients with testicular germ cell tumors for relapse. PMID:7492382

Edler von Eyben, F; Lindegaard Madsen, E; Blaabjerg, O; Hyltoft Petersen, P

1995-01-01

171

Different pressure resistance of lactate dehydrogenases from hagfish is dependent on habitat depth and caused by tetrameric structure dissociation.  

PubMed

The effects of high hydrostatic pressure on lactate dehydrogenase (LDH) activities from two species of hagfish were examined. LDH from Eptatretus okinoseanus, a deep-sea species, retained 67% of the original activity even at 100 MPa. LDH activity from Eptatretus burgeri, a shallow-sea species, was completely lost at 50 MPa but recovered to the original value at 0.1 MPa. The tetrameric structure of LDH-A(4) from E. okinoseanus did not change at 50 MPa. In contrast, almost all LDH tetramers from E. burgeri dissociated to dimers and monomers at 50 MPa but reverted to tetramers at 0.1 MPa. These results show that the dissociation of tetramers caused the inactivation of E. burgeri LDH. The difference depends on the number 6 and 10 amino acids. The mechanism of the slight, gradual inactivation of E. okinoseanus LDH at high pressure differs and is probably due to the metamorphosis of its inner structures. PMID:20514503

Nishiguchi, Yoshikazu; Abe, Fumiyoshi; Okada, Mitsumasa

2011-04-01

172

Urinary enzyme excretion and renal lactate dehydrogenase isoenzyme pattern in acute HgCl2 nephropathy of rat.  

PubMed

The activity of several enzymes with different intracellular sites was determined in urine at various times following nonfatal acute tubular necrosis induced by mercuric chloride administration. The excretion rate of all tested enzymes rose on the 1st and 2nd day; in the next observations (days 7-15) enzymatic values approached the basal values. The lactate dehydrogenase isoenzyme pattern of the renal cortical zone showed an early shift towards cathodic fractions and later (7 days) an increase of middle ones; the normal anodic zymogram recovered after a suitable time interval (30 days). The isoenzymatic changes are related both to the renal hypoxia and to the appearance of less differentiated cells. The behaviour of functional parameters (urine flow, osmolality, urea clearance, creatinine clearance) were well in agreement with the observed enzyme and renal isoenzyme changes. PMID:6121704

Emanuelli, G; Cestonaro, G; Anfossi, G; Calcamuggi, G; Gatti, G; Marcarino, C

1982-01-01

173

Effect of neem limonoids on lactate dehydrogenase (LDH) of the rice leaffolder, Cnaphalocrocis medinalis (Guenée) (Insecta: Lepidoptera: Pyralidae).  

PubMed

Neem is derived from the neem tree Azadirachta indica A. Juss. (Meliaceae), and its primary insecticidal component is the tetranortriterpenoid azadirachtin and other limonoids. The effect of neem limonoids azadirachtin, salannin, deacetylgedunin, gedunin, 17-hydroxyazadiradione and deacetylnimbin on enzyme lactate dehydrogenase (LDH) activity of the rice leaffolder (RLF) Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) larvae was investigated. There was a decrease in enzyme activity relative to the control at all concentrations tested. When fed a diet of rice leaves treated with neem limonoids in bioassays, gut tissue enzyme, LDH levels in rice leaffolder larvae are affected. These results indicate neem limonoids affect LDH activity. These effects are most pronounced in early instar larvae. Azadirachtin was the most potent in of all the limonoids in all experiments indicating strong enzyme inhibition. Clear dose-response relationships were established with respect to LDH activity. PMID:16154614

Senthil Nathan, Sengottayan; Kalaivani, Kandaswamy; Chung, Paul Gene; Murugan, Kadarkarai

2006-03-01

174

High Creatine Kinase (CK)-MB and Lactate Dehydrogenase in the Absence of Myocardial Injury or Infarction: A Case Report  

PubMed Central

Acute myocardial infarction (AMI) is a life threatening condition that needs emergency diagnosis and early treatment in the emergency room. Rapid laboratory testing for creatine kinase (CK)-MB greatly revolutionized the diagnosis and management of acute myocardial infarction. We report a case with chest pain that referred to the emergency department (ED). Laboratory data showed high serum levels of creatine kinase and lactate dehydrogenase. With diagnosis of acute myocardial infarction, he was hospitalized and angiography was performed which showed three vessels disease; the patient was referred to surgical ward for coronary artery bypass graft. Surgery was performed after one week; during the operation there was no sign of infarction over the heart. Our observation suggests that false positive laboratory result may be due to other condition which must be evaluated.

Maghamiour, Nasrollah; Safaie, Naser

2014-01-01

175

Production of l-lactic acid by the yeast Candida sonorensis expressing heterologous bacterial and fungal lactate dehydrogenases  

PubMed Central

Background Polylactic acid is a renewable raw material that is increasingly used in the manufacture of bioplastics, which offers a more sustainable alternative to materials derived from fossil resources. Both lactic acid bacteria and genetically engineered yeast have been implemented in commercial scale in biotechnological production of lactic acid. In the present work, genes encoding l-lactate dehydrogenase (LDH) of Lactobacillus helveticus, Bacillus megaterium and Rhizopus oryzae were expressed in a new host organism, the non-conventional yeast Candida sonorensis, with or without the competing ethanol fermentation pathway. Results Each LDH strain produced substantial amounts of lactate, but the properties of the heterologous LDH affected the distribution of carbon between lactate and by-products significantly, which was reflected in extra-and intracellular metabolite concentrations. Under neutralizing conditions C. sonorensis expressing L. helveticus LDH accumulated lactate up to 92 g/l at a yield of 0.94 g/g glucose, free of ethanol, in minimal medium containing 5 g/l dry cell weight. In rich medium with a final pH of 3.8, 49 g/l lactate was produced. The fermentation pathway was modified in some of the strains studied by deleting either one or both of the pyruvate decarboxylase encoding genes, PDC1 and PDC2. The deletion of both PDC genes together abolished ethanol production and did not result in significantly reduced growth characteristic to Saccharomyces cerevisiae deleted of PDC1 and PDC5. Conclusions We developed an organism without previous record of genetic engineering to produce L-lactic acid to a high concentration, introducing a novel host for the production of an industrially important metabolite, and opening the way for exploiting C. sonorensis in additional biotechnological applications. Comparison of metabolite production, growth, and enzyme activities in a representative set of transformed strains expressing different LDH genes in the presence and absence of a functional ethanol pathway, at neutral and low pH, generated a comprehensive picture of lactic acid production in this yeast. The findings are applicable in generation other lactic acid producing yeast, thus providing a significant contribution to the field of biotechnical production of lactic acid.

2013-01-01

176

Production of L-lactic acid by the yeast Candida sonorensis expressing heterologous bacterial and fungal lactate dehydrogenases.  

PubMed

BACKGROUND: Polylactic acid is a renewable raw material that is increasingly used in the manufacture of bioplastics, which offers a more sustainable alternative to materials derived from fossil resources. Both lactic acid bacteria and genetically engineered yeast have been implemented in commercial scale in biotechnological production of lactic acid. In the present work, genes encoding l-lactate dehydrogenase (LDH) of Lactobacillus helveticus, Bacillus megaterium and Rhizopus oryzae were expressed in a new host organism, the non-conventional yeast Candida sonorensis, with or without the competing ethanol fermentation pathway. RESULTS: Each LDH strain produced substantial amounts of lactate, but the properties of the heterologous LDH affected the distribution of carbon between lactate and by-products significantly, which was reflected in extra-and intracellular metabolite concentrations. Under neutralizing conditions C. sonorensis expressing L. helveticus LDH accumulated lactate up to 92 g/l at a yield of 0.94 g/g glucose, free of ethanol, in minimal medium containing 5 g/l dry cell weight. In rich medium with a final pH of 3.8, 49 g/l lactate was produced. The fermentation pathway was modified in some of the strains studied by deleting either one or both of the pyruvate decarboxylase encoding genes, PDC1 and PDC2. The deletion of both PDC genes together abolished ethanol production and did not result in significantly reduced growth characteristic to Saccharomyces cerevisiae deleted of PDC1 and PDC5. CONCLUSIONS: We developed an organism without previous record of genetic engineering to produce L-lactic acid to a high concentration, introducing a novel host for the production of an industrially important metabolite, and opening the way for exploiting C. sonorensis in additional biotechnological applications. Comparison of metabolite production, growth, and enzyme activities in a representative set of transformed strains expressing different LDH genes in the presence and absence of a functional ethanol pathway, at neutral and low pH, generated a comprehensive picture of lactic acid production in this yeast. The findings are applicable in generation other lactic acid producing yeast, thus providing a significant contribution to the field of biotechnical production of lactic acid. PMID:23706009

Ilmén, Marja; Koivuranta, Kari; Ruohonen, Laura; Rajgarhia, Vineet; Suominen, Pirkko; Penttilä, Merja

2013-05-25

177

Covalent binding of 4-hydroxy-2-nonenal to lactate dehydrogenase decreases NADH formation and metmyoglobin reducing activity.  

PubMed

Lactate dehydrogenase (LDH) activity can regenerate NADH, which is a critical component in metmyoglobin reduction. However, limited research has determined the effects of lipid oxidation products on LDH activity. The overall objective of this study was to determine the effects of 4-hydroxy-2-nonenal (HNE) on LDH activity. LDH was reacted with HNE at pH 5.6 and 7.4, and LDH activity was measured as NADH formation following the addition of lactate and NAD. The effects of HNE on NADH-dependent metmyoglobin reduction also were analyzed. Mass spectrometric examination revealed that HNE adducts to LDH at both pH 5.6 and 7.4. More specifically, HNE binds with cysteine and histidine residues of LDH at pH 5.6 and 7.4. Covalent binding of HNE decreased NADH formation and metmyoglobin reduction (P < 0.05). These results indicate that secondary lipid oxidation products can inactivate enzymes involved in metmyoglobin reduction and have the potential to increase beef discoloration. PMID:24552270

Ramanathan, Ranjith; Mancini, Richard A; Suman, Surendranath P; Beach, Carol M

2014-03-01

178

Highly stereoselective biosynthesis of (R)-?-hydroxy carboxylic acids through rationally re-designed mutation of d-lactate dehydrogenase  

PubMed Central

An NAD-dependent d-lactate dehydrogenase (d-nLDH) of Lactobacillus bulgaricus ATCC 11842 was rationally re-designed for asymmetric reduction of a homologous series of ?-keto carboxylic acids such as phenylpyruvic acid (PPA), ?-ketobutyric acid, ?-ketovaleric acid, ?-hydroxypyruvate. Compared with wild-type d-nLDH, the Y52L mutant d-nLDH showed elevated activities toward unnatural substrates especially with large substitutes at C-3. By the biocatalysis combined with a formate dehydrogenase for in situ generation of NADH, the corresponding (R)-?-hydroxy carboxylic acids could be produced at high yields and highly optical purities. Taking the production of chiral (R)-phenyllactic acid (PLA) from PPA for example, 50?mM PPA was completely reduced to (R)-PLA in 90?min with a high yield of 99.0% and a highly optical purity (>99.9% e.e.) by the coupling system. The results presented in this work suggest a promising alternative for the production of chiral ?-hydroxy carboxylic acids.

Zheng, Zhaojuan; Sheng, Binbin; Gao, Chao; Zhang, Haiwei; Qin, Tong; Ma, Cuiqing; Xu, Ping

2013-01-01

179

[Hypoxia-hypercapnic effects on lactate dehydrogenase activity in rat heart tissue].  

PubMed

Activity of lastate dehydrogenase (LDH) was studied in blood and tissues of left and right rat heart ventricles after the simultaneous effect of hypoxia and hypercapnia. The animals were kept in the closed space, where content of gases to the end of the experiment (about 1.5 hr) was equal to: O2--10%, CO2--3%. Activity of LDH was decreased within the first 3 days and the maximal enzymatic activity was observed within 15 days; after 30 days of hypoxic-hypercapnic exposure activity of LDH was significantly decreased in myocardium but it reached the control value in the serum of blood obtained from the heart. PMID:7281555

Mikhalkina, N I; Sverchkova, V S

1981-01-01

180

Kinetic and immunological differences between the retinal specific C 4- and B 4-lactate dehydrogenase of the cichlid fish Oreochromis mossambicus  

Microsoft Academic Search

The eye-specific C4-lactate dehydrogenase (LDH) and the widely distributed B4-LDH isozymes from the fish Oreochromis mossambicus were purified to homogeneity using DEAE Sepharose ion-exchange chromatography and oxamate-linked sepharose affinity-chromatography. Kinetic analysis was performed on pure B4- and C4-LDH. The Michaelis-Menten constant (Km) for B4-LDH and pyruvate was 32.3?M, for B4-LDH and lactate 717 ?M for C4-LDH and pyruvate 14.1 ?M

Jón M. Einarsson; Patrick Joyce; Yvette W. Kunz

1995-01-01

181

[Lactate dehydrogenase from the tetraploid weatherfish Misgurnus fossilis during temperature adaptation: determination of structural differences in two forms of the enzyme by molecular modeling methods].  

PubMed

A structural analysis of two lactate dehydrogenase M4 protein forms has been performed. These structures are the protein products of two lactate dehydrogenase gene (LDH-A) copies in the weatherfish Misgurnus fossilis genome after thermal adaptation (acclimation) to 5 degrees C and 18 degrees C. The localization of three earlier identified amino acid substitutions (Gly214Val, Leu304Ile, Asp312Glu) has been determined, and the molecular dynamics simulation and computer modeling of two forms of the enzyme from skeletal muscles LDH-M4 have been carried out. After molecular dynamics trajectory calculations carried out at 5, 18, and 25 degrees C, the intersubunit distances for all structures used in calculations have been determined. It has been found that the Gly214Val substitution localized in the intersubunit region leads to a new intersubunit interaction, which plays a role in the stabilization of tetrameric enzyme structure after the adaptation to 18 degrees C. PMID:21950062

Puliakhina, I V; Ozerniuk, N D

2011-01-01

182

Changes in the cytoplasmic (lactate dehydrogenase) and plasma membrane (acetylcholinesterase) marker enzymes in the synaptic and nonsynaptic mitochondria derived from rats with moderate hyperammonemia  

Microsoft Academic Search

The activities of the cytoplasmic and plasma membrane marker enzymes: lactate dehydrogenase (LDH) and acetylcholinesterase\\u000a (AChE), respectively, were measured in the cerebral homogenates, in the synaptic and nonsynaptic mitochondrial fractions,\\u000a and in the postmitochondrial supernatants derived from rats in which a 3-d, moderately hyperammonemic condition (no more than\\u000a 120% increases in blood ammonia) was produced by repeated administration of ammonium

Lidia Faff-Michalak; Jan Albrecht

1993-01-01

183

Additive effects of antitumor drugs and lymphokine-activated killer cell cytotoxic activity in tumor cell killing determined by lactate-dehydrogenase-release assay  

Microsoft Academic Search

Summary The effect of pretreatment with antitumor drugs on lymphokine-activated killer (LAK) cell cytotoxic activity, determined by lactate-dehydrogenase(LDH)-release assay, was investigated. LAK cells were induced by incubating peripheral blood lymphocytes of healthy donors in medium containing interleukin-2 (IL-2) and monoclonal anti-CD3 antibody for 6–7 days. A human lung squamous carcinoma cell line, SQ-5, was used as an adherent target. After

Koji Kawail; Tetsuji Sasakil; Kaoru Saijo-Kurita; Hideyuki Akaza; Kenkichi Koiso; Tadao Ohnol

1992-01-01

184

Measurement of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity and lactate dehydrogenase release using MTT  

Microsoft Academic Search

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and lactate dehydrogenase (LDH) release assay have been widely used for evaluating cell viability in culture. MTT reduction assay measures the redox activity of living cells, while LDH assay measures the activity of LDH released into the medium from dead cells. In this paper, we introduce a quick and simple method of measuring cellular MTT

Kazuho Abe; Norio Matsuki

2000-01-01

185

Purification and Properties of the Threespine Stickleback ( Gasterosteus aculeatus) Lactate Dehydrogenase LDH-B 4 and LDH-C 4 Isoenzymes  

Microsoft Academic Search

In the threespine stickleback (Gasterosteus aculeatus) lactate dehydrogenase (LDH, EC 1.1.1.27) is encoded by three loci, Ldh-A, Ldh-B, and Ldh-C. LDH-B4 isoenzyme restricted its function to eye and brain, while LDH-C4 isoenzyme functions in the eye. In the Dead Vistula stickleback population, none of LDH loci is polymorphic. The LDH-B4 and LDH-C4 isoenzymes from the eye were purified to homogeneity

Marek S. Zitara; Jadwiga Gronczewska; Edward F. Skorkowski

1997-01-01

186

The interaction of solutes and temperature on A4-lactate dehydrogenase orthologs from warm-adapted and cold-adapted marine fishes  

Microsoft Academic Search

We examined the effects of temperature and stabilizing solutes on A4-lactate dehydrogenase (A4-LDH) from warm- and cold-adapted fishes, to determine how extrinsic stabilizers affect orthologs with different intrinsic stabilities. Conformational changes during substrate binding are rate- limiting for A4-LDH, thus stabilization due to intrinsic or extrinsic factors leads to decreased activity. A4-LDH from a warm-temperate goby (Gillichthys mirabilis ), which

Peter A. Fields; Benjamin D. Wahlstrand; George N. Somero

187

Thermostability of lactate dehydrogenase LDH-A 4 isoenzyme: Effect of heat shock protein DnaK on the enzyme activity  

Microsoft Academic Search

Cells exposed to temperature a few degrees higher than their growth temperature synthesize heat shock proteins (hsp) which may then compose even 20% of total protein content. This paper examined the in vitro protective effect of heat shock protein DnaK (70 kDa) from Escherichia coli against the heat inactivation of lactate dehydrogenase isoenzyme LDH-A4. The LDH-A4 isoenzyme was purified from

Marek S. Zi; Edward F. Skorkowski

1995-01-01

188

Decreases in Activation Energy and Substrate Affinity in Cold-Adapted A4Lactate Dehydrogenase: Evidence from the Antarctic Notothenioid Fish Chaenocephalus aceratus  

Microsoft Academic Search

Enzyme function is strongly affected by temperature, and orthologs from species adapted to different thermal environments often show temperature compensation in kinetic properties. Antarctic notothenioid fishes live in a habitat of constant, extreme cold (-1.86 6 28C), and orthologs of the enzyme A4-lactate dehydrogenase (A4-LDH) in these species have adapted to this environment through higher catalytic rates, lower Arrhenius activation

Peter A. Fields; Daniel E. Houseman

2004-01-01

189

Comparative characterization of a temperature responsive gene (lactate dehydrogenase-B, ldh-b) in two congeneric tropical fish, Lates calcarifer and Lates niloticus  

Microsoft Academic Search

The characterization of candidate loci is a critical step in obtaining insight into adaptation and acclimation of organisms. In this study of two non-model tropical (to sub-tropical) con- generic perciformes (Lates calcarifer and Lates niloticus) we characterized both coding and non-coding regions of lactate dehydrogenase-B (ldh-b), a locus which exhibits tempera- ture-adaptive differences among temperate and sub-tropical populations of the

Richard C. Edmunds; Lynne van Herwerden; Carolyn Smith-Keune; Dean R. Jerry

190

Molecular Evolution of Mammalian Lactate Dehydrogenase-A Genes and Pseudogenes: Association of a Mouse Processed Pseudogene with a Bl Repetitive Sequence  

Microsoft Academic Search

A mouse genomic clone containing a lactate dehydrogenase-A (LDH-A) processed pseudogene and a Bl repetitive element was isolated, and a nucleotide sequence of -3 kb was determined. The pseudogene and Bl element are flanked by perfect 13-bp repeats, and the B 1 sequence starts at 14 nucleotides 3' to the presumptive polyadenylation signal of the pseudogene. The nucleotide sequences of

Kayoko M. Fukasawa; Wen-Hsiung Li; Kiyohito Yagi; Chi-Cheng Luo; Steven S.-L. Li

191

Serum lactate dehydrogenase isoenzyme 1 and relapse in patients with nonseminomatous testicular germ cell tumors clinical stage I.  

PubMed

Serum lactate dehydrogenase isoenzyme 1 catalytic concentration (S-LD-1) was measured at the time of orchiectomy in 104 patients with nonseminomatous testicular germ cell tumors (NSTGCT) clinical stage I who participated in a randomized study comparing surveillance after orchiectomy (group I) and radiotherapy (group II). For 68 patients, S-LD-1 was measured in a serum sample before or on the day of the orchiectomy. Twenty-seven patients (40%) had elevated S-LD-1; median 102 U/L (range 41-335). For the remaining 36 patients. S-LD-1 was measured in a serum sample after orchiectomy: 8 of these patients (22%) had elevated S-LD-1. S-LD-1 was normalized shortly after surgery in most patients with a preorchiectomy elevated S-LD-1. Fifteen of the 68 patients relapsed: 9 out of 27 with an elevated S-LD-1 and 6 out of 41 patients with normal S-LD-1 (p = 0.13, Fisher's exact test). In group 1, those with a preoperatively elevated S-LD-1 had a lower 8-years' relapse-free survival than those with a normal S-LD-1 (40% vs. 80%, p = 0.003, log-rank test). The role of S-LD-1 in the staging, prognostication and monitoring of patients with NSGCT clinical stage I should be further explored in a large, prospective study. PMID:11504315

von Eyben, F E; Madsen, E L; Blaabjerg, O; Petersen, P H; von der Maase, H; Jacobsen, G K; Rørth, M

2001-01-01

192

Serum Level of Lactate Dehydrogenase is a Useful Clinical Marker to Monitor Progressive Multiple Myeloma Diseases: A Case Report.  

PubMed

To follow the progression of multiple myeloma (MM) disease, serum lactate dehydrogenase (LDH) levels are as useful markers as beta-2 microglobulin and monoclonal immunoglobulin. With this study, we have presented a case of a patient with a multiple myeloma which was fulminant course, whose LDH levels were normal at the onset of diagnosis increasing as 27 times more than normal as the disease progressed and who showed the development of extramedullary plasmacytomas. The patient, an 80-year-old female, was diagnosed with stage IIIA IgA type multiple myeloma and melphalan-prednisolon (MP) treatment was started. Although the LDH levels were low during the diagnosis and chemotherapy, the LDH levels increased up to 7557 U/L following the progression and occurrence of extramedullary plasmacytomas and the patient died. During the observation of the patient with MM, if the LDH levels are abnormally high, the progression of the disease should be considered after eliminating the other causes. Bone marrow aspiration and biopsy should be examined and the progression or relapse should be shown. On the other hand, the patients with LDH levels are high should be considered to have added plasmacytomas, the whole body should be examined at an early stage before the development of clinical symptoms and early treatment should be started. PMID:24764735

Teke, Hava Üsküdar; Ba?ak, Mustafa; Teke, Deniz; Kanbay, Mehmet

2014-03-01

193

Serum Level of Lactate Dehydrogenase is a Useful Clinical Marker to Monitor Progressive Multiple Myeloma Diseases: A Case Report  

PubMed Central

To follow the progression of multiple myeloma (MM) disease, serum lactate dehydrogenase (LDH) levels are as useful markers as beta-2 microglobulin and monoclonal immunoglobulin. With this study, we have presented a case of a patient with a multiple myeloma which was fulminant course, whose LDH levels were normal at the onset of diagnosis increasing as 27 times more than normal as the disease progressed and who showed the development of extramedullary plasmacytomas. The patient, an 80-year-old female, was diagnosed with stage IIIA IgA type multiple myeloma and melphalan-prednisolon (MP) treatment was started. Although the LDH levels were low during the diagnosis and chemotherapy, the LDH levels increased up to 7557 U/L following the progression and occurrence of extramedullary plasmacytomas and the patient died. During the observation of the patient with MM, if the LDH levels are abnormally high, the progression of the disease should be considered after eliminating the other causes. Bone marrow aspiration and biopsy should be examined and the progression or relapse should be shown. On the other hand, the patients with LDH levels are high should be considered to have added plasmacytomas, the whole body should be examined at an early stage before the development of clinical symptoms and early treatment should be started.

Teke, Hava Uskudar; Basak, Mustafa; Teke, Deniz; Kanbay, Mehmet

2014-01-01

194

Large scale dynamics of the Michaelis complex in Bacillus stearothermophilus lactate dehydrogenase revealed by a single-tryptophan mutant study.  

PubMed

Large scale dynamics within the Michaelis complex mimic of Bacillus stearothermophilus thermophilic lactate dehydrogenase, bsLDH·NADH·oxamate, were studied with site specific resolution by laser-induced temperature jump relaxation spectroscopy with a time resolution of 20 ns. NADH emission and Trp emission from the wild type and a series of single-tryptophan bsLDH mutants, with the tryptophan positions different distances from the active site, were used as reporters of evolving structure in response to the rapid change in temperature. Several distinct dynamical events were observed on the millisecond to microsecond time scale involving motion of atoms spread over the protein, some occurring concomitantly or nearly concomitantly with structural changes at the active site. This suggests that a large portion of the protein-substrate complex moves in a rather concerted fashion to bring about catalysis. The catalytically important surface loop undergoes two distinct movements, both needed for a competent enzyme. Our results also suggest that what is called "loop motion" is not just localized to the loop and active site residues. Rather, it involves the motion of atoms spread over the protein, even some quite distal from the active site. How these results bear on the catalytic mechanism of bsLDH is discussed. PMID:23428201

Nie, Beining; Deng, Hua; Desamero, Ruel; Callender, Robert

2013-03-19

195

Targeting glucose metabolism in chondrosarcoma cells enhances the sensitivity to doxorubicin through the inhibition of lactate dehydrogenase-A.  

PubMed

Chondrosarcoma is a malignant cartilage-forming cancer composed of cells derived from transformed cells that produce cartilage. Conventional chemotherapy and radiotherapy have very limited efficacy in patients with advanced chondrosarcoma. In the present study, we reported a novel therapeutic approach in the treatment of chondrosarcoma cells. We detected that lactate dehydrogenase-A (LDHA) is highly active in chondrosarcoma cells and chondrosarcoma patient samples compared with normal chondrocyte cell lines and primary human chondrocyte. Moreover, chondrosarcoma cells exhibited elevated levels of LDHA expression under doxorubicin treatment. To further explore the mechanisms, we generated doxorubicin-resistant cells from SW1353 chondrosarcoma cell line. Notably, the activity and expression of LDHA are upregulated in doxorubicin-resistant cells. Moreover, our data showed a strong correlation between glucose metabolism and doxorubicin resistance in chondrosarcoma cells; doxorubicin-resistant cells displayed highly activated glucose metabolism and depended more on glucose supply. Finally, we reported a synergistic effect produced by incorporating doxorubicin with glycolysis inhibitors-oxamate in the combined treatment of chondrosarcoma cells in vitro and in vivo. In summary, the present study may aid in the development of new approaches using the combination of chemotherapeutic agents for the treatment of chondrosarcoma patients. PMID:24789077

Hua, Guojun; Liu, Yunpeng; Li, Xiangyong; Xu, Peirong; Luo, Yuchun

2014-06-01

196

Large Scale Dynamics of the Michaelis Complex in B. stearothermophilus Lactate Dehydrogenase Revealed by Single Tryptophan Mutants Study  

PubMed Central

Large scale dynamics within the Michaelis complex mimic of Bacillus stearothermophilus thermophylic lactate dehydrogenase, bsLDH•NADH•oxamate, were studied with site specific resolution by laser induced temperature jump relaxation spectroscopy having a time resolution of 20 ns. NADH emission and Trp emission from the wild type and a series of single-tryptophan bsLDH mutants, with the tryptophan positions at different distances from the active site, were used as reporters of evolving structure in response to the rapid change in temperature. Several distinct dynamical events were observed on the ms - ?s time-scale involving motion of atoms spread over the protein, some occurring concomitantly or nearly concomitantly with structural changes at the active site. This suggests that a large portion of the protein-substrate complex moves in a rather concerted fashion to bring about catalysis. The catalytically important surface loop undergoes two distinct movements, both needed for a competent enzyme. Our results also suggest that what is called `loop motion' is not just localized to the loop and active site residues. Rather, it involves the motion of atoms spread over the protein, even some quite distal from the active site. How these results bear on catalytic mechanism of bsLDH is discussed.

Nie, Beining; Deng, Hua; Desamero, Ruel; Callender, Robert

2013-01-01

197

Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider, Trachemys scripta elegans  

PubMed Central

Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD+. The present study investigated purified LDH from the muscle of 20?h anoxic and normoxic T. s. elegans, and LDH from anoxic muscle showed a significantly lower (47%) Km for L-lactate and a higher Vmax value than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a) stimulation of endogenously present protein phosphatases decreased the Km of L-lactate of control LDH to anoxic levels, whereas (b) stimulation of kinases increased the Km of L-lactate of anoxic LDH to normoxic levels, and (c) dot blot analysis shows significantly less serine (78%) and threonine (58%) phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions.

Dawson, Neal J.; Bell, Ryan A. V.; Storey, Kenneth B.

2013-01-01

198

Antigen capture immuno-chromatographic strip format in detecting parasite-specific lactate dehydrogenase to diagnose malaria in nonimmune patients.  

PubMed

Several rapid diagnostic test devices (RDT) based on detection of malaria antigen in the whole blood were developed. OptiMal test the presence of parasite-specific lactate dehydrogenase (LDH) enzyme using three monoclonal antibodies was used. Two monoclonal antibodies were pan-specific and recognized all malaria species. The third one was specific only for Plasmodium falciparum. The parasite antigens were detected using an antigen-capture immunochromatographic strip format. One hundred-nine malaria positive and 730 malaria negative cases diagnosed by microscopy were included. 75/109 were P. falciparum 26 as P. vivax, 3 P. malariae and 5 mixed infection of P. falciparum & P. vivax. The RDT showed a low sensitivity (85%, 95% Confidence Interval [CI], 79-92%) with a much lower sensitivity in detecting species other than P. falciparum as well as in mixed infections. The sensitivity was 50% for less than 200 parasites/micro. The sensitivity of OptiMal for P. falciparum was 87% (95% CI, 79-94), 81% (95% CI, 66-96) for P. vivax, and failed with P. malariae. Mixed infections were misdiagnosed as Pfalciparum. The sensitivity of OptiMal was quite good in detecting both P. falciparum & P. vivax (98%; 95% CI, 97-99 & 100%; 95% CI, 100-100 respectively) and 99% (95% CI, 98-99) for all species. The positive and negative ratio for all malaria species was: (+LR = 62.3, -LR = 0.01); for P. falciparum (+LR = 38.9, -LR = 0.01) and for P. vivax (+LR = 0.8077/0, -LR = 0.2). The test value to assess drug resistance in post treatment days was discussed. PMID:18383801

El-Moamly, Amal M Abdul-Rasheed

2007-12-01

199

Elevated lactate dehydrogenase activity and increased cardiovascular mortality in the arsenic-endemic areas of southwestern Taiwan  

SciTech Connect

Arsenic ingestion has been linked to increasing global prevalence of and mortality from cardiovascular disease (CVD); arsenic can be removed from drinking water to reduce related health effects. Lactate dehydrogenase (LDH) is used for the evaluation of acute arsenic toxicity in vivo and in vitro, but it is not validated for the evaluation of long-term, chronic arsenic exposure. The present study examined the long-term effect of chronic arsenic exposure on CVD and serum LDH levels, after consideration of arsenic metabolism capacity. A total of 380 subjects from an arseniasis-endemic area and 303 from a non-endemic area of southwestern Taiwan were recruited in 2002. Various urinary arsenic species were analyzed using high-performance liquid chromatography (HPLC) and hydride generation systems. Fasting serum was used for quantitative determination of the total LDH activity. A significant dose–response relationship was observed between arsenic exposure and LDH elevation, independent of urinary arsenic profiles (P < 0.001). Furthermore, abnormal LDH elevation was associated with CVD mortality after adjustment for Framingham risk scores for 10-year CVD and arsenic exposure (hazard ratio, 3.98; 95% confidence interval, 1.07–14.81). LDH was elevated in subjects with arsenic exposure in a dose-dependent manner. LDH is a marker of arsenic toxicity associated with CVD mortality. Results of this study have important implications for use in ascertaining long-term arsenic exposure risk of CVD. -- Highlights: ? We showed that arsenic exposure was correlated with LDH elevation. ? LDH elevation was related to arsenic methylation capacity. ? Abnormal LDH elevation can be a marker of susceptibility to CVD mortality.

Liao, Ya-Tang [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan (China) [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan (China); Graduate Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taiwan (China); Genomics Research Center, Academia Sinica, Taiwan (China); Chen, Chien-Jen [Graduate Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taiwan (China) [Graduate Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taiwan (China); Genomics Research Center, Academia Sinica, Taiwan (China); Li, Wan-Fen [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan (China)] [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan (China); Hsu, Ling-I [Genomics Research Center, Academia Sinica, Taiwan (China)] [Genomics Research Center, Academia Sinica, Taiwan (China); Tsai, Li-Yu; Huang, Yeou-Lih [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Taiwan (China)] [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Taiwan (China); Sun, Chien-Wen [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan (China)] [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan (China); Chen, Wei J., E-mail: wjchen@ntu.edu.tw [Graduate Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taiwan (China); Genetic Epidemiology Core Laboratory, National Taiwan University Center for Genomic Medicine, Taiwan (China); Wang, Shu-Li, E-mail: slwang@nhri.org.tw [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan (China) [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan (China); Department of Public Health, College of Public Health, China Medical University, Taichung, Taiwan (China)

2012-08-01

200

Impact of an acute bout of vibration on muscle contractile properties, creatine kinase and lactate dehydrogenase response.  

PubMed

The aim of this study was to assess the effects of a bout of whole body vibration (WBV) on muscle response and to determine whether this stimulus leads to muscle damage. Thirty healthy and physically active participants (mean ± SD; age: 21.8 ± 2.0 years; height: 176.7 ± 5.8 cm; body mass: 76 ± 6.8 kg and BMI: 23.1 ± 3.7 kg·m(-2)) participated in this study. Participants were randomly allocated in one of two groups, one of them performed a bout of 360 s WBV (frequency: 30 Hz; peak-to-peak displacement: 4 mm) (VIB) and the other one adopted a sham position (CON). Muscle contractile properties were analysed in the rectus femoris (RF) by using tensiomyography (TMG) 2 min before the warm-up and 2 min after intervention. Muscle damage was assessed by determining plasma creatine kinase (CK) and lactate dehydrogenase (LDH) levels at three time points; 5 min before warm-up and 1 h and 48 h after the intervention. TMG results showed a significant decrease in maximal displacement (p<0.05) and delay time (p<0.05) in VIB and in delay time (p<0.05) and relaxation time (p<0.05) in CON. Muscle damage markers showed significant group differences (p<0.05) for CK 1 h after the intervention. In addition, differences for CK 1 h after the intervention from baseline (p<0.05) were also observed in VIB. In conclusion, a 6-min bout of WBV results in an increase of muscle stiffness in RF and increased CK levels 1 h after intervention (returning to baseline within 48 h). PMID:24251744

de Hoyo, Moisés; Carrasco, Luis; Da Silva-Grigoletto, Marzo E; Sañudo, Borja; Caballero-Villarraso, Javier; Arriaza, Eva; Escobar, Maria Del Carmen

2013-01-01

201

[Diagnostic value of definition of lactate dehydrogenase in mixed saliva in children with periodontitis at diabetes mellitus, type I].  

PubMed

The problem of treatment of periodontitis remains one of the hot topics in practical stomatology. It has been established that modern adaptogenic infection is rather aggressive to whole organism of a human being. All these demands accurate approach while choosing of a conservative method of treatment for such forms as acute and chronic periodontitis. There were 27 children under observation with diabetes mellitus of type 1 (I group). Mean age was 10.5+/-0.75 years. 15 were girls and 13 boys. All patients from the I group were examined for the pathologies of oral cavity. In 100% dryness in a mouth and in 67% bleeding from the gum had been revealed. The mild form of chronic catarrhal gingivitis was revealed in 12 patients, moderate in 5, chronic hypertrophic gingivitis in 8 respectively. Studying of pH of saliva and lactate dehydrogenase (LDH) activity in children with periodontitis developed on the background of recently diagnosed type 1 diabetes mellitus has shown, that pH of saliva was equal to 5.3+/-0.18. In control group (healthy children) pH of saliva was 6.8+/-0.06. In the conclusion it should be emphasized, that we have tried to explain some aspects of multiple character of development of periodontitis at recently discovered insulin-depended diabetes mellitus. Character of changes of some properties of saliva pH and of enzyme activity of LDG promotes to carrying out medical and preventive actions, influencing the main blocks of pathogenesis of this pathological process. Besides, we consider possibility of inclusion the studied parameters of mixed saliva in the algorithm of investigation of periodontitis in children with recently diagnosed type 1 diabetes mellitus. PMID:16510913

Chidzhavadze, E M; Akhvlediani, M V; Vadachkoriia, Z O; Gordeladze, M R

2006-01-01

202

Effect of some thiocarbamate compounds on aldehyde dehydrogenase and implications for the disulfiram ethanol reaction.  

PubMed Central

The effects of S-methyl diethyldithiocarbamate, S-methyl diethylmonothiocarbamate and bis(diethylcarbamoyl) disulphide on sheep liver cytoplasmic aldehyde dehydrogenase were investigated in vitro. The first compound has negligible effect. The second one is a weak inhibitor of the esterase activity of the enzyme and a weaker inhibitor of the dehydrogenase activity. A very low concentration of the third compound, however, acts as a potent inactivator of aldehyde dehydrogenase, similar in this respect to disulfiram, although somewhat slower to react. The possible involvement of these compounds in the physiological phenomenon known as the disulfiram ethanol reaction is discussed.

Kitson, T M

1991-01-01

203

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol using resting cells of the lactate dehydrogenase-deficient recombinant Klebsiella pneumoniae overexpressing an aldehyde dehydrogenase.  

PubMed

In the present study, the lactate dehydrogenase-deficient (ldhA(-)) recombinant Klebsiella pneumoniae overexpressing an ALDH (KGSADH) was developed and the co-production of 3-HP and PDO from glycerol by this recombinant under resting cell conditions was examined. The new recombinant did not produce any appreciable lactate, which seriously inhibits the production of 3-HP and PDO. The final titers of 3-HP and PDO by the ldhA(-) recombinant strain at 60 h were 252.2 mM and 308.7 mM, respectively, which were improved by approximately 30% and 50%, respectively, compared to those by the counterpart recombinant strain, which was the wild type for ldhA. In addition, after deleting ldhA, the cumulative yield on glycerol and specific production rate of these two metabolites (3-HP and PDO) were enhanced by 41.4% and 52%, respectively. PMID:23228456

Kumar, Vinod; Sankaranarayanan, Mugesh; Durgapal, Meetu; Zhou, Shengfang; Ko, Yeounjoo; Ashok, Somasundar; Sarkar, Ritam; Park, Sunghoon

2013-05-01

204

Exposing local adaptation: synergistic stressors elicit population-specific lactate dehydrogenase-B ( ldh - b ) expression profiles in Australian barramundi, Lates calcarifer  

Microsoft Academic Search

The molecular response of fish to independently and\\/or concurrently applied ecological stressors (e.g. thermal and\\/or aerobic\\u000a stress) can be quantified at the level of transcript abundance (i.e. gene expression). In temperate fish, the expression of\\u000a the metabolic candidate gene lactate dehydrogenase-B (ldh-b) responds to both aerobic swimming challenge and extended acclimation to various ecologically relevant temperatures. We examined\\u000a hepatic ldh-b

Richard C. Edmunds; Carolyn Smith-Keune; Lynne van Herwerden; Christopher J. Fulton; Dean R. Jerry

205

Changes of Reaction Time and Blood Lactate Concentration of Elite Volleyball Players During a Game  

PubMed Central

The purpose of this study was to investigate changes in reaction time of elite volleyball players during a game. Fourteen volleyball players participated in the study. Reaction time was measured using the Optojump system. In addition, blood lactate concentration was assessed to monitor physiological load during the game. All measurements were performed during a pre-game test and during sets 1, 2, 3 and 4. Reaction time during set 1 decreased significantly by 13,3 % compared with pre-game values, from 600 ms during the pre-game test to 520 ms during set 1 (p<0,05). Blood lactate concentration increased significantly during set 1, 2, 3 and 4 compared with pre-game conditions (p<0,05). Reaction time stays in the first phase of its changes pattern and elite volleyball players do not reach psychomotor fatigue threshold throughout the game.

Mroczek, Dariusz; Kawczynski, Adam; Chmura, Jan

2011-01-01

206

Stereoselective titanium-mediated aldol reactions of a chiral lactate-derived ethyl ketone with ketones.  

PubMed

Aldol reactions of titanium enolates of lactate-derived ethyl ketone 1 with other ketones proceed in a very efficient and stereocontrolled manner provided that a further equivalent of TiCl4 is added to the reacting mixture. The scope of these reactions encompasses simple ketones such as acetone or cyclohexanone as well as other ketones that contain potential chelating groups such as pyruvate esters or ?- and ?-hydroxy ketones. PMID:24372372

Alcoberro, Sandra; Gómez-Palomino, Alejandro; Solà, Ricard; Romea, Pedro; Urpí, Fèlix; Font-Bardia, Mercè

2014-01-17

207

Serum lactate dehydrogenase isoenzyme 1 and prediction of death in patients with metastatic testicular germ cell tumors.  

PubMed

The International Germ Cell Cancer Collaborative Group study of patients with metastatic testicular germ cell tumors showed that catalytic concentration of serum lactate dehydrogenase (S-LD), serum alpha-fetoprotein concentration (S-AFP), and serum human chorionic gonadotropin concentration (S-hCG) predicted death from tumor. The recent international TNM classification (T primary tumor, N lymph node metastasis, M distant metastasis) is based on these results. The aim of our study was to evaluate whether catalytic concentration of S-LD isoenzyme 1 (S-LD-1) was a better predictor than the criteria used for the international classification. In an evaluation series of 44 patients from Odense University Hospital, Denmark, a raised S-LD-1 (>1.0 x upper limit of reference values) had a predictive value for death from tumor in 5-years observation of 46%. The predictive value was 46% for S-LD, 25% for S-AFP, and 40% for S-hCG. A normal SLD-1 had a predictive value for survival over 5-years observation of 100%. It was 81% for S-LD, 75% for SAFP, and 77% for S-hCG. The fraction of the patients who died of tumor and had a raised tumor marker value was 100% for S-LD-1, 46% for S-LD, 9% for S-AFP, and 18% for S-hCG. The fraction of patients with a normal serum tumor marker value among those who survived was 61% for S-LD-1, 81% for S-LD, 94% for SAFP, and 94% for S-hCG. A validation series of 37 patients treated at the University of Texas MD Anderson Cancer Center showed similar findings. Combining the patients in the two series, a raised value of SLD-1 classified more patients into a subgroup with an impaired survival (53%) than S-LD (35%), S-AFP (6%), or S-hCG (11%), and the high risk subgroups based on the international classification (40%). The findings have implications for the staging and treatment of patients with metastatic testicular germ cell tumors. PMID:11256799

von Eyben, F E; Blaabjerg, O; Hyltoft-Petersen, P; Madsen, E L; Amato, R; Liu, F; Fritsche, H

2001-01-01

208

Differences and similarities in binding of pyruvate and L-lactate in the active site of M4 and H4 isoforms of human lactate dehydrogenase.  

PubMed

We present QM/MM calculations that show differences in geometries of active sites of M(4) and H(4) isoforms of human LDH ligated with oxamate, pyruvate or L-lactate. As the consequence of these differences, binding isotope effects of the methyl hydrogen atoms of pyruvate and l-lactate may be used to experimentally distinguish these isoforms. Based on the FEP calculations we argue that L-lactate is a better candidate for the experimental studies. Our calculations of energies of interactions of ligands with the active site residues provide explanation for the observed experimentally sensitivity to inhibition of the M(4) isoenzyme isoform and pinpoint the differences to interactions of the ligand with the histidine residue. We conclude that pyruvate interacts much stronger in the active site of H(4) than M(4) isoform and that the latter interactions are weaker than with water molecules in the aqueous solution. PMID:20951115

Swiderek, Katarzyna; Paneth, Piotr

2011-01-01

209

Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth  

PubMed Central

L-Lactic acid, one of the most important chiral molecules and organic acids, is produced via pyruvate from carbohydrates in diverse microorganisms catalyzed by an NAD+-dependent L-lactate dehydrogenase. Naturally, Escherichia coli does not produce L-lactate in noticeable amounts, but can catabolize it via a dehydrogenation reaction mediated by an FMN-dependent L-lactate dehydrogenase. In aims to make the E. coli strain to produce L-lactate, three L-lactate dehydrogenase genes from different bacteria were cloned and expressed. The L-lactate producing strains, 090B1 (B0013-070, ?ldhA::diflldD::Pldh-ldhLca), 090B2 (B0013-070, ?ldhA::diflldD::Pldh-ldhStrb) and 090B3 (B0013-070, ?ldhA::diflldD::Pldh-ldhBcoa) were developed from a previously developed D-lactate over-producing strain, E. coli strain B0013-070 (ack-ptappspflBdldpoxBadhEfrdA) by: (1) deleting ldhA to block D-lactate formation, (2) deleting lldD to block the conversion of L-lactate to pyruvate, and (3) expressing an L-lactate dehydrogenase (L-LDH) to convert pyruvate to L-lactate under the control of the ldhA promoter. Fermentation tests were carried out in a shaking flask and in a 25-l bioreactor. Strains 090B1, 090B2 or 090B3 were shown to metabolize glucose to L-lactate instead of D-lactate. However, L-lactate yield and cell growth rates were significantly different among the metabolically engineered strains which can be attributed to a variation between temperature optimum for cell growth and temperature optimum for enzymatic activity of individual L-LDH. In a temperature-shifting fermentation process (cells grown at 37°C and L-lactate formed at 42°C), E. coli 090B3 was able to produce 142.2 g/l of L-lactate with no more than 1.2 g/l of by-products (mainly acetate, pyruvate and succinate) accumulated. In conclusion, the production of lactate by E. coli is limited by the competition relationship between cell growth and lactate synthesis. Enzymatic properties, especially the thermodynamics of an L-LDH can be effectively used as a factor to regulate a metabolic pathway and its metabolic flux for efficient L-lactate production. Highlights The enzymatic thermodynamics was used as a tool for metabolic regulation. ? minimizing the activity of L-lactate dehydrogenase in growth phase improved biomass accumulation. ? maximizing the activity of L-lactate dehydrogenase improved lactate productivity in production phase.

2014-01-01

210

Relayed (13)C magnetization transfer: detection of malate dehydrogenase reaction in vivo.  

PubMed

Malate dehydrogenase catalyzes rapid interconversion between dilute metabolites oxaloacetate and malate. Both oxaloacetate and malate are below the detection threshold of in vivo MRS. Oxaloacetate is also in rapid exchange with aspartate catalyzed by aspartate aminotransferase, the latter metabolite is observable in vivo using (13)C MRS. We hypothesized that the rapid turnover of oxaloacetate can effectively relay perturbation of magnetization between malate and aspartate. Here, we report indirect observation of the malate dehydrogenase reaction by saturating malate C2 resonance at 71.2 ppm and detecting a reduced aspartate C2 signal at 53.2 ppm due to relayed magnetization transfer via oxaloacetate C2 at 201.3 ppm. Using this strategy the rate of the cerebral malate dehydrogenase reaction was determined to be 9+/-2 micromol/g wet weight/min (means+/-SD, n=5) at 11.7 Tesla in anesthetized adult rats infused with [1,6-(13)C(2)]glucose. PMID:17126047

Yang, Jehoon; Shen, Jun

2007-02-01

211

Secondary sup 15 N isotope effects on the reactions catalyzed by alcohol and formate dehydrogenases  

SciTech Connect

Secondary {sup 15}N isotope effects at the N-1 position of 3-acetylpyridine adenine dinucleotide have been determined, by using the internal competition technique, for horse liver alcohol dehydrogenase (LADH) with cyclohexanol as a substrate and yeast formate dehydrogenase (FDH) with formate as a substrate. On the basis of less precise previous measurements of these {sup 15}N isotope effects, the nicotinamide ring of NAD has been suggested to adopt a boat conformation with carbonium ion character at C-4 during hydride transfer. If this mechanism were valid, as N-1 becomes pyramidal an {sup 15}N isotope effect for the reaction catalyzed by LADH was measured. These values suggest that a significant {sup 15}N kinetic isotope effect is not associated with hydride transfer for LADH and FDH. Thus, in contrast with the deformation mechanism previously postulated, the pyridine ring of the nucleotide apparently remains planar during these dehydrogenase reactions.

Rotberg, N.S.; Cleland, W.W. (Univ. of Wisconsin, Madison (USA))

1991-04-23

212

13C-NMR and transient kinetic studies on lactate dehydrogenase [Cys(13CN)165]. Direct measurement of a rate-limiting rearrangement in protein structure.  

PubMed

Chemical modification of cysteine-165 in pig heart lactate dehydrogenase to produce lactate dehydrogenase [Cys(13CN)165] introduces an covalently bound, enriched 13C probe at a position adjacent to the active cen. The signal from the thiocyanate probe is clearly visible at 47 ppm relative to dioxane. On formation of binary complexes with NAD+ and NADH, no signal change is detected. Formation of the ternary complexes E-NADH-oxamate and E-NAD+-oxalate results in an upfield shift of the signal of 1.2 ppm. These results interpreted as demonstrating that binding of the substrate analogue induces a conformational change a position adjacent to the active centre. Exchange experiments in which the enzyme is poised in dynamic equilibrium between binary and ternary complexes show that the rate at which the probe senses a change environment is the same as the kinetically observed unimolecular event which limits the enzyme-catalyst reduction of pyruvate. The two processes show the same dependence on temperature, solvent composition and pH. These results indicate that the rate-limiting isomerisation corresponds to a rearrangement of the protein in the region of cysteine-165. PMID:3947644

Waldman, A D; Birdsall, B; Roberts, G C; Holbrook, J J

1986-03-01

213

Regeneration of NAD(H) covalently bound to formate dehydrogenase with several second enzymes  

Microsoft Academic Search

N6-[N-(6-Aminohexyl)carbamoylmethyl]-NAD was covalently bound to formate dehydrogenase. The formate dehydrogenase-NAD complex, which contained 0.2 mol of reactive NAD moiety per subunit, functioned as an NAD(H)-regeneration system for a second coupled reaction involving one of the following enzymes; lactate, malate, alanine and leucine dehydrogenases, whose reductive reactions proceeded stoichiometrically in the absence of exogenous NAD.

Nobuo Kato; Tomohide Yamagami; Masayuki Shimao; Chikahiro Sakazawa

1987-01-01

214

Ontogenesis of Oxidative Reaction of 17?-hydroxysteroid Dehydrogenase and 11?-hydroxysteroid Dehydrogenase in Rat Leydig Cells, a Histochemical Study  

Microsoft Academic Search

The enzyme 17-hydroxysteroid dehydrogenase is required for the synthesis and 11-hydroxysteroid dehydrogenase for the regulation of androgens in rat Leydig cells. This histochemical study describes ontogenetic changes in distribution and intensity of these enzymes in Leydig cells from postnatal day (pnd) 1–90. Using NAD or NADP as the cofactor, 17-hydroxysteroid dehydrogenase (substrate: 5-androstene-3, 17-diol) peaks were observed on pnd 16

Barbara A. Schäfers; Britta G. Schlutius; Syed G. Haider

2001-01-01

215

Comparison of a parasite lactate dehydrogenase-based immunochromatographic antigen detection assay (OptiMAL) with microscopy for the detection of malaria parasites in human blood samples.  

PubMed

Microscopic examination of blood smears remains the gold standard for malaria diagnosis, but is labor-intensive and requires skilled operators. Rapid dipstick technology provides a potential alternative. A study was conducted in The Gambia to compare the performance of OptiMAL, an immunochromatographic antigen detection assay for the diagnosis of malaria using parasite lactate dehydrogenase, against standard microscopy in patients with suspected malaria. For initial diagnosis of Plasmodium falciparum, irrespective of stage, this assay had a sensitivity of 91.3%, a specificity of 92%, a positive predictive value of 87.2%, and a negative predictive value of 94.7%. The sensitivity of the test decreased markedly at parasitemias < 0.01%. This assay can be used for the diagnosis of malaria in areas where microscopy is not available and for urgent malaria diagnosis at night and at weekends, when routine laboratories are closed and when relatively inexperienced microscopists may be on duty. PMID:10072131

Cooke, A H; Chiodini, P L; Doherty, T; Moody, A H; Ries, J; Pinder, M

1999-02-01

216

The Effects of Heart and Skeletal Muscle Inflammation and Cardiomyopathy Syndrome on Creatine Kinase and Lactate Dehydrogenase Levels in Atlantic Salmon (Salmo salar L.)  

PubMed Central

Heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS) are putative viral cardiac diseases of Atlantic salmon. This study examined the levels and correlated the serum enzymes creatine kinase (CK) and lactate dehydrogenase (LDH) to the histopathology of clinical outbreaks of HSMI and chronic CMS in farmed Atlantic salmon. A total of 75 fish from 3 different HSMI outbreaks, 30 chronic CMS fish, and 68 fish from 3 nondiseased fish groups were used as the study population (N = 173). Serum CK and LDH levels correlated significantly with the total inflammation and total necrosis scores for HSMI fish (P = 0.001). However, no correlation was identified for enzyme levels and histopathology scores for chronic CMS fish. The significantly increased CK and LDH levels and their positive correlations to histopathology differentiate HSMI from CMS clinically suggesting the potential use of enzymes for screening for HSMI is promising.

Yousaf, Muhammad Naveed; Powell, Mark D.

2012-01-01

217

Analytical bias by contamination from hemolysis in determination of serum lactate dehydrogenase isoenzyme 1 in patients with testis germ cell tumors.  

PubMed

We investigated the impact of correction for contamination from hemolysis on serum lactate dehydrogenase isoenzyme 1 (S-LD-1) determinations. A study of hemolysates from 7 control patients showed a mean correction factor for the contamination of 0.1 U/L S-LD-1 for each 1 mg/L serum hemoglobin (S-Hb). S-LD-1 in a series of blood samples from 44 patients (EJC 1992;28:410-5) would decrease median 24 U/L (range 8-70 U/L) if the measurements were corrected with this factor. So we advice to correct S-LD-1 determinations for the contamination with a common correction based on the S-Hb concentrations in the samples. PMID:7974858

von Eyben, F E; Petersen, P H; Blaabjerg, O; Madsen, E L; Nørgaard-Pedersen, B; Arends, J

1993-01-01

218

The effects of heart and skeletal muscle inflammation and cardiomyopathy syndrome on creatine kinase and lactate dehydrogenase levels in Atlantic salmon (Salmo salar L.).  

PubMed

Heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS) are putative viral cardiac diseases of Atlantic salmon. This study examined the levels and correlated the serum enzymes creatine kinase (CK) and lactate dehydrogenase (LDH) to the histopathology of clinical outbreaks of HSMI and chronic CMS in farmed Atlantic salmon. A total of 75 fish from 3 different HSMI outbreaks, 30 chronic CMS fish, and 68 fish from 3 nondiseased fish groups were used as the study population (N = 173). Serum CK and LDH levels correlated significantly with the total inflammation and total necrosis scores for HSMI fish (P = 0.001). However, no correlation was identified for enzyme levels and histopathology scores for chronic CMS fish. The significantly increased CK and LDH levels and their positive correlations to histopathology differentiate HSMI from CMS clinically suggesting the potential use of enzymes for screening for HSMI is promising. PMID:22701371

Yousaf, Muhammad Naveed; Powell, Mark D

2012-01-01

219

[Leucine arylamidase, lactate dehydrogenase and alkaline phosphatase activity of the urine of normal subjects of infant age].  

PubMed

Urinary activity of Leucine arylamidase, lactate dahydrogenase and Alkaline phosphatase in 14 healt subjects, ranging from 2 to 10 years are described. Some correlations between enzymatic activities, ratios enzymatic activities/creatininuria and enzymatic activities/dayly proteic clearance are investigated. PMID:7375016

Camerini, G; Castaldi, G; Menegatti, E

1980-04-01

220

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of L-lactate dehydrogenase and its H171C mutant from Bacillus subtilis  

SciTech Connect

L-Lactate dehydrogenase (LDH) is an important enzyme involved in the last step of glycolysis that catalyzes the reversible conversion of pyruvate to L-lactate with the simultaneous oxidation of NADH to NAD{sup +}. In this study, wild-type LDH from Bacillus subtilis (BsLDH-WT) and the H171C mutant (BsLDH-H171C) were expressed in Escherichia coli and purified to near-homogeneity. BsLDH-WT was crystallized in the presence of fructose 1,6-bisphosphate (FBP) and NAD{sup +} and the crystal diffracted to 2.38 {angstrom} resolution. The crystal belonged to space group P3, with unit-cell parameters a = b = 171.04, c = 96.27 {angstrom}. BsLDH-H171C was also crystallized as the apoenzyme and in complex with NAD{sup +}, and data sets were collected to 2.20 and 2.49 {angstrom} resolution, respectively. Both BsLDH-H171C crystals belonged to space group P3, with unit-cell parameters a = b = 133.41, c = 99.34 {angstrom} and a = b = 133.43, c = 99.09 {angstrom}, respectively. Tetramers were observed in the asymmetric units of all three crystals.

Zhang, Yanfeng; Gao, Xiaoli (MSU)

2012-08-31

221

Targeting lactate dehydrogenase-a inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor-initiating cells.  

PubMed

The lactate dehydrogenase-A (LDH-A) enzyme catalyzes the interconversion of pyruvate and lactate, is upregulated in human cancers, and is associated with aggressive tumor outcomes. Here we use an inducible murine model and demonstrate that inactivation of LDH-A in mouse models of NSCLC driven by oncogenic K-RAS or EGFR leads to decreased tumorigenesis and disease regression in established tumors. We also show that abrogation of LDH-A results in reprogramming of pyruvate metabolism, with decreased lactic fermentation in vitro, in vivo, and ex vivo. This was accompanied by reactivation of mitochondrial function in vitro, but not in vivo or ex vivo. Finally, using a specific small molecule LDH-A inhibitor, we demonstrated that LDH-A is essential for cancer-initiating cell survival and proliferation. Thus, LDH-A can be a viable therapeutic target for NSCLC, including cancer stem cell-dependent drug-resistant tumors. PMID:24726384

Xie, Han; Hanai, Jun-Ichi; Ren, Jian-Guo; Kats, Lev; Burgess, Kerri; Bhargava, Parul; Signoretti, Sabina; Billiard, Julia; Duffy, Kevin J; Grant, Aaron; Wang, Xiaoen; Lorkiewicz, Pawel K; Schatzman, Sabrina; Bousamra, Michael; Lane, Andrew N; Higashi, Richard M; Fan, Teresa W M; Pandolfi, Pier Paolo; Sukhatme, Vikas P; Seth, Pankaj

2014-05-01

222

Discovery of N-hydroxyindole-based inhibitors of human lactate dehydrogenase isoform A (LDH-A) as starvation agents against cancer cells.  

PubMed

Highly invasive tumor cells are characterized by a metabolic switch, known as the Warburg effect, from "normal" oxidative phosphorylation to increased glycolysis even under sufficiently oxygenated conditions. This dependence on glycolysis also confers a growth advantage to cells present in hypoxic regions of the tumor. One of the key enzymes involved in glycolysis, the muscle isoform of lactate dehydrogenase (LDH-A), is overexpressed by metastatic cancer cells and is linked to the vitality of tumors in hypoxia. This enzyme may be considered as a potential target for new anticancer agents, since its inhibition cuts cancer energetic and anabolic supply, thus reducing the metastatic and invasive potential of cancer cells. We have discovered new and efficient N-hydroxyindole-based inhibitors of LDH-A, which are isoform-selective (over LDH-B) and competitive with both the substrate (pyruvate) and the cofactor (NADH). The antiproliferative activity of these compounds was confirmed on a series of cancer cell lines, and they proved to be particularly effective under hypoxic conditions. Moreover, NMR experiments showed that these compounds are able to reduce the glucose-to-lactate conversion inside the cell. PMID:21332213

Granchi, Carlotta; Roy, Sarabindu; Giacomelli, Chiara; Macchia, Marco; Tuccinardi, Tiziano; Martinelli, Adriano; Lanza, Mario; Betti, Laura; Giannaccini, Gino; Lucacchini, Antonio; Funel, Nicola; León, Leticia G; Giovannetti, Elisa; Peters, Godefridus J; Palchaudhuri, Rahul; Calvaresi, Emilia C; Hergenrother, Paul J; Minutolo, Filippo

2011-03-24

223

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of l--lactate dehydrogenase and its H171C mutant from Bacillus subtilis  

PubMed Central

l-Lactate dehydrogenase (LDH) is an important enzyme involved in the last step of glycolysis that catalyzes the reversible conversion of pyruvate to l-lactate with the simultaneous oxidation of NADH to NAD+. In this study, wild-type LDH from Bacillus subtilis (BsLDH-WT) and the H171C mutant (BsLDH-H171C) were expressed in Escherichia coli and purified to near-homogeneity. BsLDH-WT was crystallized in the presence of fructose 1,6-bisphosphate (FBP) and NAD+ and the crystal diffracted to 2.38?Å resolution. The crystal belonged to space group P3, with unit-cell parameters a = b = 171.04, c = 96.27?Å. BsLDH-H171C was also crystallized as the apoenzyme and in complex with NAD+, and data sets were collected to 2.20 and 2.49?Å resolution, respectively. Both BsLDH-H171C crystals belonged to space group P3, with unit-cell parameters a = b = 133.41, c = 99.34?Å and a = b = 133.43, c = 99.09?Å, respectively. Tetramers were observed in the asymmetric units of all three crystals.

Zhang, Yanfeng; Gao, Xiaoli

2012-01-01

224

Reaction mechanism for mammalian pyruvate dehydrogenase using natural lipoyl domain substrates.  

PubMed

The pyruvate dehydrogenase (E1) component of the pyruvate dehydrogenase complex (PDC) catalyzes a two-step reaction. Recombinant production of substrate amounts of the lipoyl domains of the dihydrolipoyl transacetylase (E2) component of the mammalian PDC allowed kinetic characterization of the rapid physiological reaction catalyzed by E1. Using either the N-terminal (L1) or the internal (L2) lipoyl domain of E2 as a substrate, analyses of steady state kinetic data support a ping pong mechanism. Using standard E1 preparations, Michaelis constants (Km) were 52 +/- 14 microM for L1 and 24.8 +/- 3.8 microM for pyruvate and k(cat) was 26.3 s(-1). With less common, higher activity preparations of E1, the Km values were > or =160 microM for L1 and > or =35 microM for pyruvate and k(cat) was > or =70 s(-1). Similar results were found with the L2 domain. The best synthetic lipoylated-peptide (L2 residues 163-177) was a much poorer substrate (Km > or =15 mM, k(cat) approximately equals 5 s(-1); k(cat)/Km decreased >1,500-fold) than L1 or L2, but a far better substrate in the E1 reaction than free lipoamide (k(cat)/Km increased >500-fold). Each lipoate source was an effective substrate in the dihydrolipoyl dehydrogenase (E3) reaction, but E3 had a lower Km for the L2 domain than for lipoamide or the lipoylated peptides. In contrast to measurements with slow E1 model reactions that use artificial acceptors, we confirmed that the natural E1 reaction, using lipoyl domain acceptors, was completely inhibited (>99%) by phosphorylation of E1 and the phosphorylation strongly inhibited the reverse of the second step catalyzed by E1. The mechanisms by which phosphorylation interferes with E1 activity is interpreted based on accrued results and the location of phosphorylation sites mapped onto the 3-D structure of related alpha-keto acid dehydrogenases. PMID:11368334

Liu, S; Gong, X; Yan, X; Peng, T; Baker, J C; Li, L; Robben, P M; Ravindran, S; Andersson, L A; Cole, A B; Roche, T E

2001-02-15

225

Galloflavin suppresses lactate dehydrogenase activity and causes MYC downregulation in Burkitt lymphoma cells through NAD/NADH-dependent inhibition of sirtuin-1.  

PubMed

Activation of the myc oncogene in cancer cells upregulates lactate dehydrogenase A (LDH-A) expression, leading to a sustained glycolytic flux that is needed to produce ATP under hypoxic conditions. We studied the effects of galloflavin (GF), a recently identified LDH inhibitor, on myc overexpressing Burkitt lymphoma (BL) cells. Epstein-Barr virus-infected lymphoblasts were used as a non-neoplastic control. Our results showed that myc overactivation induced a two- to seven-fold increase in LDH-A expression in BL cells compared with non-neoplastic lymphoblasts; this result is consistent with previously reported data. Moreover, GF treatment suppressed LDH activity and inhibited BL cell replication but did not affect lymphoblast viability. Surprisingly, we found that increased levels of the MYC and LDH-A proteins did not lead to a metabolic shift in BL cells toward glycolytic ATP generation. BL cells were treated with GF at doses that achieved 50% inhibition of cell growth and lactate production, and ATP levels were scarcely affected after GF treatment. The same results were also obtained by suppressing LDH activity with oxamate, an LDH specific inhibitor. Our data suggest that LDH activity is important for maintaining a correct NAD/NADH balance in BL cells. LDH inhibition led to decreased NAD cellular levels, which resulted in sirtuin-1 inhibition. Confirming previous studies, sirtuin-1 inhibition caused a reduction in MYC protein levels, depriving BL cells of their most important survival signal. This study further describes the biological functions of the LDH enzyme and suggests that LDH inhibition could be useful for the treatment of cancer. PMID:23797802

Vettraino, Marina; Manerba, Marcella; Govoni, Marzia; Di Stefano, Giuseppina

2013-09-01

226

Meso-alpha,epsilon-diaminopimelate D-dehydrogenase: distribution and the reaction product.  

PubMed Central

A high activity of meso-alpha-epsilon-diaminopimelate dehydrogenase was found in extracts of Bacillus sphaericus, Brevibacterium sp., Corynebacterium glutamicum, and Proteus vulgaris among bacteria tested. B. sphaericus IFO 3525, in which the enzyme is most abundant, was chosen to study the enzyme reaction. The enzyme was not induced by the addition of meso-alpha-epsilon-diaminopimelate to the growth medium. The reaction product was isolated and identified as alpha-amino-epsilon-ketopimelate by a comparison of the properties of its 2,4-dinitrophenylhydrazone with those of an authentic sample in silica gel thin-layer chromatography, absorption, infrared and proton nuclear magnetic resonance spectrometry, and elemental analyses. The alpha-amino-epsilon-ketopimelate formed enzymatically was decarboxylated by H2O2 to yield L-alpha-aminoadipate. This suggests that the amino group with D-configuration in the substrate is oxidatively deaminated; the enzyme is a D-amino acid dehydrogenase. L-alpha-Amino-epsilon-ketopimelate undergoes spontaneous dehydration to the cyclic delta1-piperideine-2,6-dicarboxylate. The enzyme reaction is reversible, and meso-alpha-epsilon-diaminopimelate was formed in the reductive amination of L-alpha-epsilon-ketopimelate.

Misono, H; Togawa, H; Yamamoto, T; Soda, K

1979-01-01

227

Homo-D-lactic acid fermentation from arabinose by redirection of the phosphoketolase pathway to the pentose phosphate pathway in L-lactate dehydrogenase gene-deficient Lactobacillus plantarum.  

PubMed

Optically pure d-lactic acid fermentation from arabinose was achieved by using the Lactobacillus plantarum NCIMB 8826 strain whose l-lactate dehydrogenase gene was deficient and whose phosphoketolase gene was substituted with a heterologous transketolase gene. After 27 h of fermentation, 38.6 g/liter of d-lactic acid was produced from 50 g/liter of arabinose. PMID:19502433

Okano, Kenji; Yoshida, Shogo; Tanaka, Tsutomu; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

2009-08-01

228

Rapid Reaction Kinetics of Proline Dehydrogenase in the Multifunctional Proline Utilization A Protein†  

PubMed Central

The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli catalyzes the oxidation of proline to glutamate in two reaction steps using separate proline dehydrogenase (PRODH) and ?1-pyrroline-5-carboxylate (P5C) dehydrogenase domains. Here, the kinetic mechanism of PRODH in PutA is studied by stopped-flow kinetics to determine microscopic rate constants for the proline:ubiquinone oxidoreductase mechanism. Stopped-flow data for proline reduction of the flavin cofactor (reductive half-reaction) and oxidation of reduced flavin by CoQ1 (oxidative half-reaction) were best-fit by a double exponential from which maximum observable rate constants and apparent equilibrium dissociation constants were determined. Flavin semiquinone was not observed in the reductive or oxidative reactions. Microscopic rate constants for steps in the reductive and oxidative half-reactions were obtained by globally fitting the stopped-flow data to a simulated mechanism that includes a chemical step followed by an isomerization event. A microscopic rate constant of 27.5 s?1 was determined for proline reduction of the flavin cofactor followed by an isomerization step of 2.2 s?1. The isomerization step is proposed to report on a previously identified flavin-dependent conformational change (Zhang, W. et al. (2007) Biochemistry 46, 483–491) that is important for PutA functional switching but is not kinetically relevant to the in vitro mechanism. Using CoQ1, a soluble analog of ubiquinone, a rate constant of 5.4 s?1 was obtained for the oxidation of flavin, thus indicating that this oxidative step is rate-limiting for kcat during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental kcat and kcat/Km parameters.

Moxley, Michael A.; Becker, Donald F.

2011-01-01

229

Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization A protein.  

PubMed

The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli catalyzes the oxidation of proline to glutamate in two reaction steps using separate proline dehydrogenase (PRODH) and ?(1)-pyrroline-5-carboxylate (P5C) dehydrogenase domains. Here, the kinetic mechanism of PRODH in PutA is studied by stopped-flow kinetics to determine microscopic rate constants for the proline:ubiquinone oxidoreductase mechanism. Stopped-flow data for proline reduction of the flavin cofactor (reductive half-reaction) and oxidation of reduced flavin by CoQ(1) (oxidative half-reaction) were best-fit by a double exponential from which maximum observable rate constants and apparent equilibrium dissociation constants were determined. Flavin semiquinone was not observed in the reductive or oxidative reactions. Microscopic rate constants for steps in the reductive and oxidative half-reactions were obtained by globally fitting the stopped-flow data to a simulated mechanism that includes a chemical step followed by an isomerization event. A microscopic rate constant of 27.5 s(-1) was determined for proline reduction of the flavin cofactor followed by an isomerization step of 2.2 s(-1). The isomerization step is proposed to report on a previously identified flavin-dependent conformational change [Zhang, W. et al. (2007) Biochemistry 46, 483-491] that is important for PutA functional switching but is not kinetically relevant to the in vitro mechanism. Using CoQ(1), a soluble analogue of ubiquinone, a rate constant of 5.4 s(-1) was obtained for the oxidation of flavin, thus indicating that this oxidative step is rate-limiting for k(cat) during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental k(cat) and k(cat)/K(m) parameters. PMID:22148640

Moxley, Michael A; Becker, Donald F

2012-01-10

230

A population pharmacodynamic model for lactate dehydrogenase and neuron specific enolase to predict tumor progression in small cell lung cancer patients.  

PubMed

The development of individualized therapies poses a major challenge in oncology. Significant hurdles to overcome include better disease monitoring and early prediction of clinical outcome. Current clinical practice consists of using Response Evaluation Criteria in Solid Tumors (RECIST) to categorize response to treatment. However, the utility of RECIST is restricted due to limitations on the frequency of measurement and its categorical rather than continuous nature. We propose a population modeling framework that relates circulating biomarkers in plasma, easily obtained from patients, to tumor progression levels assessed by imaging scans (i.e., RECIST categories). We successfully applied this framework to data regarding lactate dehydrogenase (LDH) and neuron specific enolase (NSE) concentrations in patients diagnosed with small cell lung cancer (SCLC). LDH and NSE have been proposed as independent prognostic factors for SCLC. However, their prognostic and predictive value has not been demonstrated in the context of standard clinical practice. Our model incorporates an underlying latent variable ("disease level") representing (unobserved) tumor size dynamics, which is assumed to drive biomarker production and to be influenced by exposure to treatment; these assumptions are in agreement with the known physiology of SCLC and these biomarkers. Our model predictions of unobserved disease level are strongly correlated with disease progression measured by RECIST criteria. In conclusion, the proposed framework enables prediction of treatment outcome based on circulating biomarkers and therefore can be a powerful tool to help clinicians monitor disease in SCLC. PMID:24740245

Buil-Bruna, Núria; López-Picazo, José-María; Moreno-Jiménez, Marta; Martín-Algarra, Salvador; Ribba, Benjamin; Trocóniz, Iñaki F

2014-05-01

231

Dynamics of a Lactate Dehydrogenase Polymorphism in the Wood Louse PORCELLIO SCABER Latr.: Evidence for Partial Assortative Mating and Heterosis in Natural Populations  

PubMed Central

Electrophoretic separation of lactate dehydrogenase (LDH) of Porcellio scaber from 14 natural populations in California, and one each in Oregon, Delaware and Massachusetts, indicates a biallelic polymorphism. Phenotypes are recovered from laboratory matings of virgin females in frequencies agreeing with simple Mendelian inheritance, and the frequency distributions of phenotypes in natural populations are typically in agreement with the appropriate Hardy-Weinberg distributions for these same populations. The same allele predominates in all natural populations examined. Temporal stability within populations suggests that the polymorphism is at, or near, equilibrium. The spatial distribution of allele frequencies, however, is apparently mosaic. Abrupt discontinuities in gene frequency over short distances (50 m to 1 km) suggest that interpopulation migration is insufficient to swamp local differences in gene frequency. Analysis of the transmission dynamics of the polymorphism in natural populations using mother-offspring genotype comparisons suggests that the allelic frequencies of transmitted male gametes are not independent of female genotype. Specifically, the observed mating scheme in natural populations appears to be partially assortative. Comparisons of progeny genotype distributions with yearling (or adult) genotype distributions from the same populations indicate a superior post-partum viability of heterozygous individuals relative to homozygotes. The distortion of progeny genotypic distributions created by assortment is thus apparently counteracted by subsequent heterosis.

Sassaman, Clay

1978-01-01

232

Function of muscle-type lactate dehydrogenase and citrate synthase of the Galápagos marine iguana, Amblyrhynchus cristatus, in relation to temperature.  

PubMed

The Galápagos marine iguana, Amblyrhynchus cristatus, is unique among lizards in foraging subtidally, leading to activity across a broad range of ambient temperatures ( approximately 14-40 degrees C). To determine whether the marine iguana shows any biochemical changes consistent with maintaining enzyme function at both warm and cold body temperatures, we examined the function of the aerobic enzyme citrate synthase (CS) and the muscle isoform of the anaerobic enzyme lactate dehydrogenase (A(4)-LDH) in A. cristatus and a confamilial species, Iguana iguana, from 14 to 46 degrees C. We also deduced amino acid sequences from cDNA of each enzyme. In CS, despite two amino acid substitutions, we found no difference in the apparent Michaelis-Menten constant K(m) of oxaloacetate at any temperature, indicating that the substrate affinity of CS in A. cristatus has not adapted to changes in thermal environment. In A(4)-LDH, we used site-directed mutagenesis to show that the substitutions T9A and I283V (A. cristatus --> I. iguana) individually have no effect on kinetics, but together significantly decrease the K(m) of pyruvate and catalytic rate constant (k(cat)) of the A. cristatus ortholog. Thus, our data show that A. cristatus A(4)-LDH has not become cold adapted in response to this species' aquatic foraging behavior, and instead may be consistent with moderate warm adaptation with respect to the I. iguana ortholog. PMID:18313960

Fields, Peter A; Strothers, Chad M; Mitchell, Mark A

2008-05-01

233

Prognostic value of pretreatment serum levels of lactate dehydrogenase in nonmetastatic nasopharyngeal carcinoma: single-site analysis of 601 patients in a highly endemic area  

PubMed Central

Background Numerous studies have generated promising but incomplete evidence for the prognostic value of pretreatment serum levels of lactate dehydrogenase (S-LDH) in nasopharyngeal carcinoma (NPC). Methods Pretreatment serum levels of S-LDH in 601 patients with NPC were measured before treatment, and their associations with overall survival and tumor-free survival were studied. Univariate and multivariate analysis of subgroups was used to evaluate the prognostic value of S-LDH in early-stage and late-stage NPC separately. Results Pretreatment S-LDH levels were significantly lower in T1+2 patients than in T3+4 patients, lower in N0+1 patients than in N2+3 ones, and lower in stage I + II patients than in III + IV ones. Multivariate analysis showed that among patients with late-stage NPC, high pretreatment S-LDH levels >225 U/L were an independent predictor of poor overall survival and tumor-free survival. Among patients with early-stage NPC, pretreatment S-LDH levels >171 U/L, which overlap with the normal range, were an independent predictor of shorter overall survival and tumor-free survival. Conclusion Pretreatment S-LDH levels may be a reliable biomarker for predicting the long-term prognosis of patients with early-stage or late-stage NPC.

Wei, Zhengbo; Zeng, Xianjie; Xu, Jian; Duan, Xuwei; Xie, Ying

2014-01-01

234

Momordica charantia seed extract reduces pre-adipocyte viability, affects lactate dehydrogenase release, and lipid accumulation in 3T3-L1 cells.  

PubMed

A triterpenoid containing bitter melon (Momordica charantia) seed (BMS) extract was found to reduce cultured 3T3-L1 cell viability. The 50% lethal concentration values were determined to be 0.78?±?0.01?mg/mL at 24 hours, 0.69?±?0.01?mg/mL at 48 hours, and 0.56?±?0.02?mg/mL at 72 hours. 3T3-L1 cells were utilized as models of pre-adipocyte to adipocyte differentiation. BMS extract also caused a G(2)/M arrest in the cell cycle reducing cells by 23.9%, 37.7%, and 34.7% compared with the control after 72 hours of treatment at concentrations of 0.4, 0.5, and 0.6?mg/mL respectively. BMS extract did not increase the release of lactate dehydrogenase from 3T3-L1 cells, which was unexpected. Furthermore, BMS extract reduced lipid accumulation during differentiation from pre-adipocyte to adipocyte corresponding to reduction in overall triglyceride of 32.4% after 72 hours compared with untreated control cells. BMS is an underutilized agricultural commodity that may have potential for nutraceutical and functional food development. PMID:21332398

Popovich, David G; Lee, Yiyu; Li, Lu; Zhang, Wei

2011-03-01

235

An assessment of combined tumour markers in patients with seminoma: placental alkaline phosphatase (PLAP), lactate dehydrogenase (LD) and beta human chorionic gonadotrophin (beta HCG).  

PubMed Central

We have assessed the tumour markers placental alkaline phosphatase (PLAP), lactate dehydrogenase (LD), and human chorionic gonadotrophin (beta HCG) using 2,000 serum samples from 286 patients with seminoma. The ROC curves show that no one marker performs adequately for the detection of disease either at initial staging or during follow-up. We used a Markov model heuristically to devise strategies, in which marker results were assessed in combination, which might be useful in clinical practice. We found that the best strategy was to consider a test result abnormal only if either the beta HCG was greater than 6 Ul-1 or the LD was greater than 400 U l-1 and the PLAP level was greater than 60 U l-1. This will detect about 50% of patients with disease and the false-positive rate is 2%. In practical terms this means that PLAP need only be estimated in patients whose beta HCG is less than 6 IU l-1 and whose LD is greater than 400 U l-1.

Munro, A. J.; Nielsen, O. S.; Duncan, W.; Sturgeon, J.; Gospodarowicz, M. K.; Malkin, A.; Thomas, G. M.; Jewett, M. A.

1991-01-01

236

Evaluation of Milk Trace Elements, Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activity of Subclinical Mastitis as and Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis)  

PubMed Central

Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu), iron (Fe), zinc (Zn), cobalt (Co) and manganese (Mn) and enzyme activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in riverine buffalo milk can be used as an indicator of subclinical mastitis (SCM) with the aim of developing suitable diagnostic kit for SCM. Trace elements and enzyme activity in milk were estimated with Atomic absorption Spectrophotometer, GBC 932 plus and biochemical methods, respectively. Somatic cell count (SCC) was done microscopically. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. A statistically significant (p<0.01) increase in SCC, Fe, Zn, Co and LDH occurred in SCM milk containing gram positive bacterial agents only. ALP was found to be elevated in milk infected by both gram positive and negative bacteria. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC?2×105 cells/ml of milk as the benchmark. Only ALP and Zn, the former being superior, were found to be suitable for diagnosis of SCM irrespective of etiological agents. LDH, Co and Fe can be introduced in the screening programs where Gram positive bacteria are omnipresent. It is recommended that both ALP and Zn be measured together in milk to diagnose buffalo SCM, irrespective of etiology.

Guha, Anirban; Gera, Sandeep; Sharma, Anshu

2012-01-01

237

Electrochemical behavior of lactate dehydrogenase immobilized on "silica sol-gel/nanometre-sized tridecameric aluminum polycation" modified gold electrode and its application.  

PubMed

This paper reports the electrochemical behavior of lactate dehydrogenase (LDH) immobilized in the silica sol-gel film on gold electrode after adding nanometre-sized tridecameric aluminium polycation (nano-Al13, also called nanopolynuclear Al13) as a promoter. A pair of surface controlled quasi-reversible cyclic voltammetry peaks with the formal potential (E0') of 154 mV (vs. SCE) was found in the presence of nano-Al13. A potential application of the nano-Al13-LDH electrode for the determination of resorcinol and p-xylene was also investigated. The experimental results showed that both resorcinol and p-xylene inhibited LDH activity, and the calibration ranges were 5.0 x 10(-6)-3.0 x 10(-4) mol L(-1) for resorcinol and 1.0 x 10(-6)-1.0 x 10(-5) mol L(-1) for p-xylene, respectively. The nano-Al13-LDH electrode can be anticipated to be applied to environmental toxic assessments. PMID:19562207

Cheng, Jiongjia; Huang, Deqian; Zhang, Jing; Yang, Wenjing; Wang, Na; Sun, Yongbo; Wang, Keyu; Mo, Xiangyin; Bi, Shuping

2009-07-01

238

Pseudotype virions formed between mouse hepatitis virus and lactate dehydrogenase-elevating virus (LDV) mediate LDV replication in cells resistant to infection by LDV virions.  

PubMed Central

Infection of cultures of peritoneal macrophages with both lactate dehydrogenase-elevating virus (LDV) and mouse hepatitis virus (MHV) resulted in the formation of pseudotype virions containing LDV RNA which productively infected cells that are resistant to infection by intact LDV virions but not to infection by MHV. These cells were mouse L-2 and 3T3-17Cl-1 cells as well as residual peritoneal macrophages from persistently LDV-infected mice. Productive LDV infection of these cells via pseudotype virions was inhibited by antibodies to the MHV spike protein or to the MHV receptor, indicating that LDV RNA entered the cells via particles containing the MHV envelope. Simultaneous exposure of L-2 cells to both LDV and MHV resulted in infection by MHV but not by LDV. The results indicate that an internal block to LDV replication is not the cause of the LDV nonpermissiveness of many cell types, including the majority of the macrophages in an adult mouse. Instead, LDV permissiveness is restricted to a subpopulation of mouse macrophages because only these cells possess a surface component that acts as an LDV receptor.

Even, C; Plagemann, P G

1995-01-01

239

A comparison of the primary structures of lactate dehydrogenase isozymes M4 from giant panda, red panda, black bear and dog.  

PubMed

Lactate dehydrogenase isozymes M4 have been isolated and purified from red panda (Ailurus fulgens), black bear (Selenarctos thibetanus) and dog (Canis familiars) by affinity chromatography and compared with that from giant panda (Ailuropoda melanoleuca). Experimental results have shown that the N-termini, C-termini and the molecular weights of LDH-M subunits of red panda, black bear and dog are the same as those of the LDH-M subunit of giant panda. Analysis and comparison of HPLC peptide maps from the tryptic digests of the isozymes of red panda, black bear and dog have shown that most of their peptide fragments had the same retention time and amino acid composition as the corresponding peptide fragments from giant panda. Fragments with different retention times and/or amino acid compositions were sequenced. Careful examination of those variant amino acid residues demonstrated clearly that the primary structure of giant panda LDH-M subunit is unique and it appears that the giant panda might be classified as an independent family. PMID:3629217

Liang, S P; Zhang, L X

1987-03-01

240

Reaction between sheep liver mitochondrial aldehyde dehydrogenase and various thiol-modifying reagents.  

PubMed Central

Sheep liver mitochondrial aldehyde dehydrogenase reacts with 2,2'-dithiodipyridine and 4,4'-dithiodipyridine in a two-step process: an initial rapid labelling reaction is followed by slow displacement of the thiopyridone moiety. With the 4,4'-isomer the first step results in an activated form of the enzyme, which then loses activity simultaneously with loss of the label (as has been shown to occur with the cytoplasmic enzyme). With 2,2'-dithiodipyridine, however, neither of the two steps of the reaction has any effect on the enzymic activity, showing that the mitochondrial enzyme possesses two cysteine residues that must be more accessible or reactive (to this reagent at least) than the postulated catalytically essential residue. The symmetrical reagent 5,5'-dithiobis-(1-methyltetrazole) activates mitochondrial aldehyde dehydrogenase approximately 4-fold, whereas the smaller related compound methyl l-methyltetrazol-5-yl disulphide is a potent inactivator. These results support the involvement of mixed methyl disulphides in causing unpleasant physiological responses to ethanol after the ingestion of certain antibiotics.

Loomes, K M; Kitson, T M

1989-01-01

241

Production of a covalent flavin linkage in lipoamide dehydrogenase. Reaction with 8-Cl-FAD.  

PubMed

A method is described for preparation of apolipoamide dehydrogenase which gives quantitative removal of FAD. Active holoenzyme can be reconstituted by incubation with FAD. Reconstitution of apoenzyme with 8-Cl-FAD results in the fixation of most of the flavin to the protein in a covalently bound form. The portion noncovalently bound was shown to be unmodified 8-Cl-FAD. The covalently bound flavin has an absorption spectrum quite different from that of 8-Cl-FAD. It has a single band in the visible with a maximum at 459 nm (extinction coefficient of 22 mM-1 cm-1) and a shoulder at 480 nm. Model reactions between 8-Cl-Flavin (riboflavin or FAD) and organic thiols (thiophenol, beta-mercaptoethanol, or N-acetylcysteine) give products with spectra which are similar to that of FAD covalently bound to lipoamide dehydrogenase. The products of the model reactions have a single visible band with a maximum at 480 nm (extinction coefficient of 23.6 mM-1 cm-1 to 28.4 mM-1 cm-1) and a shoulder at 460 nm. The products of the model reaction and the covalently bound FAD of lipoamide dehydrogenase appear to be the result of a nucleophilic attack on the carbon at position 8 of the flavin ring by a thiolate anion, displacing the chloride. Thus, the product of the model reaction is 8-(RS)-flavin, and the product of the reaction between 8-Cl-FAD and protein probably has a cysteinyl residue covalently attacked at position 8 of FAD. Reconstitution of apoliopoamide dehydrogenase with 8-Cl-FAD gives two enzyme products which are fractionated by ammonium sulfate. Enzyme fractionating between 20% and 45% ammonium sulfate is monomeric and contains covanently bound FAD. Enzyme fractionating between 55% and 75% ammonium sulfate is dimeric and contains both covalently bound FAD and noncovalently bound 8-Cl-FAD. Both protein fractions contain one FAD per protein subunit and both are active with physiological substrates with Km values for NAD and dihydrolipoamide similar to those of native lipoamide dehydrogenase. The maximum turnover rates differ dramatically. Enzyme fractionating between 55% and 75% ammonium sulfate has a Vmax which is 61 times slower than native enzyme. Enzyme fractionating between 20% and 45% ammonium sulfate has a Vmax which is 7400 times slower than native enzyme. These slower rates are partially explainable by the oxidation-reduction potentials of the modified enzymes. Both covalently bound FAD and noncovalently bound FAD appear to reside in the native flavin binding site of the enzyme. However, once dimerization of the protien has taken place, the noncovalently bound 8-Cl-FAD cannot be induced to form a covalent bond with the protein except under protein denaturing conditions. The implications of these findings are discussed. PMID:681358

Moore, E G; Cardemil, E; Massey, V

1978-09-25

242

Relationship of creatine kinase, aspartate aminotransferase, lactate dehydrogenase, and proteinuria to cardiomyopathy in the owl monkey (Aotus vociferans)  

SciTech Connect

The purpose of this study was to determine serum reference values for crea- tine kinase (CK), aspartate aminotransferase (AST), and lactate dehydroge- nase (LDH) in captive-born and wild-caught owl monkeys to assess their usefulness for diagnosing myocardial disease. Urine samples were also collected and semi-quantitative tests performed. There was no statistically significant difference between CK, AST, and LDH when comparing both groups. However, when comparing monkeys with proteinuria to those without proteinuria, a statistically significant difference in CK value was observed (P = 0.021). In addition, the CK/AST ratio revealed that 29% of the animals included in this study had values suggesting cardiac infarction. Grossly, cardiac concentric hypertrophy of the left ventricle and small, pitted kidneys were the most common findings. Microscopically, myocardial fibrosis, contraction band necrosis, hypertrophy and hyperplasia of coronary arteries, medium-sized renal arteries, and afferent glomerular arteriolae were the most significant lesions, along with increased mesangial matrix and hypercellularity of glomeruli, Bowman’s capsule, and peritubular space fibroplasia. These findings suggest that CK, AST, and LDH along with urinalysis provide a reliable method for diagnosing cardiomyopathies in the owl monkey. In addition, CK/AST ratio, proteinuria, and the observed histological and ultrastructural changes suggest that Aotus vociferans suffer from arterial hypertension and chronic myocardial infarction.

Gozalo, Alfonso S.; Chavera, Alfonso; Montoya, Enrique J.; Takano, Juan; Weller, Richard E.

2008-02-01

243

Effect Of Compartmentalization of the Sensing Layer on the Sensitivity of a Multienzyme-Based Bioluminescent Sensor For L-Lactate  

Microsoft Academic Search

A bioluminescent fiber optic sensor for the analysis of L-lactate is described. It is based on a reaction sequence catalyzed by three different enzymes: luciferase from V. harveyi, NAD(P) H: FMN oxidoreductase from V. fischeri and lactate dehydrogenase from rabbit muscle covalently immobilized on polyamide membranes. Two kinds of sensing layer were studied, one consisting of only one membrane on

Philippe E. Michel; Sabine M. Gautier; Loïc J. Blum

1996-01-01

244

Dacarbazine with or without oblimersen (a Bcl-2 antisense oligonucleotide) in chemotherapy-naive patients with advanced melanoma and low-normal serum lactate dehydrogenase: 'The AGENDA trial'.  

PubMed

In a previous large randomized, open-label study, retrospective subset analysis revealed that the addition of the Bcl-2 antisense oligonucleotide oblimersen to dacarbazine (Dac) significantly improved overall survival, progression-free survival, and the response rate in chemotherapy-naive patients with advanced melanoma and normal baseline serum lactate dehydrogenase (LDH) levels. To confirm and expand on this observation, we conducted a prospective double-blind, placebo-controlled study to determine whether oblimersen augmented the efficacy of Dac in advanced melanoma patients with low-normal baseline LDH levels. A total of 314 chemotherapy-naive patients were randomly assigned to receive Dac (1000 mg/m) preceded by a 5-day continuous intravenous infusion of either oblimersen sodium (7 mg/kg/day) or placebo every 21 days for less than eight cycles. Co-primary efficacy endpoints were overall survival and progression-free survival. Response and progression of the disease were assessed by independent blinded review of computed tomography scan images. No difference in overall nor progression-free survival was observed between the Dac-oblimersen and Dac-placebo groups. Although the overall (17.2 vs. 12.1%) and durable (10.8 vs. 7.6%) response rates numerically favored Dac-oblimersen over Dac-placebo, they did not differ significantly (P=0.19 and 0.32, respectively). The incidence of hematologic adverse events, particularly thrombocytopenia and neutropenia, was higher in the Dac-oblimersen group than in the Dac-placebo group. Withdrawals from the study because of treatment-related adverse events were low (i.e. <2.5%) in both groups. The addition of oblimersen to Dac did not significantly improve overall survival nor progression-free survival in patients with advanced melanoma and low-normal levels of LDH at baseline. PMID:24667300

Bedikian, Agop Y; Garbe, Claus; Conry, Robert; Lebbe, Celeste; Grob, Jean J

2014-06-01

245

Bioactivity-guided identification and cell signaling technology to delineate the lactate dehydrogenase A inhibition effects of Spatholobus suberectus on breast cancer.  

PubMed

Aerobic glycolysis is an important feature of cancer cells. In recent years, lactate dehydrogenase A (LDH-A) is emerging as a novel therapeutic target for cancer treatment. Seeking LDH-A inhibitors from natural resources has been paid much attention for drug discovery. Spatholobus suberectus (SS) is a common herbal medicine used in China for treating blood-stasis related diseases such as cancer. This study aims to explore the potential medicinal application of SS for LDH-A inhibition on breast cancer and to determine its bioactive compounds. We found that SS manifested apoptosis-inducing, cell cycle arresting and anti-LDH-A activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cell. Oral herbal extracts (1 g/kg/d) administration attenuated tumor growth and LDH-A expression in both breast cancer xenografts. Bioactivity-guided fractionation finally identified epigallocatechin as a key compound in SS inhibiting LDH-A activity. Further studies revealed that LDH-A plays a critical role in mediating the apoptosis-induction effects of epigallocatechin. The inhibited LDH-A activities by epigallocatechin is attributed to disassociation of Hsp90 from HIF-1? and subsequent accelerated HIF-1? proteasome degradation. In vivo study also demonstrated that epigallocatechin could significantly inhibit breast cancer growth, HIF-1?/LDH-A expression and trigger apoptosis without bringing toxic effects. The preclinical study thus suggests that the potential medicinal application of SS for inhibiting cancer LDH-A activity and the possibility to consider epigallocatechin as a lead compound to develop LDH-A inhibitors. Future studies of SS for chemoprevention or chemosensitization against breast cancer are thus warranted. PMID:23457597

Wang, Zhiyu; Wang, Dongmei; Han, Shouwei; Wang, Neng; Mo, Feizhi; Loo, Tjing Yung; Shen, Jiangang; Huang, Hui; Chen, Jianping

2013-01-01

246

Upregulation of lactate dehydrogenase A by ErbB2 through heat shock factor 1 promotes breast cancer cell glycolysis and growth.  

PubMed

ErbB2 has been shown to activate signaling molecules that may regulate glucose metabolism. However, there is no evidence reported to directly link ErbB2 to glycolysis, and the mechanism underlying ErbB2-enhanced glycolysis is poorly understood. In this study, we investigated the role and mechanism of ErbB2 in regulating glycolysis. We found that ErbB2-overexpressing cells possessed a significantly higher level of glycolysis when compared to the ErbB2-low-expressing cells, and the downregulation of ErbB2 markedly decreased glycolysis. Overexpression of ErbB2 increased the expression of glycolysis-regulating molecules lactate dehydrogenase A (LDH-A) and heat shock factor 1 (HSF1). ErbB2 activated HSF1, indicated by the increased HSF1 trimer formation, and promoted HSF1 protein synthesis. HSF1 bound to LDH-A promoter and the downregulation of HSF1 reduced the expression of LDH-A and subsequently decreased cancer cell glycolysis and growth. Moreover, the glycolysis inhibitors, 2-deoxyglucose and oxamate, selectively inhibited the growth of ErbB2-overexpressing cells. Taken together, this study shows that in human breast cancer cells, ErbB2 promotes glycolysis at least partially through the HSF1-mediated upregulation of LDH-A. This pathway may have a major role in regulating glucose metabolism in breast cancer cells. These novel findings have important implications for the design of new approaches to target ErbB2-overexpressing breast cancers. PMID:19668225

Zhao, Y H; Zhou, M; Liu, H; Ding, Y; Khong, H T; Yu, D; Fodstad, O; Tan, M

2009-10-22

247

Preventive effect of glycosaminoglycans from Amussium pleuronectus (Linne) on biomolecules, lactate dehydrogenase-isoenzyme and electrocardiographic patterns in isoproterenol-induced myocardial infarction in Wistar rats  

PubMed Central

Objectives: This study was aimed to assess the cardioprotective role of low molecular weight glycosaminoglycans (LMW-GAG) in isoproterenol (ISO)-induced myocardial infarction (MI) in male Wistar rats. Effect of LMW-GAG on biomolecules, lactate dehydrogenase (LDH)-isoenzyme, and electrocardiographic (ECG)-patterns was studied as evidence of cardioprotection. Materials and Methods: Male Wistar rats (140 ± 10 g) were divided into four groups; untreated control (group I), LMW-GAG treated (300 ?g/day s. c. for 2 weeks—group II), ISO (85 mg s.c. injected on 13th and 14th days—group III), and LMW-GAG plus ISO (300 ?g/day s. c. for 12 days followed by 85 mg/kg ISO on the end of 13th and 14th days—group IV). At the end of the experimental period, all animals were terminated. Results: Rats treated with LMW-GAG (300 ?g/kg) for 12 days showed significant increasing levels of triglyceride (TG) (both serum and heart tissue), low density lipoprotein (LDL), very low density lipoprotein (VLDL), total cholesterol, uric acid, creatinine, and glucose. However, it significantly decreased the levels of high density lipoprotein (HDL) (serum), plasma total protein, and albumin/globulin (A/G) ratio. ISO also adversely affected the LDH-isoenzymes and caused marked elevation in ST segment. Pretreatment with LMW-GAG (300 ?g/kg) daily for a period of 2 weeks prevented the ISO-treated changes. Conclusions: The results indicate that LMW-GAG exhibits a cardioprotective effect in ISO-induced MI in rats, by maintaining the biomolecules and LDH-isoenzymes.

Saravanan, Ramachandran; Shanmugam, Annaian; Rajkumar, Devaraj

2012-01-01

248

Karnofsky Performance Status and Lactate Dehydrogenase Predict the Benefit of Palliative Whole-Brain Irradiation in Patients With Advanced Intra- and Extracranial Metastases From Malignant Melanoma  

SciTech Connect

Purpose: To determine prognostic factors that allow the selection of melanoma patients with advanced intra- and extracerebral metastatic disease for palliative whole-brain radiation therapy (WBRT) or best supportive care. Methods and Materials: This was a retrospective study of 87 patients who underwent palliative WBRT between 1988 and 2009 for progressive or multiple cerebral metastases at presentation. Uni- and multivariate analysis took into account the following patient- and tumor-associated factors: gender and age, Karnofsky performance status (KPS), neurologic symptoms, serum lactate dehydrogenase (LDH) level, number of intracranial metastases, previous resection or stereotactic radiosurgery of brain metastases, number of extracranial metastasis sites, and local recurrences as well as regional lymph node metastases at the time of WBRT. Results: In univariate analysis, KPS, LDH, number of intracranial metastases, and neurologic symptoms had a significant influence on overall survival. In multivariate survival analysis, KPS and LDH remained as significant prognostic factors, with hazard ratios of 3.3 (95% confidence interval [CI] 1.6-6.5) and 2.8 (95% CI 1.6-4.9), respectively. Patients with KPS ?70 and LDH ?240 U/L had a median survival of 191 days; patients with KPS ?70 and LDH >240 U/L, 96 days; patients with KPS <70 and LDH ?240 U/L, 47 days; and patients with KPS <70 and LDH >240 U/L, only 34 days. Conclusions: Karnofsky performance status and serum LDH values indicate whether patients with advanced intra- and extracranial tumor manifestations are candidates for palliative WBRT or best supportive care.

Partl, Richard, E-mail: richard.partl@medunigraz.at [Department of Therapeutic Radiology and Oncology, Medical University of Graz, Graz (Austria)] [Department of Therapeutic Radiology and Oncology, Medical University of Graz, Graz (Austria); Richtig, Erika [Department of Dermatology, Medical University of Graz, Graz (Austria)] [Department of Dermatology, Medical University of Graz, Graz (Austria); Avian, Alexander; Berghold, Andrea [Institute for Medical Informatics, Statistics and Documentation, Medical University of Graz, Graz (Austria)] [Institute for Medical Informatics, Statistics and Documentation, Medical University of Graz, Graz (Austria); Kapp, Karin S. [Department of Therapeutic Radiology and Oncology, Medical University of Graz, Graz (Austria)] [Department of Therapeutic Radiology and Oncology, Medical University of Graz, Graz (Austria)

2013-03-01

249

(1-3)-Beta-D-glucan in association with lactate dehydrogenase as biomarkers of Pneumocystis pneumonia (PcP) in HIV-infected patients.  

PubMed

Pneumocystis pneumonia (PcP) is a major HIV-related illness caused by Pneumocystis jirovecii. Definitive diagnosis of PcP requires microscopic detection of P. jirovecii in pulmonary specimens. The objective of this study was to evaluate the usefulness of two serum markers in the diagnosis of PcP. Serum levels of (1-3)-beta-d-glucan (BG) and lactate dehydrogenase (LDH) were investigated in 100 HIV-positive adult patients and 50 healthy blood donors. PcP cases were confirmed using indirect immunofluorescence with monoclonal anti-Pneumocystis antibodies and nested-PCR to amplify the large subunit mitochondrial rRNA gene of P. jirovecii in pulmonary specimens. BG and LDH levels in serum were measured using quantitative microplate-based assays. BG and LDH positive sera were statistically associated with PcP cases (P???0.001). Sensitivity, specificity, positive/negative predictive values (PPV/NPV), and positive/negative likelihood ratios (PLR/NLR) were 91.3 %, 61.3 %, 85.1 %, 79.2 %, 2.359, and 0.142, respectively, for the BG kit assay, and 91.3 %, 35.5 %, 75.9 %, 64.7 %, 1.415 and 0.245, respectively, for the LDH test. Serologic markers levels combined with the clinical diagnostic criteria for PcP were evaluated for their usefulness in diagnosis of PcP. The most promising cutoff levels for diagnosis of PcP were determined to be 400 pg/ml of BG and 350 U/l of LDH, which combined with clinical data presented 92.8 % sensitivity, 83.9 % specificity, 92.8 % PPV, 83.9 % NPV, 5.764 PLR and 0.086 NLR (P?

Esteves, F; Lee, C-H; de Sousa, B; Badura, R; Seringa, M; Fernandes, C; Gaspar, J F; Antunes, F; Matos, O

2014-07-01

250

Central lactate metabolism regulates food intake.  

PubMed

The central nervous system regulates food intake (FI) and body weight (BW), but the associated mechanisms remain to be elucidated. Here we report that central injections of lactate reduced FI and BW in rodents. Inhibition of central lactate metabolism to pyruvate with the lactate dehydrogenase inhibitor oxamate abolished the central effects of lactate on FI and BW. Conversely, central injections of pyruvate recapitulated the effects of lactate. Finally, inhibition of central lactate metabolism prevented the ability of circulating lactate to lower FI and BW. Together, the data indicate that activation of central lactate metabolism lowers FI and BW. PMID:18577696

Lam, Carol K L; Chari, Madhu; Wang, Penny Y T; Lam, Tony K T

2008-08-01

251

Site-specific incorporation of 5-fluorotryptophan as a probe of the structure and function of the membrane-bound D-lactate dehydrogenase of Escherichia coli: A sup 19 F nuclear magnetic resonance study  

SciTech Connect

The structure and function of the membrane-bound D-lactate dehydrogenase of Escherichia coli have been investigated by fluorine-19 nuclear magnetic resonance spectroscopy of 5-fluorotryptophan-labeled enzyme in conjunction with oligonucleotide-directed, site-specific mutagenesis. 5-Fluorotryptophan has been substituted for nine phenylalanine, tyrosine, and leucine residues in the enzyme molecule without loss of activity. The {sup 19}F signals from these additional tryptophan residues have been used as markers for sensitivity to substrate, exposure to aqueous solvent, and proximity to a lipid-bound spin-label. The nuclear magnetic resonance data show that two mutational sites, at amino acid residues 340 and 361, are near the lipid environment used to stabilize the enzyme. There are a number of amino acid residues on the carboxyl side of this region that are strongly sensitive to the aqueous solvent. The environment of the wide-type tryptophan residue at position 469 changes as a result of two of the substitution mutations, suggesting some amino acid residue-residue interactions. Secondary structure prediction methods indicate a possible binding site for the flavin adenine dinucleotide cofactor in the carboxyl end of the enzyme molecule. These results suggest that the membrane-bound D-lactate dehydrogenase may have the two-domain structure of many cytoplasmic dehydrogenases but with the addition of a membrane-binding domain between the catalytic and cofactor-binding domains. This type of three-domain structure may be of general significance for understanding the structure of membrane-bound proteins which do not traverse the lipid bilayer of membranes.

Peersen, O.B.; Pratt, E.A.; Truong, H.T. N.; Ho, C. (Carnegie Mellon Univ., Pittsburgh, PA (USA)); Rule, G.S. (Univ. of Virginia, Charlottesville (USA))

1990-04-03

252

Lactic acid conversion to 2,3-pentanedione and acrylic acid over silica-supported sodium nitrate: Reaction optimization and identification of sodium lactate as the active catalyst  

SciTech Connect

Lactic acid is converted to 2,3-pentanedione, acrylic acid, and other products in vapor-phase reactions over silica-supported sodium lactate formed from sodium nitrate. Multiparameter optimization of reaction conditions using a Box-Benkhen experimental design shows that the highest yield and selectivity to 2,3-pentanedione are achieved at low temperature, elevated pressure, and long contact time, while yield and selectivity to acrylic acid are most favorable at high temperature, low pressure, and short contact time. Post-reaction Fourier transform infrared spectroscopic analyses of the catalyst indicate that sodium nitrate as the initial catalyst material is transformed to sodium lactate at the onset of reaction via proton transfer from lactic acid to nitrate. The resultant nitric acid vaporizes as it is formed, leaving sodium lactate as the sole sodium-bearing species on the catalyst during reaction. 19 refs., 8 figs., 5 tabs.

Wadley, D.C.; Tam, M.S.; Miller, D.J. [Michigan State Univ., East Lansing, MI (United States)] [and others] [Michigan State Univ., East Lansing, MI (United States); and others

1997-01-15

253

Amino acid sequence differences cannot fully explain interspecific variation in thermal sensitivities of gobiid fish A4-lactate dehydrogenases (A4-LDHs)  

PubMed

We compared the deduced amino acid sequences, heat stabilities and thermal sensitivities of a kinetic property, the apparent Michaelis­Menten constant (Km) of pyruvate, of A4-lactate dehydrogenase (A4-LDH) in four species of goby fishes (Family Gobiidae), adapted to different temperatures, to examine how changes in primary structure influence the adaptation of enzymes. The effect of temperature on Km of pyruvate reflected each species' environmental temperature. For the most eurythermal species, Gillichthys seta, which is endemic to shallow intertidal regions of the upper Gulf of California and encounters temperatures between approximately 9 and 40 °C, Km of pyruvate was minimally affected by temperature, compared with the A4-LDH orthologues from a less eurythermal congener, G. mirabilis (9­30 °C), a cold temperate goby, Coryphopterus nicholsi (10­18 °C) and a tropical species, C. personatus (25­32 °C). Heat denaturation profiles failed to correlate with habitat temperature; G. mirabilis A4-LDH was most thermally stable, followed by the orthologues of C. nicholsi and G. seta. Complementary DNAs (cDNAs) encoding LDH-As of G. seta, Gulf of California and Pacific coast populations of G. mirabilis and C. nicholsi were isolated and sequenced, and the corresponding amino acid sequences deduced. The nucleotide sequences of LDH-A of the two populations of G. mirabilis were identical. Five nucleotide differences in the coding region and one amino acid substitution (at position 78) distinguished LDH-As of G. mirabilis and C. nicholsi. The substitution of a glycyl residue (C. nicholsi) for an alanyl residue (G. mirabilis) may account for the difference in thermal stability between these two orthologues. Comparisons of the LDH-A cDNAs of G. mirabilis and G. seta revealed four differences in nucleotide sequence in the coding region, but all nucleotide substitutions were synonymous. The identical deduced primary structures of the two enzymes suggested the possibility of different protein conformational variants ('conformers') in the two species. This hypothesis is supported by electrospray ionization mass spectrometry, which indicates that the masses of the A4-LDH orthologues of the two species are the same within the resolution of the technique. To explore the possibility that the two enzymes were different conformers of the same primary structure, we treated purified G. seta and G. mirabilis A4-LDHs with 3.0 mol l-1 urea or 6 mol l-1 guanidine­HCl and, after removing the denaturant, compared their kinetic properties and heat stabilities. Neither treatment had an effect on the A4-LDH of G. mirabilis, but both converted the Km versus temperature profile of the G. seta enzyme to that of the G. mirabilis A4-LDH. The thermal stability of neither enzyme was affected. We propose, as has been suggested in several previous studies of A4-LDH, that this enzyme can fold into a number of conformers with different stabilities and functional properties. The A4-LDH of G. seta furnishes evidence that such conformers may provide an important mechanism for adaptation of proteins to temperature. PMID:9319749

Fields; Somero

1997-01-01

254

Lack of effect of strain type on detection of toxigenic Clostridium difficile by glutamate dehydrogenase and polymerase chain reaction.  

PubMed

Glutamate dehydrogenase (GDH) is popular as a preliminary test for the detection of Clostridium difficile. Recent work has suggested that GDH sensitivity may vary according to ribotype and may be lower for ribotypes 002, 027, and 106 compared with polymerase chain reaction (PCR). We investigated this effect using a dilution series of 64 isolates tested by GDH and Cepheid GeneXpert PCR. PCR was significantly more sensitive than GDH overall; however, there was no difference in detection according to specific ribotype. PMID:21683272

Goldenberg, Simon D; Gumban, Maria; Hall, Anthony; Patel, Amita; French, Gary L

2011-07-01

255

Internal controls for quantitative polymerase chain reaction of swine mammary glands during pregnancy and lactation.  

PubMed

High-throughput microarray analysis is an efficient means of obtaining a genome-wide view of transcript profiles across physiological states. However, quantitative PCR (qPCR) remains the chosen method for high-precision mRNA abundance analysis. Essential for reliability of qPCR data is normalization using appropriate internal control genes (ICG), which is now, more than ever before, a fundamental step for accurate gene expression profiling. We mined mammary tissue microarray data on >13,000 genes at -34, -14, 0, 7, 14, 21, and 28 d relative to parturition in 27 crossbred primiparous gilts to identify suitable ICG. Initial analysis revealed TBK1, PCSK2, PTBP1, API5, VAPB, QTRT1, TRIM41, TMEM24, PPP2R5B, and AP1S1 as the most stable genes (sample/reference = 1 +/- 0.2). We also included 9 genes previously identified as ICG in bovine mammary tissue. Gene network analysis of the 19 genes identified AP1S1, API5, MTG1, VAPB, TRIM41, MRPL39, and RPS15A as having no known co-regulation. In addition, UXT and ACTB were added to this list, and mRNA abundance of these 9 genes was measured by qPCR. Expression of all 9 of these genes was decreased markedly during lactation. In a previous study with bovine mammary tissue, mRNA of stably expressed genes decreased during lactation due to a dilution effect brought about by large increases in expression of highly abundant genes. To verify this effect, highly abundant mammary genes such as CSN1S2, SCD, FABP3, and LTF were evaluated by qPCR. The tested ICG had a negative correlation with these genes, demonstrating a dilution effect in the porcine mammary tissue. Gene stability analysis identified API5, VABP, and MRPL39 as the most stable ICG in porcine mammary tissue and indicated that the use of those 3 genes was most appropriate for calculating a normalization factor. Overall, results underscore the importance of proper validation of internal controls for qPCR and highlight the limitations of using absence of time effects as the criteria for selection of appropriate ICG. Further, we showed that use of the same ICG from one organism might not be suitable for qPCR normalization in other species. PMID:18650282

Tramontana, S; Bionaz, M; Sharma, A; Graugnard, D E; Cutler, E A; Ajmone-Marsan, P; Hurley, W L; Loor, J J

2008-08-01

256

Computational study of the mechanism of half-reactions in class 1A dihydroorotate dehydrogenase from Trypanosoma cruzi.  

PubMed

Chagas' disease is considered to be a health problem affecting millions of people in Latin America. This disease is caused by the parasite Trypanosoma cruzi. Recently dihydroorotate dehydrogenase class 1A from Trypanosoma cruzi (TcDHODA) was shown to be essential for the survival and growth of T. cruzi and proposed as a drug target against Chagas' disease. This enzyme catalyzes the oxidation of (S)-dihydroorotate to orotate, with a proposed catalytic cycle consisting of two half-reactions. In the first half-reaction dihydroorotate is oxidized to orotate, with the consequent reduction of the flavin mononucleotide cofactor. In the second half-reaction fumarate is reduced to succinate. The first oxidation half-reaction may occur via a concerted or a stepwise mechanism. Herein, the catalytic mechanism of TcDHODA has been studied using hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) Molecular Dynamics (MD) simulations. The free energy profiles derived from the bidimensional potential of mean force reveal more details for two half-reaction processes. PMID:24084894

Silva, Natália de Farias; Lameira, Jerônimo; Alves, Cláudio Nahum; Martí, Sergio

2013-11-21

257

Stopped flow kinetic studies on reductive half-reaction of histamine dehydrogenase from Nocardioides simplex with histamine.  

PubMed

Histamine dehydrogenase from Nocardioides simplex (HmDH) which catalyzes the oxidative deamination of histamine is an iron-sulphur-containing flavoprotein. For our further understanding on the intramolecular electron transfer process, the reductive half reaction of HmDH with histamine has been studied by stopped flow spectrophotometry at pH 7.5 and 10. The reaction at pH 7.5 is found to be analysed on a kinetic model composed of three sequential first-order reactions. The first fast phase, of which the rate constant shows a hyperbolic dependence on the histamine concentration, is assigned to a direct two-electron reduction of the oxidized flavin (CFMN(O)) by histamine with no involvement of the semiquinone form of the flavin (CFMN(S)). The second moderate process is the substrate-independent intramolecular single-electron transfer from the reduced flavin to the oxidized iron-sulphur cluster. The third slow process is considered to reflect the second binding of histamine to CFMN(S), which is responsible for the substrate inhibition. At pH 10, the reaction is analysed with one pseudo-first-order reaction phase which is substrate-dependent two-electron reduction of CFMN(O) coupled with the subsequent fast intersubunit single-electron transfer. The UV-vis spectroscopy of HmDH suggests the deprotonation of Tyr residues, which seems to cause the switching of the electron transfer property. PMID:20305273

Tsutsumi, Maiko; Tsujimura, Seiya; Shirai, Osamu; Kano, Kenji

2010-07-01

258

Genetic polymorphism of blood proteins in the troops of Japanese macaques, Macaca fuscata : II. Erythrocyte lactate dehydrogenase polymorphism in Macaca fuscata  

Microsoft Academic Search

In investigating the genetic marker for population genetics of Japanese macaques by electrophoresis, the author found the erythrocyte lacate dehydrogenase (LDH) polymorphism existing in some troops. There were four kinds of variations which seemed to be controlled by two loci, controlling A and B subunits of this enzyme. The variant phenotypes were named LDH-Amac2-1 LDH-Bmac1-1, LDH-Amac3-1 LDH-Bmac1-1, LDH-Amac 1-1 LDH-Bmac2-1,

Takayoshi Shotake

1974-01-01

259

A multiple reaction monitoring method for absolute quantification of the human liver alcohol dehydrogenase ADH1C1 isoenzyme.  

PubMed

Although significant progress has been made in protein quantification using mass spectrometry during recent years, absolute protein quantification in complex biological systems remains a challenging task in proteomics. The use of stable isotope-labeled standard peptide is the most commonly used strategy for absolute quantification, but it might not be suitable in all instances. Here we report an alternative strategy that employs a stable isotope-labeled intact protein as an internal standard to absolutely quantify the alcohol dehydrogenase (ADH) expression level in a human liver sample. In combination with a new targeted proteomics approach employing the method of multiple reaction monitoring (MRM), we precisely and quantitatively measured the absolute protein expression level of an ADH isoenzyme, ADH1C1, in human liver. Isotope-labeled protein standards are predicted to be particularly useful for measurement of highly homologous isoenzymes such as ADHs where multiple signature peptides can be examined by MRM in a single experiment. PMID:17692277

Janecki, Dariusz J; Bemis, Kerry G; Tegeler, Tony J; Sanghani, Paresh C; Zhai, Lanmin; Hurley, Thomas D; Bosron, William F; Wang, Mu

2007-10-01

260

Reaction mechanism of the heterotetrameric (alpha2beta2) E1 component of 2-oxo acid dehydrogenase multienzyme complexes.  

PubMed

Pyruvate decarboxylase (E1) catalyzes the first two reactions of the four involved in oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase (PDH) multienzyme complex. It requires thiamin diphosphate to bring about the decarboxylation of pyruvate, which is followed by the reductive acetylation of a lipoyl group covalently bound to the N(6) amino group of a lysine residue in the second catalytic component, a dihydrolipoyl acetyltransferase (E2). Replacement of two histidine residues in the E1alpha and E1beta chains of the heterotetrameric E1 (alpha(2)beta(2)) component of the PDH complex of Bacillus stearothermophilus, considered possible proton donors at the active site, was carried out. Subsequent characterization of the mutants permitted different roles to be assigned to these two particular residues in the reaction catalyzed by E1: E1alpha His271 to stabilize the dianion formed during decarboxylation of the 2-oxo acid and E1beta His128 to provide the proton required to protonate the incoming dithiolane ring in the subsequent reductive acetylation of the lipoyl goup. On the basis of these and other results from a separate investigation into the roles of individual residues in a loop region in the E1alpha chain close to the active site of E1 [Fries, M., Chauhan, H. J., Domingo, G. J., Jung, H., and Perham, R. N. (2002) Eur. J. Biochem. 270, 861-870] together with work from other laboratories, a detailed mechanism for the E1 reaction can be formulated. PMID:12795594

Fries, Markus; Jung, Hyo-Il; Perham, Richard N

2003-06-17

261

Serum lactate dehydrogenase isoenzyme 1 activity in patients with testicular germ cell tumors correlates with the total number of copies of the short arm of chromosome 12 in the tumor.  

PubMed

The aim of our study was to assess the relationship between the serum lactate dehydrogenase isoenzyme 1 (S-LDH-1) activity in patients with testicular germ cell tumors and the number of copies of the short arm of chromosome 12 (12p) present in tumor. Twenty-seven adult patients with measurable tumor lesions were studied. Twenty-five had three or more copies of chromosome 12 per cell in the tumors. Nineteen had one or more copies of a specific chromosomal abnormality, an isochromosome of the short arm of chromosome 12, i(12p). Fourteen had increased S-LDH-1 levels. S-LDH-1 activity correlated significantly with the product of total tumor volume and the total number of copies of the short arm of chromosome 12 present per cell (total tumor 12p). We conclude that the total number of copies of the short arm of chromosome 12 in the tumors is most probably a factor contributing to the LDH-1 activity released from the tumors. PMID:1435725

von Eyben, F E; de Graaff, W E; Marrink, J; Blaabjerg, O; Sleijfer, D T; Koops, H S; Oosterhuis, J W; Petersen, P H; van Echten-Arends, J; de Jong, B

1992-10-01

262

Substrate flexibility and reaction specificity of tropinone reductase-like short-chain dehydrogenases.  

PubMed

Annotations of protein or gene sequences from large scale sequencing projects are based on protein size, characteristic binding motifs, and conserved catalytic amino acids, but biochemical functions are often uncertain. In the large family of short-chain dehydrogenases/reductases (SDRs), functional predictions often fail. Putative tropinone reductases, named tropinone reductase-like (TRL), are SDRs annotated in many genomes of organisms that do not contain tropane alkaloids. SDRs in vitro often accept several substrates complicating functional assignments. Cochlearia officinalis, a Brassicaceae, contains tropane alkaloids, in contrast to the closely related Arabidopsis thaliana. TRLs from Arabidopsis and the tropinone reductase isolated from Cochlearia (CoTR) were investigated for their catalytic capacity. In contrast to CoTR, none of the Arabidopsis TRLs reduced tropinone in vitro. NAD(H) and NADP(H) preferences were relaxed in two TRLs, and protein homology models revealed flexibility of amino acid residues in the active site allowing binding of both cofactors. TRLs reduced various carbonyl compounds, among them terpene ketones. The reduction was stereospecific for most of TRLs investigated, and the corresponding terpene alcohol oxidation was stereoselective. Carbonyl compounds that were identified to serve as substrates were applied for modeling pharmacophores of each TRL. A database of commercially available compounds was screened using the pharmacophores. Compounds identified as potential substrates were confirmed by turnover in vitro. Thus pharmacophores may contribute to better predictability of biochemical functions of SDR enzymes. PMID:24583623

Reinhardt, Nicole; Fischer, Juliane; Coppi, Ralph; Blum, Elke; Brandt, Wolfgang; Dräger, Birgit

2014-04-01

263

Analysis of Conformationally Restricted ?-Ketoglutarate Analogues as Substrates of Dehydrogenases and Aminotransferases  

Microsoft Academic Search

Five synthetic, conformationally restricted ?-ketoglutarate analogues were tested as substrates of a variety of dehydrogenases and aminotransferases. The compounds were found not to be detectable substrates of glutamate dehydrogenase, l-leucine dehydrogenase, l-phenylalanine dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glutamine transaminase K, aspartate aminotransferase, alanine aminotransferase, and ?-ketoglutarate dehydrogenase complex. However, two thermostable aminotransferases were identified that catalyze transamination between several l-amino

Travis T. Denton; Charles M. Thompson; Arthur J. L. Cooper

2001-01-01

264

Elucidating the reaction mechanism of the benzoate oxidation pathway encoded aldehyde dehydrogenase from Burkholderia xenovorans LB400  

PubMed Central

Oxidation of cis-3,4-dehydroadipyl-CoA semialdehyde to cis-3,4-dehydroadipyl-CoA by the aldehyde dehydrogenase, ALDHC (EC.1.2.1.77), is an essential step in the metabolism of benzoate in Burkholderia xenovorans LB400. In a previous study, we established a structural blueprint for this novel group of ALDH enzymes. Here, we build significantly on this initial work and propose a detailed reaction mechanism for ALDHC based on comprehensive structural and functional investigations of active site residues. Kinetic analyses reveal essential roles for C296 as the nucleophile and E257 as the associated general base. Structural analyses of E257Q and C296A variants suggest a dynamic charge repulsion relationship between E257 and C296 that contributes to the inherent flexibility of E257 in the native enzyme, which is further regulated by E496 and E167. A proton relay network anchored by E496 and supported by E167 and K168 serves to reset E257 for the second catalytic step. We also propose that E167, which is unique to ALDHC and its homologs, serves a critical role in presenting the catalytic water to the newly reset E257 such that the enzyme can proceed with deacylation and product release. Collectively, the reaction mechanism proposed for ALDHC promotes a greater understanding of these novel ALDH enzymes, the ALDH super-family in general, and benzoate degradation in B. xenovorans LB400.

Bains, Jasleen; Leon, Rafael; Temke, Kevin G; Boulanger, Martin J

2011-01-01

265

Ontogenetic changes and developmental adjustments in lactate dehydrogenase isozymes of an obligate air-breathing fish Channa punctatus during deprivation of air access.  

PubMed

In air-breathing snakehead Channa punctatus, Ldh-B is expressed at all ontogenetic and developmental stages, while Ldh-A is expressed temporally in pre-hatchlings 12-13 days ahead of bimodal respiration marked by air-breathing. Remarkable differences are observed in the LDH isozyme expression among various ontogenetic and developmental stages upon denying air access. When denied air access, water-breathing larvae show two distinct characteristics: (i) they survive longer than transitory air-breathers due to independence from air-breathing and (ii) there is more transient induction of Ldh-B than Ldh-A. Transition to bimodal breathing, which occurred post-hatching in 15-day old larvae, is coincidental with inducibility of Ldh-A and concomitant down-regulation of Ldh-B. Heart tissue from air-breathing adults denied air access shows a preferential expression of LDH-A subunit and slight down-regulation of LDH-B. Heterotetramers of A and B subunits participate in adjusting LDH levels among those stages which either precede air-breathing switchover, or are subsequent to this transition. The contribution of heterotetramers depends on the stage-specific levels of LDH homotetramers A(4) or B(4). Scaling of muscle mass during growth, tolerance to extended deprivation of air access and induction of Ldh-A are correlated. Response to restoring air contact indicated that advanced air-breathing stages of C. punctatus possess an inherent capacity to sense surface air. In kinetic properties, LDH isozymes of C. punctatus are teleost-like but species specificity is displayed in oxidative potential by cardiac muscle and in L-lactate reduction by skeletal muscle. PMID:15649774

Ahmad, Riaz; Hasnain, Absar-Ul

2005-02-01

266

On the rate of proton exchange with solvent of the catalytic histidine in flavocytochrome b2 (yeast L-lactate dehydrogenase).  

PubMed Central

The family of FMN-dependent, alpha-hydroxy acid-oxidizing enzymes catalyzes substrate dehydrogenation by a mechanism the first step of which is abstraction of the substrate alpha-proton (so-called carbanion mechanism). For flavocytochrome b2 and lactate oxidase, it was shown that once on the enzyme this proton is lost only slowly to the solvent (Lederer F, 1984, In: Bray RC, Engel PC, Mayhew SG, eds, Flavins & flavoproteins, Berlin: Walter de Gruyter & Co., pp 513-526; Urban P, Lederer F, 1985, J Biol Chem 260:11115-11122). This suggested the occurrence of a pKa increase of the catalytic histidine upon enzyme reduction by substrate. For flavocytochrome b2, the crystal structure indicated 2 possible origins for the stabilization of the imidazolium form of His 373: either a network of hydrogen bonds involving His 373, Tyr 254, flavin N5 and O4, a heme propionate, and solvent molecules, and/or electrostatic interactions with Asp 282 and with the reduced cofactor N1 anion. In this work, we probe the effect of the hydrogen bond network at the active site by studying proton exchange with solvent for 2 mutants: Y254F and the recombinant flavodehydrogenase domain, in which this network should be disrupted. The rate of proton exchange, as determined by intermolecular hydrogen transfer experiments, appears identical in the flavodehydrogenase domain and the wild-type enzyme, whereas it is about 3-fold faster in the Y254F mutant. It thus appears that specific hydrogen bonds to the solvent do not play a major role in stabilizing the acid form of His 373 in reduced flavocytochrome b2. Removal of the Y254 phenol group induces a pKa drop of about half a pH unit for His 373 in the reduced enzyme. Even then, the rate of exchange of the imidazolium proton with solvent is still lower by several orders of magnitude than that of a normally ionizing histidine. Other factors must then also contribute to the pKa increase, such as the electrostatic interactions with D282 and the anionic reduced cofactor, as suggested by the crystal structure.

Balme, A.; Lederer, F.

1994-01-01

267

Continuous electrochemical monitoring of extracellular lactate production from neonatal rat cardiomyocytes following myocardial hypoxia.  

PubMed

Continuous monitoring of lactate production from cardiomyocytes is of great physiological and pathological importance since the level of lactate in extracellular fluid is closely associated with myocardial energy metabolism with implication in the diagnosis and therapeutics of myocardial hypoxia and ischemia. This study demonstrates an electrochemical approach to continuous monitoring of lactate production from neonatal rat cardiomyocytes following myocardial hypoxia with a dehydrogenase-based electrochemical biosensor and a negative pressure driven culture sampling. To eliminate the effect of pH variation occurring following the cardiomyocyte hypoxia on the biosensor response and to supply nicotinamide adenine dinucleotide (NAD(+)) cofactor necessary for the enzymatic reaction of lactate dehydrogenase (LDH), artificial cerebrospinal fluid (aCSF) containing NAD(+) cofactor is externally perfused and mixed online with cell culture before the culture goes to the detector. The method exhibits a high selectivity against the electrochemically active species endogenously existing in the extracellular culture of cardiomyocytes and a high tolerance against the variation of pH following cardiomyocyte hypoxia. The dynamic linear range for lactate detection is from 0.20 to 10 mM (I (nA) = 25.6 C(Lactate) (mM) + 20.1, ? = 0.996) with a detection limit of 0.16 mM (S/N = 3). The physiological level of the extracellular lactate of neonatal rat cardiomyocytes is determined to be 1.1 ± 0.1 mM (n = 3) with the cell density of about 0.5 × 10(3) cells/mm(2). When the cardiomyocytes are subject to hypoxia induced with anoxic reagents, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), the extracellular lactate increases to 255 ± 30.3% (n = 3), relative to the physiological level, following 20 min of the hypoxia. This study essentially offers a new and effective electrochemical platform for investigating energy metabolism during cardiac physiological and pathological processes. PMID:22607532

Li, Xianchan; Zhao, Lingzhi; Chen, Zhenling; Lin, Yuqing; Yu, Ping; Mao, Lanqun

2012-06-19

268

Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat.  

PubMed

1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates. PMID:212016

Hutson, N J; Kerbey, A L; Randle, P J; Sugden, P H

1978-08-01

269

Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat.  

PubMed Central

1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates.

Hutson, N J; Kerbey, A L; Randle, P J; Sugden, P H

1978-01-01

270

Reactions of Glyceraldehyde 3-Phosphate Dehydrogenase Sulfhydryl Groups with bis-Electrophiles Produce DNA-Protein Crosslinks but Not Mutations  

PubMed Central

The environmental contaminant 1,2-dibromoethane and diepoxybutane, an oxidation product of the important industrial chemical butadiene, are bis-functional electrophiles and known to be mutagenic and carcinogenic. One mechanism by which bis-electrophiles can exert their toxic effects is through the induction of genotoxic and mutagenic DNA–peptide crosslinks. This mechanism has been shown in systems overexpressing the DNA repair protein O6-alkylguanine DNA-alkyltransferase (AGT) or glutathione S-transferase and involves reactions with nucleophilic cysteine residues. The hypothesis that DNA-protein crosslink formation is a more general mechanism for genotoxicity by bis-electrophiles was investigated by screening nuclear proteins for reactivity with model monofunctional electrophiles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was identified as a candidate due to the nucleophilicity of two cysteine residues (Cys152 and Cys246) in reaction screens with model electrophiles (Dennehy, M. K. et al. (2006) Chem. Res. Toxicol. 19, 20–29). Incubation of GAPDH with bis-electrophiles resulted in inhibition of its catalytic activity, but only at high concentrations of diepoxybutane. In vitro assays indicated DNA-GAPDH crosslink formation in the presence of diepoxybutane, and bis-electrophile reactivity at Cys246 was confirmed using mass spectral analysis. In contrast to AGT, overexpression of human GAPDH in Escherichia coli did not enhance mutagenesis by diepoxybutane. We propose that the lack of mutational enhancement is in part due to the inherently lower reactivity of GAPDH toward bis-electrophiles as well as the reduced DNA binding ability relative to AGT, preventing the in vivo formation of DNA-protein crosslinks and enhanced mutagenesis.

Loecken, Elisabeth M.; Guengerich, F. Peter

2014-01-01

271

Lactation Consultant  

MedlinePLUS

... consultants educate women, families, health professionals, and the community about breast feeding and human lactation; facilitate the development of policies which protect, promote, and support breastfeeding; ...

272

Specificity determinants for the pyruvate dehydrogenase component reaction mapped with mutated and prosthetic group modified lipoyl domains.  

PubMed

Efficient catalysis in the second step of the pyruvate dehydrogenase (E1) component reaction requires a lipoyl group to be attached to a lipoyl domain that displays appropriately positioned specificity residues. As substrates, the human dihydrolipoyl acetyltransferase provides an N-terminal (L1) and an inner (L2) lipoyl domain. We evaluated the specificity requirements for the E1 reaction with 27 mutant L2 (including four substitutions for the lipoylated lysine, Lys(173)), with three analogs substituted for the lipoyl group on Lys(173), and with selected L1 mutants. Besides Lys(173) mutants, only E170Q mutation prevented lipoylation. Based on analysis of the structural stability of mutants by differential scanning calorimetry, alanine substitutions of residues with aromatic side chains in terminal regions outside the folded portion of the L2 domain significantly decreased the stability of mutant L2, suggesting specific interactions of these terminal regions with the folded domain. E1 reaction rates were markedly reduced by the following substitutions in the L2 domain (equivalent site-L1): L140A, S141A (S14A-L1), T143A, E162A, D172N, and E179A (E52A-L1). These mutants gave diverse changes in kinetic parameters. These residues are spread over >24 A on one side of the L2 structure, supporting extensive contact between E1 and L2 domain. Alignment of over 40 lipoyl domain sequences supports Ser(141), Thr(143), and Glu(179) serving as specificity residues for use by E1 from eukaryotic sources. Extensive interactions of the lipoyl-lysine prosthetic group within the active site are supported by the limited inhibition of E1 acetylation of native L2 by L2 domains altered either by mutation of Lys(173) or enzymatic addition of lipoate analogs to Lys(173). Thus, efficient use by mammalian E1 of cognate lipoyl domains derives from unique surface residues with critical interactions contributed by the universal lipoyl-lysine prosthetic group, key specificity residues, and some conserved residues, particularly Asp(172) adjacent to Lys(173). PMID:10788482

Gong, X; Peng, T; Yakhnin, A; Zolkiewski, M; Quinn, J; Yeaman, S J; Roche, T E

2000-05-01

273

L-lactate metabolism in potato tuber mitochondria.  

PubMed

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. PMID:17489101

Paventi, Gianluca; Pizzuto, Roberto; Chieppa, Gabriella; Passarella, Salvatore

2007-03-01

274

Six hours of resting platelet concentrates stored at 22-24 ?C for 48 hours in permeable bags preserved pH, swirling and lactate dehydrogenase better and caused less platelet activation  

PubMed Central

Background During transportation, platelet concentrates (PC) usually undergo a long period without agitation. Whether this interruption improves quality and viability or, contrariwise, has deleterious effects on PC stored for 48 hours (h) is unknown. The aim of this study was to investigate the effects of metabolic resting (6 h of interruption of agitation) vs continue agitation of PC stored for 48 h in the blood bank of Tehran. Materials and methods PC were prepared from platelet-rich plasma and stored in permeable bags in a shaker/incubator for 42 h at room temperature (20–24 ºC). Then, simply by stopping the agitator, the PC remained stationary (“resting”) without agitation for 6 h (WCA6h), prior to transfusion. In vitro measurements of platelet quality were carried out just after completion of the resting period and the results were compared with those of PC continuously agitated in the same day (designated as the control group, CA6h). The in vitro variables measured were swirling, ristocetin-induced aggregation (GPIb-related function), lactate dehydrogenase (LDH) concentration, platelet factor 4 (PF4) release and P-selectin expression (activation markers). Results The mean platelet counts of the control group (CA6h) and rested (WCA6h) PC were not statistically different (P =0.548). Likewise, the mean pH values were not significantly different: WCA6h (7.16±0.08) and CA6h (7.22±0.16) (P =0.300). Although ristocetin-induced aggregation did not differ significantly between CA6h (79.2±4.4) and WCA6h (66.65±28.55) (P =0.186), WCA6h showed significantly less PFA release (P =0.015) and lower P-selectin expression (P =0.006). Conclusions We observed that PC stored under agitation for 42 h at 22–24 ºC in permeable bags and then rested for 6 h had better preserved pH, swirling and LDH and less platelet activation then PC kept under continuous agitation for the whole 48 h storage period.

Naghadeh, Hossin T.; Badlou, Bahram A.; Ferizhandy, Ali S.; Mohammadreza, Tabatabai S.; Shahram, Vaeli

2013-01-01

275

Combining Parasite Lactate Dehydrogenase-Based and Histidine-Rich Protein 2-Based Rapid Tests To Improve Specificity for Diagnosis of Malaria Due to Plasmodium knowlesi and Other Plasmodium Species in Sabah, Malaysia.  

PubMed

Plasmodium knowlesi causes severe and fatal malaria in Malaysia. Microscopic misdiagnosis is common and may delay appropriate treatment. P. knowlesi can cross-react with "species-specific" parasite lactate dehydrogenase (pLDH) monoclonal antibodies used in rapid diagnostic tests (RDTs) to detect P. falciparum and P. vivax. At one tertiary-care hospital and two district hospitals in Sabah, we prospectively evaluated two combination RDTs for malaria diagnosis by using both a pan-Plasmodium-pLDH (pan-pLDH)/P. falciparum-specific-pLDH (Pf-pLDH) RDT (OptiMAL-IT) and a non-P. falciparum VOM-pLDH/Pf-HRP2 RDT (CareStart). Differential cross-reactivity among these combinations was hypothesized to differentiate P. knowlesi from other Plasmodium monoinfections. Among 323 patients with PCR-confirmed P. knowlesi (n = 193), P. falciparum (n = 93), and P. vivax (n = 37) monoinfections, the VOM-pLDH individual component had the highest sensitivity for nonsevere (35%; 95% confidence interval [CI], 27 to 43%) and severe (92%; CI, 81 to 100%) P. knowlesi malaria. CareStart demonstrated a P. knowlesi sensitivity of 42% (CI, 34 to 49%) and specificity of 74% (CI, 65 to 82%), a P. vivax sensitivity of 83% (CI, 66 to 93%) and specificity of 71% (CI, 65 to 76%), and a P. falciparum sensitivity of 97% (CI, 90 to 99%) and specificity of 99% (CI, 97 to 100%). OptiMAL-IT demonstrated a P. knowlesi sensitivity of 32% (CI, 25 to 39%) and specificity of 21% (CI, 15 to 29%), a P. vivax sensitivity of 60% (CI, 42 to 75%) and specificity of 97% (CI, 94 to 99%), and a P. falciparum sensitivity of 82% (CI, 72 to 89%) and specificity of 39% (CI, 33 to 46%). The combination of CareStart plus OptiMAL-IT for P. knowlesi using predefined criteria gave a sensitivity of 25% (CI, 19 to 32%) and specificity of 97% (CI, 92 to 99%). Combining two RDT combinations was highly specific for P. knowlesi malaria diagnosis; however, sensitivity was poor. The specificity of pLDH RDTs was decreased for P. vivax and P. falciparum because of P. knowlesi cross-reactivity and cautions against their use alone in areas where P. knowlesi malaria is endemic. Sensitive P. knowlesi-specific RDTs and/or alternative molecular diagnostic tools are needed in areas where P. knowlesi malaria is endemic. PMID:24696029

Grigg, Matthew J; William, Timothy; Barber, Bridget E; Parameswaran, Uma; Bird, Elspeth; Piera, Kim; Aziz, Ammar; Dhanaraj, Prabakaran; Yeo, Tsin W; Anstey, Nicholas M

2014-06-01

276

Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa.  

PubMed

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)(+) to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD(+)- and NADP(+)-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)(+) and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)(+) binds first and NAD(P)H leaves last, particularly in the NADP(+)-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP(+)-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose. PMID:11104673

Velasco-García, R; González-Segura, L; Muñoz-Clares, R A

2000-12-15

277

Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa.  

PubMed Central

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)(+) to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD(+)- and NADP(+)-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)(+) and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)(+) binds first and NAD(P)H leaves last, particularly in the NADP(+)-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP(+)-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose.

Velasco-Garcia, R; Gonzalez-Segura, L; Munoz-Clares, R A

2000-01-01

278

Colour remediation of pulp mill effluent using purified fungal cellobiose dehydrogenase: reaction optimisation and mechanism of degradation.  

PubMed

Cellobiose dehydrogenase purified from two different fungal sources was assessed for its ability to remove and/or reduce colour from pulp mill bleach plant effluent. Cellobiose dehydrogenase purified from Phanerochaete chrysosporium was shown to prefer acidic conditions and was consequently used to treat the acid effluent stream discharged from a pulp mill bleach plant, while an analogous enzyme originating from Humicola insolens preferred alkaline conditions, and was applied to the effluent discharged from the caustic sewer of the bleach plant. Both enzyme preparations were able to remove colour from their respective effluent sources to a comparable extent. Up to 50% of the effluent colour was removed within 4 days when treated under optimised conditions. Furthermore, it was also shown that this enzymatic approach was effective at removing colour generated by both softwood and hardwood resources. Mechanistically, it was shown that colour was removed from all molecular weight fractions, and the higher molecular weight material (>300 kDa) was concurrently preferentially degraded. Cellobiose dehydrogenase treatment of effluent did not target phenolic, stilbene, or alpha-carbonyl structures, but did affect the quinone content. Further investigations using model compounds confirmed these results, and subsequently showed that only the para-quinones with low substitution were reduced with cellobiose dehydrogenase. PMID:15726583

Wingate, Kathryn G; Stuthridge, Trevor; Mansfield, Shawn D

2005-04-01

279

Efficacy of Dichloroacetate as a Lactate-Lowering Drug  

Microsoft Academic Search

Dichloroacetate (DCA) decreases blood, cerebral spinal fluid, and intracellular lactate concentrations by activating the mitochondrial pyruvate dehydrogenase enzyme complex. The authors reviewed the efficacy of this investigational drug in the treatment of acquired or congenital forms of lactic acidosis from data in 40 English-language publications. The hypolactatemic effect of DCA occurs over a broad range of pretreatment lactate concentrations and

Peter W. Stacpoole; Nelamangala V. Nagaraja; Alan D. Hutson

2003-01-01

280

Towards catalyst compartimentation in combined chemo- and biocatalytic processes: immobilization of alcohol dehydrogenases for the diastereoselective reduction of a ?-hydroxy ketone obtained from an organocatalytic aldol reaction.  

PubMed

The alcohol dehydrogenases (ADHs) from Lactobacillus kefir and Rhodococcus sp., which earlier turned out to be suitable for a chemoenzymatic one-pot synthesis with organocatalysts, were immobilized with their cofactors on a commercially available superabsorber based on a literature known protocol. The use of the immobilized ADH from L. kefir in the reduction of acetophenone as a model substrate led to high conversion (>95%) in the first reaction cycle, followed by a slight decrease of conversion in the second reaction cycle. A comparable result was obtained when no cofactor was added although a water rich reaction media was used. The immobilized ADHs also turned out to be suitable catalysts for the diastereoselective reduction of an organocatalytically prepared enantiomerically enriched aldol adduct, leading to high conversion, diastereomeric ratio and enantioselectivity for the resulting 1,3-diols. However, at a lower catalyst and cofactor amount being still sufficient for biotransformations with "free" enzymes the immobilized ADH only showed high conversion and >99% ee for the first reaction cycle whereas a strong decrease of conversion was observed already in the second reaction cycle, thus indicating a significant leaching effect of catalyst and/or cofactor. PMID:24036136

Rulli, Giuseppe; Heidlindemann, Marcel; Berkessel, Albrecht; Hummel, Werner; Gröger, Harald

2013-11-01

281

Dinosaur lactation?  

PubMed

Lactation is a process associated with mammals, yet a number of birds feed their newly hatched young on secretions analogous to the milk of mammals. These secretions are produced from various sections (crop organ, oesophageal lining and proventriculus) of the upper digestive tract and possess similar levels of fat and protein, as well as added carotenoids, antibodies and, in the case of pigeons and doves, epidermal growth factor. Parental care in avian species has been proposed to originate from dinosaurs. This study examines the possibility that some dinosaurs used secretory feeding to increase the rate of growth of their young, estimated to be similar to that of present day birds and mammals. Dinosaur 'lactation' could also have facilitated immune responses as well as extending parental protection as a result of feeding newly hatched young in nest environments. While the arguments for dinosaur lactation are somewhat generic, a case study for lactation in herbivorous site-nesting dinosaurs is presented. It is proposes that secretory feeding could have been used to bridge the gap between hatching and establishment of the normal diet in some dinosaurs. PMID:23325856

Else, Paul L

2013-02-01

282

Characterization of electron tunneling and hole hopping reactions between different forms of MauG and methylamine dehydrogenase within a natural protein complex  

PubMed Central

Respiration, photosynthesis and metabolism require the transfer of electrons through and between proteins over relatively long distances. It is critical that this electron transfer (ET) occur with specificity to avoid cellular damage, and at a rate which is sufficient to support the biological activity. A multi-step hole hopping mechanism could, in principle, enhance the efficiency of long range ET through proteins as it does in organic semiconductors. To explore this possibility, two different ET reactions that occur over the same distance within the protein complex of the diheme enzyme MauG and different forms of methylamine dehydrogenase (MADH) were subjected to kinetic and thermodynamic analysis. An ET mechanism of single-step direct electron tunneling from diferrous MauG to the quinone form of MADH is consistent with the data. In contrast, the biosynthetic ET from preMADH, which contains incompletely synthesized tryptophan tryptophylquinone, to the bis-Fe(IV) form of MauG is best described by a two-step hole hopping mechanism. Experimentally-determined values of ET distance matched the distances determined from the crystal structure that would be expected for single-step tunneling and multi-step hopping, respectively. Experimentally-determined relative values of electronic coupling (HAB) for the two reactions correlated well with the relative HAB values predicted from computational analysis of the structure. The rate of the hopping-mediated ET reaction is also ten-fold greater than that of the single-step tunneling reaction despite having a smaller overall driving force for the reaction. These data provide insight into how the intervening protein matrix and redox potentials of the electron donor and acceptor determine whether the ET reaction proceeds via single-step tunneling or multi-step hopping.

Choi, Moonsung; Shin, Sooim; Davidson, Victor L.

2012-01-01

283

Nitroreductase activity of heart lipoamide dehydrogenase.  

PubMed Central

A novel reaction catalysed by lipoamide dehydrogenase is described. In the presence of NADH, lipoamide dehydrogenase reduces the nitro group of 4-nitropyridine and 4-nitropyridine N-oxide. The elution profiles from a DEAE-cellulose column for the dehydrogenase and nitroreductase activities are identical. Chemical modifications of critical amino acid residues suggest that the two activities share a common catalytic domain. Nitro reduction catalysed by lipoamide dehydrogenase was monitored spectrophotometrically and chromatographically. The major product from the enzymic reduction of 4-nitropyridine was isolated and characterized structurally as NN-bis(pyridinyl)hydroxylamine, which is formed presumably via 4-hydroxyaminopyridine in a four-electron redox reaction. Images Fig. 4.

Tsai, C S

1987-01-01

284

Three overlapping lct genes involved in L-lactate utilization by Escherichia coli.  

PubMed Central

In Escherichia coli, the lct locus at min 80 on the chromosome map is associated with ability to grow on L-lactate and to synthesize a substrate-inducible flavin-linked dehydrogenase. Similar to that of the glpD-encoded aerobic glycerol-3-phosphate dehydrogenase, the level of induced enzyme activity is elevated by aerobiosis. Both of these controls are mediated by the two-component signal transduction system ArcB/ArcA, although sensitivity to the control is much more striking for L-lactate dehydrogenase. This study disclosed that the lct locus contained three overlapping genes in the clockwise order of lctD (encoding a flavin mononucleotide-dependent dehydrogenase), lctR (encoding a putative regulator), and lctP (encoding a permease) on the chromosomal map. These genes, however, are transcribed in the counterclockwise direction. No homology in amino acid sequence was found between aerobic glycerol-3-phosphate dehydrogenase and L-lactate dehydrogenase. A phi (lctD-lac) mutant was inducible by L-lactate but not D-lactate. Although the mutant lost the ability to grow on L-lactate, growth on D-lactate, known to depend on a different enzyme, remained normal. Images

Dong, J M; Taylor, J S; Latour, D J; Iuchi, S; Lin, E C

1993-01-01

285

Heavy atom isotope effects on enzymatic reactions  

NASA Astrophysics Data System (ADS)

The theory of isotope effects, which has proved to be extremely useful in providing geometrical details of transition states in a variety of chemical reactions, has recently found an application in studies of enzyme-catalyzed reactions. These reactions are multistep in nature with few steps being partially rate-limiting, thus interpretation of these isotope effects is more complex. The theoretical framework of heavy-atom isotope effects on enzymatic reactions is critically analyzed on the basis of recent results of: carbon kinetic isotope effects on carbonic anhydrase and catalytic antibodies; multiple carbon, deuterium isotope effects on reactions catalyzed by formate decarboxylase; oxygen isotope effects on binding processes in reactions catalyzed by pyruvate kinase; and equilibrium oxygen isotope effect on binding an inhibitor to lactate dehydrogenase. The advantages and disadvantages of reaction complexity in learning details of formal and molecular mechanisms are discussed in the examples of reactions catalyzed by phosphoenolpyruvate carboxylase, orotidine decarboxylase and glutamine synthetase.

Paneth, Piotr

1994-05-01

286

Hypothalamic sensing of circulating lactate regulates glucose production.  

PubMed

Emerging studies indicate that hypothalamic hormonal signalling pathways and nutrient metabolism regulate glucose homeostasis in rodents. Although hypothalamic lactate-sensing mechanisms have been described to lower glucose production (GP), it is currently unknown whether the hypothalamus senses lactate in the blood circulation to regulate GP and maintain glucose homeostasis in vivo. To examine whether hypothalamic sensing of circulating lactate is required to regulate GP, we infused intravenous (i.v.) lactate in the absence or presence of inhibition of central/hypothalamic lactate-sensing mechanisms in normal rodents. Inhibition of central/hypothalamic lactate-sensing mechanisms was achieved by three independent approaches. Tracer-dilution methodology in combination with the pancreatic clamp technique was used to assess the effect of i.v. and central/hypothalamic administrations on glucose metabolism in vivo. In the presence of physiologically relevant increases in the levels of plasma lactate, inhibition of central lactate-sensing mechanisms by lactate dehydrogenase inhibitor oxamate (OXA) or ATP-sensitive potassium channels blocker glibenclamide increased GP. Furthermore, direct administration of OXA into the mediobasal hypothalamus increased GP in the presence of similar elevation of circulating lactate. Together, these data indicate that hypothalamic sensing of circulating lactate regulates GP and is required to maintain glucose homeostasis. PMID:19040414

Kokorovic, Andrea; Cheung, Grace W C; Rossetti, Luciano; Lam, Tony K T

2009-01-01

287

Characterization of the Ni-Fe-C complex formed by reaction of carbon monoxide with the carbon monoxide dehydrogenase from Clostridium thermoaceticum by Q-band ENDOR  

SciTech Connect

Q-Band ENDOR studies on carbon monoxide dehydrogenase (CODH) from the acetogenic bacterium Clostridium thermoaceticum provided unambiguous evidence that the reaction of CO with CODH produces a novel metal center that includes at least one nickel, at least three iron sites, and the carbon of one CO. The {sup 57}Fe hyperfine couplings determined by ENDOR are similar to the values used in simulation of the Mossbauer spectra. EPR simulation using these A{sup Fe} values is equally good for a 4Fe or a 3Fe center. The {sup 13}C ENDOR data are consistent with the binding of a carbon atom to either the Ni or the Fe component of the spin-coupled cluster. The {sup 13}C hyperfine couplings are similar to those determined earlier for the CO-bound form of the H cluster of the Clostridium pasteurianum hydrogenase, proposed to be the active site of hydrogen activation. The {sup 61} Ni ENDOR data are the first nickel ENDOR recorded for an enzyme. The EPR simulation using the ENDOR-derived hyperfine values for {sup 61}Ni is consistent with a single nickel site in the Ni-Fe-C complex. On the basis of our results and the Mossbauer data the authors propose the stoichiometry of the components of the Ni-Fe-C complex to be Ni{sub 1}Fe{sub 3-4}S{sub {ge}}4C{sub 1}, with four acid-labile sulfides.

Fan, Chaoliang; Hoffman, B.M. (Northwestern Univ., Evanston, IL (USA)); Gorst, C.M.; Ragsdale, S.W. (Univ. of Wisconsin, Milwaukee (USA))

1991-01-01

288

d-3-Hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Kinetic mechanism from steady-state kinetics of the reaction catalysed by the enzyme in solution and covalently attached to diethylaminoethylcellulose  

PubMed Central

1. The reversible NAD+-linked oxidation of d-3-hydroxybutyrate to acetoacetate in 0.1m-sodium pyrophosphate buffer, pH8.5, at 25.0°C, catalysed by d-3-hydroxybutyrate dehydrogenase (d-3-hydroxybutyrate–NAD+ oxidoreductase, EC 1.1.1.30), was studied by initial-velocity, dead-end inhibition and product-inhibition analysis. 2. The reactions were carried out on (a) the soluble enzyme from Rhodopseudomonas spheroides and (b) an insoluble derivative of this enzyme prepared by its covalent attachment to DEAE-cellulose by using 2-amino-4,6-dichloro-s-triazine as coupling agent. 3. The insolubilized enzyme preparation contained 5mg of protein/g wet wt. of total material, and when freshly prepared its specific activity was 1.2?mol/min per mg of protein, which is 67% of that of the soluble dialysed enzyme. 4. The reactions catalysed by both the enzyme in solution and the insolubilized enzyme were shown to follow sequential pathways in which the nicotinamide nucleotides bind obligatorily first to the enzyme. Evidence is presented for kinetically significant ternary complexes and that the rate-limiting step(s) of both catalyses probably involves isomerization of the enzyme–nicotinamide nucleotide complexes and/or dissociation of the nicotinamide nucleotides from the enzyme. Both catalyses therefore are probably best described as ordered Bi Bi mechanisms, possibly with multiple enzyme–nicotinamide nucleotide complexes. 5. The kinetic parameters and the calculable rate constants for the catalysis by the soluble enzyme are similar to the corresponding parameters and rate constants for the catalysis by the insolubilized enzyme.

Preuveneers, M. J.; Peacock, D.; Crook, E. M.; Clark, J. B.; Brocklehurst, K.

1973-01-01

289

The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli.  

PubMed

The effect of gene knockout on metabolism in the pflA-, pflB-, pflC-, and pflD- mutants of Escherichia coli was investigated. Batch cultivations of the pfl- mutants and their parent strain were conducted using glucose as a carbon source. It was found that pflA- and pflB- mutants, but not pflC- and pflD- mutants, produced large amounts of D-lactate from glucose under the microaerobic condition, and the maximum yield was 73%. In order to investigate the metabolic regulation mechanism, we measured enzyme activities for the following eight enzymes: glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase, lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, acetate kinase, and alcohol dehydrogenase. Intracellular metabolite concentrations of glucose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate, pyruvate, acetyl coenzyme A as well as ATP, ADP, AMP, NADH, and NAD+ were also measured. It was shown that the GAPDH and LDH activities were considerably higher in pflA- and pflB- mutants, which implies coupling between NADH production and consumption between the two corresponding reactions. The urgent energy requirement was shown by the lower ATP/AMP level due to both oxygen limitation and pfl gene knockout, which promoted significant stepping-up of glycolysis when using glucose as a carbon source. It was shown that the demand for energy is more important than intracellular redox balance, thus excess NADH produced through GAPDH resulted in a significantly higher intracellular NADH/NAD+ ratio in pfl- mutants. Consequently, the homolactate production was achieved to meet the requirements of the redox balance and the energy production through glycolysis. The effect of using different carbon sources such as gluconate, pyruvate, fructose, and glycerol was investigated. PMID:14673546

Zhu, J; Shimizu, K

2004-04-01

290

Enzymatic synthesis of l-?-chloroalanine using amino acid dehydrogenase  

Microsoft Academic Search

l-ß-Chloroalanine is a useful intermediate for the synthesis of several l-amino acids. Conditions for synthesizing optically pure l-ß-chloroalanine from 3-chloropyruvate using alanine dehydrogenase (AlaDH), leucine dehydrogenase and phenylalanine dehydrogenase with a regeneration of NADH by formate dehydrogenase (FDH) were investigated. The enzymatic reaction was carried out at neutral pH because of a chemical instability of 3-chloropyruvate on the alkaline side.

Yasuo Kato; Kenji Fukumoto; Yasuhisa Asano

1993-01-01

291

[High-efficiency L-lactate production from glycerol by metabolically engineered Escherichia coli].  

PubMed

High-efficient conversion of glycerol to L-lactate is beneficial for the development of both oil hydrolysis industry and biodegradable materials manufacturing industry. In order to construct an L-lactate producer, we first cloned a coding region of gene BcoaLDH encoding an L-lactate dehydrogenase from Bacillus coagulans CICIM B1821 and the promoter sequence (P(ldhA)) of the D-lactate dehydrogenase (LdhA) from Escherichia coli CICIM B0013. Then we assembled these two DNA fragments in vitro and yielded an expression cassette, P(ldhA)-BcoaLDH. Then, the cassette was chromosomally integrated into an ldhA mutant strain, Escherichia coli CICIM B0013-080C, by replacing lldD encoding an FMN-dependent L-lactate dehydrogenase. An L-lactate higher-producer strain, designated as E. coli B0013-090B, possessing genotype of lldD::P(ldhA)-BcoaLDH, deltaack-pta deltapps deltapflB deltadld deltapoxB deltaadhE deltafrdA and deltaldhA, was generated. Under the optimal condition, 132.4 g/L L-lactate was accumulated by B0013-090B with the lactate productivity of 4.90 g/Lh and the yield of 93.7% in 27 h from glycerol. The optical purity of L-lactate in broth is above 99.95%. PMID:24409690

Tian, Kangming; Shi, Guiyang; Lu, Fuping; Singh, Suren; Wang, Zhengxiang

2013-09-01

292

NADH electrochemical sensor coupled with dehydrogenase enzymes  

SciTech Connect

A graphite electrode assembled in a flow cell has shown to be a good detector for NADH. Current is linearly dependent on concentration in the range 10{sup {minus}7}-10{sup {minus}3} M without any mediator at the potential applied of 300 mV vs Ag/AgCl. Lactate and alcohol dehydrogenases were immobilized near to the electrode surface or in a reactor to obtain an NADH-based biosensor for lactate or ethanol. With lactate the authors succeeded to obtain a response only if the reactor was used and for alcohol a current proportional to the concentration was obtained either if the enzyme was immobilized in a membrane and placed near the electrode surface or when the enzyme was immobilized in a reactor form. By FIA procedures fast responses and recoveries were obtained, but with a short linear range.

Yamanaka, Hideko; Mascini, Marco (Epidemiologia e Chimica Analitica Ambientale, Firenze (Italy))

1992-06-01

293

Cis-Chlorobenzene dihydrodiol dehydrogenase (TcbB) from Pseudomonas sp. strain P51, expressed in Escherichia coli DH5{alpha}(pTCB149), catalyzes enantioselective dehydrogenase reactions  

SciTech Connect

cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5{alpha}(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (-)-cis-(S,2R) enantiomer remained unchanged, CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (-)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enatiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols.

Raschke, H.; Fleischmann, T.; Meer, J.R. van der; Kohler, H.P.E.

1999-12-01

294

Homofermentative Lactate Production Cannot Sustain Anaerobic Growth of Engineered Saccharomyces cerevisiae: Possible Consequence of Energy-Dependent Lactate Export  

PubMed Central

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.

van Maris, Antonius J. A.; Winkler, Aaron A.; Porro, Danilo; van Dijken, Johannes P.; Pronk, Jack T.

2004-01-01

295

Multiple Forms of D(--)3-Hydroxybutyrate Dehydrogenase in Rhizobium  

Microsoft Academic Search

SUMMARY 3-Hydroxybutyrate dehydrogenase was studied in 14 Rhizobium strains representing six species. Cell-free extracts of the bacteria were subjected to electrophoresis in starch or polyacrylamide gels and 3-hydroxybutyrate dehydrogenase was located in situ on the gels. Also, the rate of reaction of the enzyme, from different Rhizobium strains, with NAD was compared with its rate of reaction with various analogues

P. F. Fottrell; A. O'Hora

1969-01-01

296

Hematite nanoparticles larger than 90 nm show no sign of toxicity in terms of lactate dehydrogenase release, nitric oxide generation, apoptosis, and comet assay in murine alveolar macrophages and human lung epithelial cells.  

PubMed

Three hematite samples were synthesized by precipitation from a FeCl? solution under controlled pH and temperature conditions in different morphology and dimensions: (i) microsized (average diameter 1.2 ?m); (ii) submicrosized (250 nm); and (iii) nanosized (90 nm). To gain insight into reactions potentially occurring in vivo at the particle-lung interface following dust inhalation, several physicochemical features relevant to pathogenicity were measured (free radical generation in cell-free tests, metal release, and antioxidant depletion), and cellular toxicity assays on human lung epithelial cells (A549) and murine alveolar macrophages (MH-S) were carried out (LDH release, apoptosis detection, DNA damage, and nitric oxide synthesis). The decrease in particles size, from 1.2 ?m to 90 nm, only caused a slight increase in structural defects (disorder of the hematite phase and the presence of surface ferrous ions) without enhancing surface reactivity or cellular responses in the concentration range between 20 and 100 ?g cm?². PMID:22324577

Freyria, Francesca Stefania; Bonelli, Barbara; Tomatis, Maura; Ghiazza, Mara; Gazzano, Elena; Ghigo, Dario; Garrone, Edoardo; Fubini, Bice

2012-04-16

297

Potential of Mean Force Calculation for the Proton and Hydride Transfer Reactions Catalyzed by Medium-Chain Acyl-CoA Dehydrogenase:  Effect of Mutations on Enzyme Catalysis †  

Microsoft Academic Search

ABSTRACT: Potential of mean,force calculations have been,performed,on the wild-type medium-chain acyl-CoA dehydrogenase,(MCAD) and two of its mutant,forms. Initial simulation and analysis of the active site of the enzyme reveal that an arginine residue (Arg256), conserved in the substrate-binding domain of this group of enzymes, exists in two alternate conformations, only one of which makes the enzyme,active. This active conformation,was used

Sudeep Bhattacharyya; Shuhua Ma; Marian T. Stankovich; Donald G. Truhlar; Jiali Gao

2005-01-01

298

Enzymes involved in l-lactate metabolism in humans.  

PubMed

l-lactate formation occurs via the reduction of pyruvate catalyzed by lactate dehydrogenase. l-lactate removal takes place via its oxidation into pyruvate, which may be oxidized or converted into glucose. Pyruvate oxidation involves the cooperative effort of pyruvate dehydrogenase, the tricarboxylic acid cycle, and the mitochondrial respiratory chain. Enzymes of the gluconeogenesis pathway sequentially convert pyruvate into glucose. In addition, pyruvate may undergo reversible transamination to alanine by alanine aminotransferase. Enzymes involved in l-lactate metabolism are crucial to diabetes pathophysiology and therapy. Elevated plasma alanine aminotransferase concentration has been associated with insulin resistance. Polymorphisms in the G6PC2 gene have been associated with fasting glucose concentration and insulin secretion. In diabetes patients, pyruvate dehydrogenase is down-regulated and the activity of pyruvate carboxylase is diminished in the pancreatic islets. Inhibitors of fructose 1,6-bisphosphatase are being investigated as potential therapy for type 2 diabetes. In addition, enzymes implicated in l-lactate metabolism have revealed to be important in cancer cell homeostasis. Many human tumors have higher LDH5 levels than normal tissues. The LDHC gene is expressed in a broad range of tumors. The activation of PDH is a potential mediator in the body response that protects against cancer and PDH activation has been observed to reduce glioblastoma growth. The expression of PDK1 may serve as a biomarker of poor prognosis in gastric cancer. Mitochondrial DNA mutations have been detected in a number of human cancers. Genes encoding succinate dehydrogenase have tumor suppressor functions and consequently mutations in these genes may cause a variety of tumors. PMID:24029012

Adeva, M; González-Lucán, M; Seco, M; Donapetry, C

2013-11-01

299

Investigating the effectiveness of DNA microarray analysis for identifying the genes involved in l -lactate production by Saccharomyces cerevisiae  

Microsoft Academic Search

In order to determine whether transcriptome data obtained by DNA microarray analysis could be used to identify the genes involved\\u000a in target metabolite production, we tried to identify the genes involved in l-lactate production by l-lactate-producing recombinant Saccharomyces cerevisiae strains. We obtained DNA microarray data for these strains. Plasmids carrying lactic acid bacteria, bovine, and human l-lactate dehydrogenase (LDH) genes

Takashi Hirasawa; Aki Ookubo; Katsunori Yoshikawa; Keisuke Nagahisa; Chikara Furusawa; Hideki Sawai; Hiroshi Shimizu

2009-01-01

300

Involvement of pyruvate dehydrogenase in product formation in pyruvate-limited anaerobic chemostat cultures of Enterococcus faecalis NCTC 775  

Microsoft Academic Search

Enterococcus faecalis NCTC 775 was grown anaerobically in chemostat culture with pyruvate as the energy source. At low culture pH values, high in vivo and in vitro activities were found for both pyruvate dehydrogenase and lactate dehydrogenase. At high culture pH values the carbon flux was shifted towards pyruvate formate lyase. Some mechanisms possibly involved in this metabolic switch are

Jacky L. Snoep; M. Joost Teixeira de Mattos; Pieter W. Postma; Oense M. Neijssel

1990-01-01

301

Partial reconstruction of in vitro gluconeogenesis arising from mitochondrial l-lactate uptake/metabolism and oxaloacetate export via novel L-lactate translocators.  

PubMed Central

In the light of the occurrence of L-lactate dehydrogenase inside the mitochondrial matrix, we looked at whether isolated rat liver mitochondria can take up and metabolize L-lactate, and provide oxaloacetate outside mitochondria, thus contributing to a partial reconstruction of gluconeogenesis in vitro. We found that: (1) L-lactate (10 mM), added to mitochondria in the presence of a cocktail of glycolysis/gluconeogenesis enzymes and cofactors, can lead to synthesis of glyceraldehyde-3-phosphate at a rate of about 7 nmol/min per mg mitochondrial protein. (2) Three novel translocators exist to mediate L-lactate traffic across the inner mitochondrial membrane. An L-lactate/H+ symporter was identified by measuring fluorimetrically the rate of endogenous pyridine nucleotide reduction. Consistently, L-lactate oxidation was found to occur with P/O ratio=3 (where P/O ratio is the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation) and with generation of membrane potential. Proton uptake, which occurred as a result of addition of L-lactate to RLM together with electron flow inhibitors, and mitochondrial swelling in ammonium L-lactate solutions were also monitored. L-Lactate/oxaloacetate and L-lactate/pyruvate anti-porters were identified by monitoring photometrically the appearance of L-lactate counter-anions outside mitochondria. These L-lactate translocators, which are distinct from the monocarboxylate carrier, were found to differ from each other in V(max) values and in inhibition and pH profiles, and proved to regulate mitochondrial L-lactate metabolism in vitro. The role of lactate/mitochondria interactions in gluconeogenesis is discussed.

De Bari, Lidia; Atlante, Anna; Valenti, Daniela; Passarella, Salvatore

2004-01-01

302

Partial reconstruction of in vitro gluconeogenesis arising from mitochondrial l-lactate uptake/metabolism and oxaloacetate export via novel L-lactate translocators.  

PubMed

In the light of the occurrence of L-lactate dehydrogenase inside the mitochondrial matrix, we looked at whether isolated rat liver mitochondria can take up and metabolize L-lactate, and provide oxaloacetate outside mitochondria, thus contributing to a partial reconstruction of gluconeogenesis in vitro. We found that: (1) L-lactate (10 mM), added to mitochondria in the presence of a cocktail of glycolysis/gluconeogenesis enzymes and cofactors, can lead to synthesis of glyceraldehyde-3-phosphate at a rate of about 7 nmol/min per mg mitochondrial protein. (2) Three novel translocators exist to mediate L-lactate traffic across the inner mitochondrial membrane. An L-lactate/H+ symporter was identified by measuring fluorimetrically the rate of endogenous pyridine nucleotide reduction. Consistently, L-lactate oxidation was found to occur with P/O ratio=3 (where P/O ratio is the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation) and with generation of membrane potential. Proton uptake, which occurred as a result of addition of L-lactate to RLM together with electron flow inhibitors, and mitochondrial swelling in ammonium L-lactate solutions were also monitored. L-Lactate/oxaloacetate and L-lactate/pyruvate anti-porters were identified by monitoring photometrically the appearance of L-lactate counter-anions outside mitochondria. These L-lactate translocators, which are distinct from the monocarboxylate carrier, were found to differ from each other in V(max) values and in inhibition and pH profiles, and proved to regulate mitochondrial L-lactate metabolism in vitro. The role of lactate/mitochondria interactions in gluconeogenesis is discussed. PMID:14960150

De Bari, Lidia; Atlante, Anna; Valenti, Daniela; Passarella, Salvatore

2004-05-15

303

Aerobically derived lactate stimulates revascularization and tissue repair via redox mechanisms.  

PubMed

Hypoxia serves as a physiologic cue to drive an angiogenic response via HIF-dependent mechanisms. Interestingly, minor elevation of lactate levels in the tissue produces the same effect under aerobic conditions. Aerobic glycolysis contributes to lactate accumulation in the presence of oxygen, especially under inflammatory conditions. We previously postulated that aerobic lactate accumulation, already known to stimulate collagen deposition, will also stimulate angiogenesis. If substantiated, this concept would advance understanding of wound healing and aerobic angiogenesis because lactate accumulation has many aerobic sources. In this study, Matrigel plugs containing a powdered, hydrolyzable lactate polymer were implanted into the subcutaneous space of mice. Lactate monomer concentrations in the implant were consistent with wound levels for more than 11 days. They induced little inflammation but considerable VEGF production and were highly angiogenic, as opposed to controls. Arterial hypoxia abrogated angiogenesis. Furthermore, inhibition of lactate dehydrogenase by using oxamate also prevented the angiogenic effects of lactate. Lactate monomer, at concentrations found in cutaneous wounds, stabilized HIF-1alpha and increased VEGF levels in aerobically cultured human endothelial cells. Accumulated lactate, therefore, appears to convey the impression of "metabolic need" for vascularization, even in well-oxygenated and pH-neutral conditions. Lactate and oxygen together stimulate angiogenesis and matrix deposition. PMID:17567242

Hunt, Thomas K; Aslam, Rummana S; Beckert, Stefan; Wagner, Silvia; Ghani, Q Perveen; Hussain, M Zamirul; Roy, Sashwati; Sen, Chandan K

2007-08-01

304

Aerobically-Derived Lactate Stimulates Revascularization and Tissue Repair via Redox Mechanisms  

PubMed Central

Hypoxia serves as a physiological cue to drive angiogenic response via HIF-dependent mechanisms. Interestingly, minor elevation of lactate levels in the tissue produces the same effect under aerobic conditions. Aerobic glycolysis contributes to lactate accumulation in the presence of oxygen especially under inflammatory conditions. We have previously postulated that aerobic lactate accumulation, already known to stimulate collagen deposition, will also stimulate angiogenesis. If substantiated, this concept would advance understanding of wound healing and aerobic angiogenesis because lactate accumulation has many aerobic sources. In this study, Matrigel® plugs containing a powdered, hydrolysable lactate polymer were implanted into the subcutaneous space of mice. Lactate monomer concentrations in the implant were consistent with wound levels for over 11 days. They induced little inflammation but considerable VEGF production and were highly angiogenic as opposed to controls. Arterial hypoxia abrogated angiogenesis. Furthermore, inhibition of lactate dehydrogenase using oxamate also prevented the angiogenic effects of lactate. Lactate monomer, at concentrations found in cutaneous wounds, stabilized HIF-1? and increased VEGF levels in aerobically cultured human endothelial cells. Accumulated lactate, therefore, appears to convey the impression of “metabolic need” for vascularization even in well-oxygenated and pH-neutral conditions. Lactate and oxygen both stimulate angiogenesis and matrix deposition.

HUNT, THOMAS K; ASLAM, RUMMANA S.; BECKERT, STEFAN; WAGNER, SILVIA; GHANI, Q. PERVEEN; HUSSAIN, M. ZAMIRUL; ROY, SASHWATI; SEN, CHANDAN K.

2008-01-01

305

Lactate Dehydrogenase Catalysis: Roles of Keto, Hydrated, and Enol Pyruvate  

ERIC Educational Resources Information Center

Many carbonyl substrates of oxidoreductase enzymes undergo hydration and enolization so that these substrate systems are partitioned between keto, hydrated (gem-diol), and enol forms in aqueous solution. Some oxidoreductase enzymes are subject to inhibition by high concentrations of substrate. For such enzymes, two questions arise pertaining to…

Meany, J. E.

2007-01-01

306

Stability of Lactate Dehydrogenase Isozyme Patterns in Penguins  

Microsoft Academic Search

PENGUINS were chosen for this investigation because of their highly specialized adaptation to an aquatic life and because of the extreme physiological conditions they are subjected to during the breeding season. One of the most abundant species is the Adelie penguin, Pygoscelis adeliae, which has a circumpolar distribution south of the Antarctic pack-ice. Unlike other birds this species endures a

Clement L. Markert; William J. L. Sladen

1966-01-01

307

Lactate Dehydrogenase Polymorphism of Sockeye Salmon (Oncorhynchus nerka).  

National Technical Information Service (NTIS)

Samples of sockeye salmon from the Naknek River system, Alaska, exhibited marked heterogeneity in LDH phenotype. Similar, though less marked, heterogeneity is apparent in sockeye salmon samples from other Bristol Bay area rivers. The B allele was almost e...

H. O. Hodgins F. M. Utter

1971-01-01

308

Inefficient lactate dehydrogenases of deep-sea fishes  

Microsoft Academic Search

The respiratory rates of deep-sea animals are extremely low. Deep-sea fishes may consume oxygen at rates only 5-10% those characteristic of shallow-water species1-4. These low metabolic rates are probably an adaptation to the presumed scarcity of food in deeper water4. The biochemical basis of this metabolic adaptation is a low level of enzyme activity in the skeletal muscle (but not

George N. Somero; Joseph F. Siebenaller

1979-01-01

309

The Partial Purification and Characterization of Lactate Dehydrogenase.  

ERIC Educational Resources Information Center

Offers several advantages over other possibilities as the enzyme of choice for a student's first exposure to a purification scheme. Uses equipment and materials normally found in biochemistry laboratories. Incorporates several important biochemical techniques including spectrophotometry, chromatography, centrifugation, and electrophoresis. (MVL)

Wolf, Edward C.

1988-01-01

310

Oxytocin and prolactin suppress cortisol responses to acute stress in both lactating and non-lactating sheep.  

PubMed

Cortisol response to stress appears to differ between lactating and non-lactating animals. Lactating (14 d post partum) and non-lactating sheep were fitted with probes so that drugs and hormones could be infused directly into the posterior pituitary and paraventricular nucleus of the hypothalamus. The animals were also fitted with instruments to allow monitoring of heart rate, body temperature and blood cortisol levels. Their reactions to a source of acute stress (a barking dog) were then followed, with or without drug and hormone manipulation. Results in both lactating and non-lactating animals indicated shortcomings in the use of cortisol as a stress indicator. Infusing prolactin and oxytocin into either the posterior pituitary or the paraventricular nucleus of the hypothalamus suppressed cortisol responsiveness to stress in both lactating and non-lactating animals (the latter to a greater extent). In the absence of drugs, lactating animals had a slightly higher basal level of cortisol and a lower cortisol response to stress than their non-lactating counterparts. Despite suppression of cortisol responses, with or without drugs, other indicators of stress still changed with the presence of a barking dog, suggesting the complexity of control involved in stress responses. PMID:9275253

Cook, C J

1997-08-01

311

Mouse lacking NAD+-linked glycerol phosphate dehydrogenase has normal pancreatic beta cell function but abnormal metabolite pattern in skeletal muscle.  

PubMed

We surveyed the BALB/cHeA mouse, which lacks cytosolic glycerol phosphate dehydrogenase an enzyme that catalyzes a reaction in the glycerol phosphate shuttle. The other enzyme of this shuttle, mitochondrial glycerol phosphate dehydrogenase, is abundant in skeletal muscle and pancreatic islets suggesting that the shuttle's activity is high in these tissues. Levels of glycerol phosphate (low) and dihydroxyacetone phosphate (high) were very abnormal in nonislet tissue, especially in skeletal muscle. Intermediates situated before the triose phosphates in the glycolysis pathway were increased and those after the triose phosphates were generally low, depending on the tissue. The lactate/pyruvate ratio in muscle was low signifying a low cytosolic NAD/NADH ratio. This suggests that a nonfunctional glycerol phosphate shuttle caused a block in glycolysis at the step catalyzed by glyceraldehyde phosphate dehydrogenase. When exercised, mice were unable to maintain normal ATP levels in skeletal muscle. Blood glucose, serum insulin levels, and pancreatic islet mass were normal. In isolated pancreatic islets insulin release, glucose metabolism and ATP levels were normal, but lactate levels and lactate/pyruvate ratios with a glucose load were slightly abnormal. The BALB/cHeA mouse can maintain NAD/ NADH ratios sufficient to function normally under most conditions, but the redox state is not normal. Glycerol phosphate is apparently formed at a slow rate. Skeletal muscle is severely affected probably because it is dependent on the glycerol phosphate shuttle more than other tissues. It most likely utilizes glycerol phosphate rapidly and, due to the absence of glycerol kinase in muscle, is unable to rapidly form glycerol phosphate from glycerol. Glycerol kinase is also absent in the pancreatic insulin cell, but this cell's function is essentially normal probably because of redundancy of NAD(H) shuttles. PMID:11147825

MacDonald, M J; Marshall, L K

2000-12-01

312

Aerobic and anaerobic metabolism in oxygen minimum layer fishes: the role of alcohol dehydrogenase.  

PubMed

Zones of minimum oxygen form at intermediate depth in all the world's oceans as a result of global circulation patterns that keep the water at oceanic mid-depths out of contact with the atmosphere for hundreds of years. In areas where primary production is very high, the microbial oxidation of sinking organic matter results in very low oxygen concentrations at mid-depths. Such is the case with the Arabian Sea, with O(2) concentrations reaching zero at 200 m and remaining very low (<0.1 ml O(2)l(-1)) for hundreds of meters below this depth, and in the California borderland, where oxygen levels reach 0.2 ml O(2)l(-1) at 700 m with severely hypoxic (<1.0 ml O(2)l(-1)) waters at depths 300 m above and below that. Despite the very low oxygen, mesopelagic fishes (primarily lanternfishes: Mytophidae) inhabiting the Arabian Sea and California borderland perform a daily vertical migration into the low-oxygen layer, spending daylight hours in the oxygen minimum zone and migrating upward into normoxic waters at night. To find out how fishes were able to survive their daily sojourns into the minimum zone, we tested the activity of four enzymes, one (lactate dehydrogenase, LDH) that served as a proxy for anaerobic glycolysis with a conventional lactate endpoint, a second (citrate synthase, CS) that is indicative of aerobic metabolism, a third (malate dehydrogenase) that functions in the Krebs' cycle and as a bridge linking mitochondrion and cytosol, and a fourth (alcohol dehydrogenase, ADH) that catalyzes the final reaction in a pathway where pyruvate is reduced to ethanol. Ethanol is a metabolic product easily excreted by fish, preventing lactate accumulation. The ADH pathway is rarely very active in vertebrate muscle; activity has previously been seen only in goldfish and other cyprinids capable of prolonged anaerobiosis. Activity of the enzyme suite in Arabian Sea and California fishes was compared with that of ecological analogs in the same family and with the same lifestyle but living in systems with much higher oxygen concentrations: the Gulf of Mexico and the Southern Ocean. ADH activities in the Arabian Sea fishes were similar to those of goldfish, far higher than those of confamilials from the less severe minimum in the Gulf of Mexico, suggesting that the Arabian Sea fishes are capable of exploiting the novel ethanol endpoint to become competent anaerobes. In turn, the fishes of California exhibited a higher ADH activity than their Antarctic relatives. It was concluded that ADH activity is more widespread in fishes than previously believed and that it may play a role in allowing vertically migrating fishes to exploit the safe haven afforded by severe oxygen minima. PMID:22573769

Torres, Joseph J; Grigsby, Michelle D; Clarke, M Elizabeth

2012-06-01

313

Control by phospho-adenosinediphospho-ribose of NADP-dependent isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase in Streptomyces griseus.  

PubMed

The metabolic function of NAD(P)-glycohydrolase in the streptomycin-producing Streptomyces griseus was investigated. Phospho-adenosinediphospho-ribose, the product of NAD(P)-glycohydrolase reaction was shown to interfere as a competitive inhibitor not only with the glucose-6-phosphate dehydrogenase (VORONINA et al. 1978) but also with the NADP-dependent isocitrate and 6-phospho-gluconate dehydrogenases. Inhibition kinetics were studied with isocitrate dehydrogenase from pig heart and 6-phosphogluconate dehydrogenase from yeast as well as with mycelial extracts of a mutant of S. griseus lacking NAD(P)-glycohydrolase. PMID:6784352

Gräfe, U; Bormann, E J; Truckenbrodt, G

1980-01-01

314

Primary Structure and Phylogenetic Relationships of a Malate Dehydrogenase Gene from Giardia lamblia  

Microsoft Academic Search

.   The lactate and malate dehydrogenases comprise a complex protein superfamily with multiple enzyme homologues found in eubacteria,\\u000a archaebacteria, and eukaryotes. In this study we describe the sequence and phylogenetic relationships of a malate dehydrogenase\\u000a (MDH) gene from the amitochondriate diplomonad protist, Giardia lamblia. Parsimony, distance, and maximum-likelihood analyses of the MDH protein family solidly position G. lamblia MDH within

Andrew J. Roger; Hilary G. Morrison; Mitchell L. Sogin

1999-01-01

315

D-fructose detection based on the direct heterogeneous electron transfer reaction of fructose dehydrogenase adsorbed onto multi-walled carbon nanotubes synthesized on platinum electrode.  

PubMed

Multi-walled carbon nanotubes (MWCNTs) were synthesized on platinum plate electrodes by the chemical vapor deposition (CVD) method. From the results of X-ray photoelectron spectroscopy and voltammetric investigation, the iron nanoparticles used as a catalyst for the MWCNT synthesis were enclosed with MWCNTs. The MWCNTs synthesized on the Pt plate (MWCNTs/Pt) electrode were immediately immersed into solutions of d-fructose dehydrogenase (FDH) to immobilize the enzyme onto the MWCNTs/Pt electrode surfaces. After the FDH was immobilized onto the MWCNTs/Pt electrode, a well-defined catalytic oxidation current based on FDH was observed from ca. -0.15V (versus Ag/AgCl/sat'd KCl), which was close to the redox potential of heme c as a prosthetic group of FDH. From an analysis of a plot of the catalytic current versus substrate, the calibration range for the fructose concentration was up to ca. 40mmoldm(-3), and the apparent Michaelis-Menten constant was evaluated to be 11+/-1mmoldm(-3). PMID:18707862

Tominaga, Masato; Nomura, Shinya; Taniguchi, Isao

2009-01-01

316

Reference values of blood parameters in beef cattle of different ages and stages of lactation.  

PubMed Central

Reference (normal) values for 12 blood serum components were determined for 48 Shorthorn cows (2-10 years old) and their 48 calves, 357 crossbred cows (12-14 years old), 36 feedlot bulls and 36 feedlot steers. In addition, hemoglobin, hematocrit, triiodothyronine, thyroxine and cortisol levels were determined for the crossbred cows, and feedlot bulls and steers. Reference values were tabulated according to sex, age and stage of lactation. Serum concentrations of urea, total protein and bilirubin, and serum activity of aspartate aminotransferase and lactate dehydrogenase increased with age (P less than 0.05), while calcium, phosphorus and alkaline phosphatase decreased with age (P less than 0.05) from birth to the age of ten years. The Shorthorn cows had the highest levels of glucose at parturition (P less than 0.05) with decreasing levels during lactation. Creatinine concentration decreased during lactation and increased during postweaning. Both lactate dehydrogenase and aspartate aminotransferase levels increased (P less than 0.05) during lactation. Urea and uric acid were present at higher concentrations in lactating than nonlactating cows (P less than 0.05). The values reported, based on a wide age range and large number of cattle, could serve as clinical guides and a basis for further research.

Doornenbal, H; Tong, A K; Murray, N L

1988-01-01

317

Lactate, not pyruvate, is neuronal aerobic glycolysis end product: an in vitro electrophysiological study.  

PubMed

For over 60 years, a distinction has been made between aerobic and anaerobic glycolysis based on their respective end products: pyruvate of the former, lactate of the latter. Recently we hypothesized that, in the brain, both aerobic and anaerobic glycolysis terminate with the formation of lactate from pyruvate by the enzyme lactate dehydrogenase (LDH). If this hypothesis is correct, lactate must be the mitochondrial substrate for oxidative energy metabolism via its oxidation to pyruvate, plausibly by a mitochondrial LDH. Here we employed electrophysiology of the rat hippocampal slice preparation to test and monitor the effects of malonate and oxamate, two different LDH inhibitors, and glutamate, a neuronal activator, in experiments, the results of which support the hypothesis that lactate, at least in this in vitro setting, is indeed the principal end product of neuronal aerobic glycolysis. PMID:17560727

Schurr, A; Payne, R S

2007-07-13

318

Wheat Alcohol Dehydrogenase Isozymes  

PubMed Central

Evidence in support of the hypothesis of gene expression and subunit association suggested earlier for Triticum alcohol dehydrogenase has been obtained through purification and partial characterization of the enzyme from tetraploid wheat. Three isozymes of alcohol dehydrogenase were separated and purified to apparent homogeneity using streptomycin sulfate precipitation, gel filtration chromatography, and anion exchange chromatography. The isozymes are dimers with the same molecular weight (116,000 ± 2,000), but significantly different isoelectric pH values. The Michaelis constants for NAD+ and ethanol are 0.1 millimolar and 12 millimolar, respectively. The substrate specificity of the three alcohol dehydrogenase isozymes was investigated. Images

Langston, Pat J.; Pace, C. Nick; Hart, Gary E.

1980-01-01

319

Production of panic-like symptoms by lactate is associated with increased neural firing and oxidation of brain redox in the rat hippocampus.  

PubMed

Lactate uses an unknown mechanism to induce panic attacks in people and panic-like symptoms in rodents. We tested whether intraperitoneal (IP) lactate injections act peripherally or centrally to induce panic-like symptoms in rats by examining whether IP lactate directly affects the CNS. In Long-Evans rats, IP lactate (2 mmol/kg) injection increased lactate levels in the plasma and the cerebrospinal fluid. IP lactate also induced tachycardia and behavioral freezing suggesting the production of panic-like behavior. To enter intermediate metabolism, lactate is oxidized by lactate dehydrogenase (LDH) to pyruvate with co-reduction of NAD(+) to NADH. Therefore, we measured the ratio of NADH/NAD(+) to test whether IP lactate altered lactate metabolism in the CNS. Lactate metabolism was studied in the hippocampus, a brain region believed to contribute to panic-like symptoms. IP lactate injection lowered the ratio of NADH/NAD(+) without altering the total amount of NADH and NAD(+) suggesting oxidation of hippocampal redox state. Lactate oxidized hippocampal redox since intrahippocampal injection of the LDH inhibitor, oxamate (50mM) prevented the oxidation of NADH/NAD(+) by IP lactate. In addition to oxidizing hippocampal redox, IP lactate rapidly increased the firing rate of hippocampal neurons. Similar IP pyruvate injections had no effect. Neural discharge also increased following intrahippocampal lactate injection suggesting that increased discharge was a direct action of lactate on the hippocampus. These studies show that oxidation of brain redox and increased hippocampal firing are direct actions of lactate on the CNS that may contribute to the production of lactate-induced panic. PMID:19429039

Bergold, Peter J; Pinkhasova, Valariya; Syed, Maryam; Kao, Hsin-Yi; Jozwicka, Anna; Zhao, Ning; Coplan, Jeremy D; Dow-Edwards, Diana; Fenton, André A

2009-04-10

320

Plant Formate Dehydrogenase  

SciTech Connect

The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

John Markwell

2005-01-10

321

Lactate-induced release of GABA in the ventromedial hypothalamus contributes to counterregulatory failure in recurrent hypoglycemia and diabetes.  

PubMed

Suppression of GABAergic neurotransmission in the ventromedial hypothalamus (VMH) is crucial for full activation of counterregulatory responses to hypoglycemia, and increased ?-aminobutyric acid (GABA) output contributes to counterregulatory failure in recurrently hypoglycemic (RH) and diabetic rats. The goal of this study was to establish whether lactate contributes to raising VMH GABA levels in these two conditions. We used microdialysis to deliver artificial extracellular fluid or L-lactate into the VMH and sample for GABA. We then microinjected a GABAA receptor antagonist, an inhibitor of lactate transport (4CIN), or an inhibitor of lactate dehydrogenase, oxamate (OX), into the VMH prior to inducing hypoglycemia. To assess whether lactate contributes to raising GABA in RH and diabetes, we injected 4CIN or OX into the VMH of RH and diabetic rats before inducing hypoglycemia. L-lactate raised VMH GABA levels and suppressed counterregulatory responses to hypoglycemia. While blocking GABAA receptors did not prevent the lactate-induced rise in GABA, inhibition of lactate transport or utilization did, despite the presence of lactate. All three treatments restored the counterregulatory responses, suggesting that lactate suppresses these responses by enhancing GABA release. Both RH and diabetic rats had higher baseline GABA levels and were unable to reduce GABA levels sufficiently to fully activate counterregulatory responses during hypoglycemia. 4CIN or OX lowered VMH GABA levels in both RH and diabetic rats and restored the counterregulatory responses. Lactate likely contributes to counterregulatory failure in RH and diabetes by increasing VMH GABA levels. PMID:23939392

Chan, Owen; Paranjape, Sachin A; Horblitt, Adam; Zhu, Wanling; Sherwin, Robert S

2013-12-01

322

2-Acetylthiamin pyrophosphate (acetyl-TPP) pH-rate profile for hydrolysis of acetyl-TPP and isolation of acetyl-TPP as a transient species in pyruvate dehydrogenase catalyzed reactions  

SciTech Connect

Rate constants for the hydrolysis of acetyl-TPP were measured pH values of 2.5 and 7.5 and plotted as log k{sub obs} versus pH. The pH-rate profile defined two legs, each with a slope of +1 but separated by a region of decreased slope between pH 4 and pH 6. The rates were insensitive to buffer concentrations. Each leg of the profile reflected specific-base-catalyzed hydrolysis of acetyl-TPP, analogous to the hydrolysis of 2-acetyl-3,4-dimethylthiazolium ion. The separation of the two legs of this profile has been shown to be caused by the ionization of a group exhibiting a pK{sub a} of 4.73 within acetyl-TPP that is remote from the acetyl group, the aminopyrimidine ring, which is promoted below pH 4.73. The protonation level of this ring has been shown to control the equilibrium partitioning of acetyl-TPP among its carbinolamine, keto, and hydrate forms. The differential partitioning of these species is a major factor causing the separation between the two legs of the pH-rate profile. The characteristic pH-rate profile and the availability of synthetic acetyl-TPP have facilitated the isolation and identification of (1-{sup 14}C)acetyl-TPP from acid-quenched enymatic reaction mixtures at steady states. (1-{sup 14}C)Acetyl-TPP was identified as a transient species in reactions catalyzed by the PDH complex or the pyruvate dehydrogenase component of the complex (E{sub 1}). The pH-rate profile for hydrolysis of (1-{sup 14}C)-acetyl-TPP, isolated from enzymatic reactions was found to be indistinguishable from that for authentic acetyl-TPP, which constituted positive identification of the {sup 14}C-labeled enzymic species.

Gruys, K.J.; Datta, A.; Frey, P.A. (Univ. of Wisconsin, Madison (USA))

1989-11-14

323

The Essential Function of Genes for a Hydratase and an Aldehyde Dehydrogenase for Growth of Pseudomonas sp. Strain Chol1 with the Steroid Compound Cholate Indicates an Aldolytic Reaction Step for Deacetylation of the Side Chain  

PubMed Central

In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C5 acyl side chain of the C24 steroid compound cholate involves the C22 intermediate 7?,12?-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C24 steroid (22E)-7?,12?-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7?,12?,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7?,12?-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD+ as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage.

Holert, Johannes; Jagmann, Nina

2013-01-01

324

The essential function of genes for a hydratase and an aldehyde dehydrogenase for growth of Pseudomonas sp. strain Chol1 with the steroid compound cholate indicates an aldolytic reaction step for deacetylation of the side chain.  

PubMed

In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C5 acyl side chain of the C24 steroid compound cholate involves the C22 intermediate 7?,12?-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C24 steroid (22E)-7?,12?-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7?,12?,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7?,12?-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD(+) as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage. PMID:23708132

Holert, Johannes; Jagmann, Nina; Philipp, Bodo

2013-08-01

325

Isolation and properties of pyruvate dehydrogenase complex mutants of Pseudomonas aeruginosa PAO.  

PubMed

Four independent ace mutants of Pseudomonas aeruginosa PAO lacking the activity of the pyruvate dehydrogenase complex have been isolated. They resembled ace mutants of Escherichia coli and Salmonella typhimurium in requiring acetate as an essential supplement for aerobic growth on glucose, succinate or lactate and in their ability to utilize acetate as sole carbon and energy source. Assays for the individual components of the pyruvate dehydrogenase complex indicated that they lacked the pyruvate dehydrogenase component (El) or the pyruvate dehydrogenase and dihydrolipoamide acetyltransferase components (E1 and E2) but not the lipoamide dehydrogenase component (E3). Genetic studies with plasmid R68.45-mediated conjugation and phage F116L-mediated transduction indicated that the ace mutations are located at approximately 15 min in the P. aeruginosa PAO linkage map. PMID:6785385

Jeyaseelan, K; Guest, J R

1980-10-01

326

L-lactate transport in Ehrlich ascites-tumour cells.  

PubMed Central

Ehrlich ascites-tumour cells were investigated with regard to their stability to transport L-lactate by measuring either the distribution of [14C]lactate or concomitant H+ ion movements. The movement of lactate was dependent on the pH difference across the cell membrane and was electroneutral, as evidenced by an observed 1:1 antiport for OH- ions or 1:1 symport with H+ ions. 2. Kinetic experiments showed that lactate transport was saturable, with an apparent Km of approx. 4.68 mM and a Vmax. as high as 680 nmol/min per mg of protein at pH 6.2 and 37 degrees C. 3. Lactate transport exhibited a high temperature dependence (activation energy = 139 kJ/mol). 4. Lactate transport was inhibited competitively by (a) a variety of other substituted monocarboxylic acids (e.g. pyruvate, Ki = 6.3 mM), which were themselves transported, (b) the non-transportable analogues alpha-cyano-4-hydroxycinnamate (Ki = 0.5 mM), alpha-cyano-3-hydroxycinnamate (Ki = 2mM) and DL-p-hydroxyphenyl-lactate (Ki = 3.6 mM) and (c) the thiol-group reagent mersalyl (Ki = 125 muM). 5. Transport of simple monocarboxylic acids, including acetate and propionate, was insensitive to these inhibitors; they presumably cross the membrane by means of a different mechanism. 6. Experiments using saturating amounts of mersalyl as an "inhibitor stop" allowed measurements of the initial rates of net influx and of net efflux of [14C]lactate. Influx and efflux of lactate were judged to be symmetrical reactions in that they exhibited similar concentration dependence. 7. It is concluded that lactate transport in Ehrlich ascites-tumour cells is mediated by a carrier capable of transporting a number of other substituted monocarboxylic acids, but not unsubstituted short-chain aliphatic acids.

Spencer, T L; Lehninger, A L

1976-01-01

327

Nondecarboxylating and Decarboxylating Isocitrate Dehydrogenases: Oxalosuccinate Reductase as an Ancestral Form of Isocitrate Dehydrogenase  

Microsoft Academic Search

Isocitrate dehydrogenase (ICDH) from Hydrogenobacter thermophilus catalyzes the reduction of oxalosucci- nate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. In this study, the oxidation reaction catalyzed by H. thermophilus ICDH was kinetically analyzed. As a result, a rapid equilibrium random-order mechanism was suggested. The affinities of both substrates (isocitrate

Miho Aoshima; Yasuo Igarashi

2008-01-01

328

Glucose-6-phosphate dehydrogenase deficiency  

MedlinePLUS

Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency is a hereditary condition in which red blood cells ... doesn't have enough of an enzyme called glucose-6-phosphate dehydrogenase, which helps red blood cells ...

329

Concomitant administration of sodium dichloroacetate and thiamine in West syndrome caused by thiamine-responsive pyruvate dehydrogenase complex deficiency  

Microsoft Academic Search

We treated a female patient with West syndrome caused by thiamine-responsive pyruvate dehydrogenase complex (PDHC) deficiency. Infantile spasms occurred in association with elevated blood and CSF lactate concentrations; these symptoms disappeared when lactate concentrations had been lowered by treatment with concomitant sodium dichloroacetate (DCA) and high dose thiamine. Sequencing the patient’s PDHC E1? subunit revealed a substitution of serine for

Etsuo Naito; Michinori Ito; Ichiro Yokota; Takahiko Saijo; Shuli Chen; Mitsuo Maehara; Yasuhiro Kuroda

1999-01-01

330

L-lactate production from seaweed hydrolysate of Laminaria japonica using metabolically engineered Escherichia coli.  

PubMed

Renewable and carbon neutral, marine algal biomass could be an attractive alternative substrate for the production of biofuel and various biorefinery products. Thus, the feasibility of brown seaweed (Laminaria japonica) hydrolysate as a carbon source was investigated here for L-lactate production. This work reports the homofermentative route for L-lactate production by introducing Streptococcus bovis/equinus L-lactate dehydrogenase in an engineered Escherichia coli strain where synthesis of the competing by-product was blocked. The engineered strain utilized both glucose and mannitol present in the hydrolysate under microaerobic condition and produced 37.7 g/L of high optical purity L-lactate at 80 % of the maximum theoretical value. The result shown in this study implies that algal biomass would be as competitive with lignocellulosic biomass in terms of lactic acid production and that brown seaweed can be used as a feedstock for the industrial production of other chemicals. PMID:24297185

Mazumdar, Suman; Bang, Junho; Oh, Min-Kyu

2014-02-01

331

Structure and function of Plasmodium falciparum malate dehydrogenase: Role of critical amino acids in co-substrate binding pocket  

Microsoft Academic Search

The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal glycine motif, which forms a characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic

Anupam Pradhan; Abhai K. Tripathi; Prashant V. Desai; Prasenjit K. Mukherjee; Mitchell A. Avery; Larry A. Walker; Babu L. Tekwani

2009-01-01

332

Histidine Modifying Agents Abolish Pyruvate Dehydrogenase Kinase Activity  

Microsoft Academic Search

Pyruvate dehydrogenase kinase (PDK) specifically phosphorylates the E1? subunit of the pyruvate dehydrogenase complex (PDC). Sequence analysis of cloned PDKs led to the proposal that they are mechanistically related to prokaryotic 2-component His-kinases. The reaction mechanism of protein His-kinases involves autophosphorylation of a specific His residue followed by phosphotransfer to an Asp residue. Treatment of recombinant Arabidopsis thaliana PDK with

Brian P. Mooney; Nancy R. David; Jay J. Thelen; Jan A. Miernyk; Douglas D. Randall

2000-01-01

333

Hormonal contraception and lactation.  

PubMed

Hormonal contraceptive measures can be used immediately postpartum if the patient so desires. Progestin-only contraceptives are preferable to estrogen-containing methods if initiated during the first six months after delivery. Progestin only contraceptives do not appear to affect milk volume, composition, or to cause deleterious effects in the infant. Ideally for women who desire a form of contraception in addition to lactation-induced amenorrhea, progestin-only methods should be started at six weeks postpartum if the woman is fully breastfeeding. Since contraception protection is provided by lactation amenorrhea, the six week delay will decrease infant exposure to exogenous hormones and decrease the incidence of irregular postpartum bleeding. Milk volume may decrease with the use of estrogen; however, no detrimental effects have been shown on infant growth or development. For women who are planning to gradually wean their infant, use of COCs may provide an easier transition to bottle-feeding. COCs should be used with caution by women who are not able to obtain supplemental milk. A decrease in milk volume can lead to earlier discontinuation of the hormonal contraceptive in an attempt to increase milk quantity. Supplementation is often needed, and then the woman ovulates again, possibly resulting in an unintended pregnancy. Many women are motivated immediately postpartum to accept contraception. For other women, lack of access to health care may provide barriers in obtaining adequate contraception later. In either case, there are adequate data to show no detriments of starting progestin-only contraceptives within days of delivery. Therefore, the best method for the patient should be employed to ensure adequate contraception while preserving optimal lactation. PMID:9025449

Kelsey, J J

1996-12-01

334

Evidence that adrenal hexose-6-phosphate dehydrogenase can effect microsomal P450 cytochrome steroidogenic enzymes  

PubMed Central

The role of adrenal hexose-6-phosphate dehydrogenase in providing reducing equivalents to P450 cytochrome steroidogenic enzymes in the endoplasmic reticulum is uncertain. Hexose-6-phosphate dehydrogenase resides in the endoplasmic reticulum lumen and co-localizes with the bidirectional enzyme 11?-hydroxysteroid dehydrogenase 1. Hexose-6-phosphate dehydrogenase likely provides 11?-hydroxysteroid dehydrogenase 1 with NADPH electrons via channeling. Intracellularly, two compartmentalized reactions generate NADPH upon oxidation of glucose-6-phosphate: cytosolic glucose-6-phosphate dehydrogenase and microsomal hexose-6-phosphate dehydrogenase. Because some endoplasmic reticulum enzymes require an electron donor (NADPH), it is conceivable that hexose-6-phosphate dehydrogenase serves in this capacity for these pathways. Besides 11?-hydroxysteroid dehydrogenase 1, we examined whether hexose-6-phosphate dehydrogenase generates reduced pyridine nucleotide for pivotal adrenal microsomal P450 enzymes. 21-hydroxylase activity was increased with glucose-6-phosphate and, also, glucose and glucosamine-6-phosphate. The latter two substrates are only metabolized by hexose-6-phosphate dehydrogenase, indicating that requisite NADPH for 21-hydroxylase activity was not via glucose-6-phosphate dehydrogenase. Moreover, dihydroepiandrostenedione, a non-competitive inhibitor of glucose-6-phosphate dehydrogenase, but not hexose-6-phosphate dehydrogenase, did not curtail activation by glucose-6-phosphate. Finally, the most compelling observation was that the microsomal glucose-6-phosphate transport inhibitor, chlorogenic acid, blunted the activation by glucose-6-phosphate of both 21-hydroxylase and 17-hydroxylase indicating that luminal hexose-6-phosphate dehydrogenase can supply NADPH for these enzymes. Analogous kinetic observations were found with microsomal 17-hydroxylase. These findings indicate that hexose-6-phosphate dehydrogenase can be a source, but not exclusively so, of NADPH for several adrenal P450 enzymes in the steroid pathway. Although the reduced pyridine nucleotides are produced intra-luminally, these compounds may also slowly transverse the endoplasmic reticulum membrane by unknown mechanisms.

Foster, Christy A.; Mick, Gail J.; Wang, Xudong; McCormick, Kenneth

2014-01-01

335

Pyruvate Dehydrogenase Complex: Metabolic Link to Ischemic Brain Injury and Target of Oxidative Stress  

PubMed Central

The mammalian pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme complex (greater than 7 million Daltons) that catalyzes the oxidative decarboxylation of pyruvate to form acetyl CoA, nicotinamide adenine dinucleotide (the reduced form, NADH), and CO2. This reaction constitutes the bridge between anaerobic and aerobic cerebral energy metabolism. PDHC enzyme activity and immunoreactivity are lost in selectively vulnerable neurons after cerebral ischemia and reperfusion. Evidence from experiments carried out in vitro suggests that reperfusion-dependent loss of activity is caused by oxidative protein modifications. Impaired enzyme activity may explain the reduced cerebral glucose and oxygen consumption that occurs after cerebral ischemia. This hypothesis is supported by the hyperoxidation of mitochondrial electron transport chain components and NAD(H) that occurs during reperfusion, indicating that NADH production, rather than utilization, is rate limiting. Additional support comes from the findings that immediate postischemic administration of acetyl-l-carnitine both reduces brain lactate/pyruvate ratios and improves neurologic outcome after cardiac arrest in animals. As acetyl-l-carnitine is converted to acetyl CoA, the product of the PDHC reaction, it follows that impaired production of NADH is due to reduced activity of either PDHC or one or more steps in glycolysis. Impaired cerebral energy metabolism and PDHC activity are associated also with neurodegenerative disorders including Alzheimer's disease and Wernicke-Korsakoff syndrome, suggesting that this enzyme is an important link in the pathophysiology of both acute brain injury and chronic neurodegeneration.

Martin, Erica; Rosenthal, Robert E.; Fiskum, Gary

2008-01-01

336

Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution: chemometrics and experimental models.  

PubMed

Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution (SDESA) is proposed. SDESA requires the following: (a) Enzyme A acts on Substrate A to release Product A bearing the longest difference absorbance peak (?(A)) much larger than that of Product B (?(B)) formed by Enzyme B action on Substrate B; ?(B) is close to the longest isoabsorbance wavelength of Product A and Substrate A (?(0)); (b) absorbance at ?(A) and ?(0) is quantified via swift alternation of detection wavelengths and corrected on the basis of absorbance additivity; (c) inhibition/activation on either enzyme by any substance is eliminated; (d) Enzyme A is quantified via an integration strategy if levels of Substrate A are lower than the Michaelis constant. Chemometrics of SDESA was tested with ?-glutamyltransferase and lactate-dehydrogenase of complicated kinetics. ?-Glutamyltransferase releases p-nitroaniline from ?-glutamyl-p-nitroaniline with ?(0) at 344 nm and ?(A) close to 405 nm, lactate-dehydrogenase consumes reduced nicotinamide dinucleotide bearing ?(B) at 340 nm. Kinetic analysis of reaction curve yielded lactate-dehydrogenase activity free from inhibition by p-nitroaniline; the linear range of initial rates of ?-glutamyltransferase via the integration strategy, and that of lactate-dehydrogenase after interference elimination, was comparable to those by separate assays, respectively; the quantification limit of either enzyme by SDESA at 25-fold higher activity of the other enzyme remained comparable to that by a separate assay. To test potential application, SDESA of alkaline phosphatase (ALP) and ?-D-galactosidase as enzyme-linked-immunoabsorbent assay (ELISA) labels were examined. ALP releases 4-nitro-1-naphthol from 4-nitronaphthyl-1-phosphate with ?(0) at 405 nm and ?(A) at 458 nm, ?-D-galactosidase releases 4-nitrophenol from ?-D-(4-nitrophenyl)-galactoside with ?(B) at 405 nm. No interference from substrates/products made SDESA of ?-galactosidase and ALP simple for ELISA of penicillin G and clenbuterol in one well, and the quantification limit of either hapten was comparable to that via a separate assay. Hence, SDESA is promising. PMID:23305208

Liu, Hongbo; Yang, Xiaolan; Liu, Lin; Dang, Jizheng; Xie, Yanling; Zhang, Yi; Pu, Jun; Long, Gaobo; Li, Yuanli; Yuan, Yonghua; Liao, Juan; Liao, Fei

2013-02-19

337

Characterization of lactate utilization and its implication on the physiology of Haemophilus influenzae  

PubMed Central

Haemophilus influenzae is a Gram-negative bacillus and a frequent commensal of the human nasopharynx. Earlier work demonstrated that in H. influenzae type b, l-lactate metabolism is associated with serum resistance and in vivo survival of the organism. To further gain insight into lactate utilization of the non-typeable (NTHi) isolate 2019 and laboratory prototype strain Rd KW20, deletion mutants of the l-lactate dehydrogenase (lctD) and permease (lctP) were generated and characterized. It is shown, that the apparent KM of l-lactate uptake is 20.1 ?M as determined for strain Rd KW20. Comparison of the COPD isolate NTHi 2019-R with the corresponding lctP knockout strain for survival in human serum revealed no lactate dependent serum resistance. In contrast, we observed a 4-fold attenuation of the mutant strain in a murine model of nasopharyngeal colonization. Characterization of lctP transcriptional control shows that the lactate utilization system in H. influenzae is not an inductor inducible system. Rather negative feedback regulation was observed in the presence of l-lactate and this is dependent on the ArcAB regulatory system. Additionally, for 2019 it was found that lactate may have signaling function leading to increased cell growth in late log phase under conditions where no l-lactate is metabolized. This effect seems to be ArcA independent and was not observed in strain Rd KW20. We conclude that l-lactate is an important carbon-source and may act as host specific signal substrate which fine tunes the globally acting ArcAB regulon and may additionally affect a yet unknown signaling system and thus may contribute to enhanced in vivo survival.

Lichtenegger, Sabine; Bina, Isabelle; Roier, Sandro; Bauernfeind, Stilla; Keidel, Kristina; Schild, Stefan; Anthony, Mark; Reidl, Joachim

2014-01-01

338

Mammalian alpha-Keto Acid Dehydrogenase Complexes. Iv. Substrate Specificities and Kinetic Properties of the Pig Heart Pyruvate and 2-Oxoglutarate Dehydrogenase Complexes (U).  

National Technical Information Service (NTIS)

It was found that the pig heart pyruvate dehydrogenase complex is capable of oxidatively decarboxylating alpha-keto-butyrate at about the rate of 62% of that of pyruvate in the over-all reaction assay and about 79% in the dehydrogenase assay. The complex ...

T. Kanzaki T. Hayakawa M. Hamada Y. Fukuyoshi M. Koike

1968-01-01

339

Central lactate metabolism suppresses food intake via the hypothalamic AMP kinase/malonyl-CoA signaling pathway.  

PubMed

Previous studies showed that centrally administered glucose and fructose exert different effects on food intake--glucose decreasing and fructose increasing food intake. Because of the uncertainty of whether fructose can cross the blood-brain-barrier, the question is raised; can dietary fructose directly enter the CNS? Evidence is presented that fructose administered by intraperitoneal (ip) injection to mice is rapidly (<10 min) converted to lactate in the hypothalamus. Thus, fructose can cross the blood-brain-barrier to enter the CNS/hypothalamus for conversion to lactate without prior (slower) conversion to glucose in the liver. Fructose-derived hypothalamic lactate is not, however, responsible for the orexigenic effect of fructose. Ip lactate administered at a level equivalent to that of fructose generates a higher level of hypothalamic lactate, which rapidly triggers dephosphorylation/inactivation of AMP-kinase. Thereby, ACC--a substrate of AMP-kinase that catalyzes malonyl-CoA formation--is dephosphorylated and activated. Consistent with these findings, ip or centrally (icv) administered lactate rapidly increases (<10 min) hypothalamic malonyl-CoA. Increasing hypothalamic malonyl-CoA suppresses the expression of the orexigenic and increases the expression of the anorexigenic neuropeptides, which decrease food intake. All downstream effects of hypothalamic lactate are blocked by icv administered oxamate, a potent inhibitor of lactate dehydrogenase, thus verifying the central action of lactate. PMID:19523445

Cha, Seung Hun; Lane, M Daniel

2009-08-14

340

Mammary Candidosis in Lactating Women  

Microsoft Academic Search

Though perceived to be a growing problem by lactation professionals, fungal infection of the breast (mammary candidosis) is largely unstudied. Candida albicans, a commensal organism encountered frequently in the vagina and gastrointestinal tract of humans, has been reported to be responsible for both superficial (cutaneous) and localized (ductal) infection of the mammary gland in lactating women, though the latter association

M. Jane Heinig; Jimi Francis; Demosthenes Pappagianis

1999-01-01

341

INTERPRETATION OF THE LACTATION CURVE  

PubMed Central

The validity of the assumption of a substance determining the rate of milk secretion and undergoing monomolecular destruction, based on group behavior, is questioned on the evidence from a large number of individual lactation curves. It seems probable that the rate of decrease in the rate of milk secretion with advance in lactation is dependent upon factors of a nutritional nature.

Gaines, W. L.

1926-01-01

342

Crystal Structure of a Novel Shikimate Dehydrogenase from Haemophilus influenzae*  

PubMed Central

To date two classes of shikimate dehydrogenases have been identified and characterized, YdiB and AroE. YdiB is a bifunctional enzyme that catalyzes the reversible reductions of dehydroquinate to quinate and dehydroshikimate to shikimate in the presence of either NADH or NADPH. In contrast, AroE catalyzes the reversible reduction of dehydroshikimate to shikimate in the presence of NADPH. Here we report the crystal structure and biochemical characterization of HI0607, a novel class of shikimate dehydrogenase annotated as shikimate dehydrogenase-like. The kinetic properties of HI0607 are remarkably different from those of AroE and YdiB. In comparison with YdiB, HI0607 catalyzes the oxidation of shikimate but not quinate. The turnover rate for the oxidation of shikimate is ~1000-fold lower compared with that of AroE. Phylogenetic analysis reveals three independent clusters representing three classes of shikimate dehydrogenases, namely AroE, YdiB, and this newly characterized shikimate dehydrogenase-like protein. In addition, mutagenesis studies of two invariant residues, Asp-103 and Lys-67, indicate that they are important catalytic groups that may function as a catalytic pair in the shikimate dehydrogenase reaction. This is the first study that describes the crystal structure as well as mutagenesis and mechanistic analysis of this new class of shikimate dehydrogenase.

Singh, Sasha; Korolev, Sergey; Koroleva, Olga; Zarembinski, Thomas; Collart, Frank; Joachimiak, Andrzej; Christendat, Dinesh

2009-01-01

343

Elevated plasma citrulline: look for dihydrolipoamide dehydrogenase deficiency.  

PubMed

The E3 subunit of the pyruvate dehydrogenase complex (dihydrolipoamide dehydrogenase/dihydrolipoyl dehydrogenase/DLD/lipoamide dehydrogenase/LAD), is a mitochondrial matrix enzyme and also a part of the branched-chain ketoacid dehydrogenase and alpha-ketoglutarate dehydrogenase complexes. DLD deficiency (MIM #246900), is relatively frequent in the Ashkenazi Jewish population but occurs in other populations as well. Early diagnosis is important to prevent episodes of metabolic decompensation, liver failure, and encephalopathy. The clinical presentations are varied and may include Reye-like syndrome, hepatic failure, myopathy, and myoglobinuria. Laboratory markers, such as elevated urinary alpha-ketoglutarate, blood pyruvate, lactate, and ammonia, are mostly nonspecific and not always present, making the diagnosis difficult. Since we observed elevated plasma citrulline levels in a number of confirmed cases, we retrospectively examined the value of citrulline as a biochemical marker for DLD deficiency. Data was gathered from the files of 17 pediatric patients with DLD deficiency, confirmed by enzymatic and genetic analysis. The control group included 19 patients in whom urea cycle defects were ruled out but DLD deficiency was suspected. Seven of the DLD-deficient patients presented with elevated plasma citrulline levels (median value 205 ?M, range 59-282 ?M) (normal range 1-45 ?M) while none in the control patient group. In five patients, elevated citrulline was associated with elevated plasma glutamine and metabolic acidosis. Interestingly, elevated plasma citrulline was associated with the common G229C mutation. In conclusion, we suggest that elevated plasma citrulline in the absence of urea cycle defects warrants an investigation for DLD deficiency. PMID:23995961

Haviv, Ruby; Zeharia, Avraham; Belaiche, Corinne; Haimi Cohen, Yishai; Saada, Ann

2014-02-01

344

Breast abscesses in Nigeria: lactational versus non-lactational.  

PubMed

This review of 299 cases of breast abscesses seen over a 10-year period (1981-1990) at the University of Calabar Teaching Hospital in Nigeria seeks to establish the current status of breast abscesses in the tropics. Lactational breast abscess constitutes 95% of breast abscesses while non-lactational breast abscess constitutes only 5% in this review. The commonest pathogen cultured from lactational breast abscess is Staphylococcus aureus and the disease responds to incision and drainage and systemic antibiotics, while non-lactational breast abscess is caused mostly by anaerobic organisms, usually with underlying mammary duct ectasia. The low incidence of non-lactational breast abscess corresponds to the low incidence of cigarette smoking and mammary duct ectasia in Nigerian women. While the high incidence of lactational breast abscess corresponds to the high rate of breast feeding and low level of personal hygiene in the low income group Nigerian women in which the disease is commonest. Economic recession has also reduced patronage of artificial feeds thus intensifying breast feeding and consequent lactational breast abscess. PMID:7738892

Efem, S E

1995-02-01

345

Efficiency of superoxide anions in the inactivation of selected dehydrogenases  

NASA Astrophysics Data System (ADS)

The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as rad OH and ONOO -. In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH). We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion. Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.

Rodacka, Aleksandra; Serafin, Eligiusz; Puchala, Mieczyslaw

2010-09-01

346

N-Acylethanolamines as Novel Alcohol Dehydrogenase 3 Substrates  

PubMed Central

N-Acylethanolamines (NAEs) are members of the fatty acid amide family. The NAEs have been proposed to serve as metabolic precursors to N-acylglycines (NAGs). The sequential oxidation of the NAEs by an alcohol dehydrogenase and an aldehyde dehydrogenase would yield the N-acylglycinals and/or the NAGs. Alcohol dehydrogenase 3 (ADH3) is one enzyme that might catalyze this reaction. To define a potential role for ADH3 in NAE catabolism, we synthesized a set of NAEs and evaluated these as ADH3 substrates. NAEs were oxidized by ADH3, yielding the N-acylglycinals as the product. The (V/K)app values for the NAEs included here were low relative to cinnamyl alcohol. Our data show that the NAEs can serve as alcohol dehydrogenase substrates.

Ivkovic, Milena; Dempsey, Daniel R.; Handa, Sumit; Hilton, Joshua H.; Lowe, Edward W.; Merkler, David J.

2011-01-01

347

Negligible direct lactate oxidation in subsarcolemmal and intermyofibrillar mitochondria obtained from red and white rat skeletal muscle.  

PubMed

We examined the controversial notion of whether lactate is directly oxidized by subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria obtained from red and white rat skeletal muscle. Respiratory control ratios were normal in SS and IMF mitochondria. At all concentrations (0.18-10 mm), and in all mitochondria, pyruvate oxidation greatly exceeded lactate oxidation, by 31- to 186-fold. Pyruvate and lactate oxidation were inhibited by alpha-cyano-4-hydroxycinnamate, while lactate oxidation was inhibited by oxamate. Excess pyruvate (10 mm) inhibited the oxidation of palmitate (1.8 mm) as well as lactate (1.8 mm). In contrast, excess lactate (10 mm) failed to inhibit the oxidation of either palmitate (1.8 mm) or pyruvate (1.8 mm). The cell-permeant adenosine analogue, AICAR, increased pyruvate oxidation; in contrast, lactate oxidation was not altered. The monocarboxylate transporters MCT1 and 4 were present on SS mitochondria, but not on IMF mitochondria, whereas, MCT2, a high-affinity pyruvate transporter, was present in both SS and IMF mitochondria. The lactate dehydrogenase (LDH) activity associated with SS and IMF mitochondria was 200- to 240-fold lower than in whole muscle. Addition of LDH increased the rate of lactate oxidation, but not pyruvate oxidation, in a dose-dependent manner, such that lactate oxidation approached the rates of pyruvate oxidation. Collectively, these studies indicate that direct mitochondrial oxidation of lactate (i.e. an intracellular lactate shuttle) does not occur within the matrix in either IMF or SS mitochondria obtained from red or white rat skeletal muscle, because of the very limited quantity of LDH within mitochondria. PMID:17556391

Yoshida, Yuko; Holloway, Graham P; Ljubicic, Vladimir; Hatta, Hideo; Spriet, Lawrence L; Hood, David A; Bonen, Arend

2007-08-01

348

Biochemistry and biotechnology of amino acid dehydrogenases  

Microsoft Academic Search

Over the last decade, amino acid dehydrogenases such as alanine dehydrogenase (Ala DH), leucine dehydrogenase (Leu DH), and phenylalanine dehydrogenase (Phe DH) have been applied to the enantiomer-specific synthesis and analysis of various amino acids. In perticular, amino acid dehydrogenases from thermophiles have received much attention because of their high stability. Their productivity was enhanced and the purification facilitated by

Toshihisa Ohshima; Kenji Soda

349

Localization of dehydrogenases, reductases, and electron transfer components in the sulfate-reducing bacterium Desulfovibrio gigas.  

PubMed Central

Various dehydrogenases, reductases, and electron transfer proteins involved in respiratory sulfate reduction by Desulfovibrio gigas have been localized with respect to the periplasmic space, membrane, and cytoplasm. This species was grown on a lactate-sulfate medium, and the distribution of enzyme activities and concentrations of electron transfer components were determined in intact cells, cell fractions prepared with a French press, and lysozyme spheroplasts. A significant fraction of formate dehydrogenase was demonstrated to be localized in the periplasmic space in addition to hydrogenase and some c-type cytochrome. Cytochrome b, menaquinone, fumarate reductase, and nitrite reductase were largely localized on the cytoplasmic membrane. Fumarate reductase was situated on the inner aspect on the membrane, and the nitrite reductase appeared to be transmembraneous. Adenylylsulfate reductase, bisulfite reductase (desulfoviridin), pyruvate dehydrogenase, and succinate dehydrogenase activities were localized in the cytoplasm. Significant amounts of hydrogenase and c-type cytochromes were also detected in the cytoplasm. Growth of D. gigas on a formate-sulfate medium containing acetate resulted in a 10-fold increase in membrane-bound formate dehydrogenase and a doubling of c-type cytochromes. Growth on fumarate with formate resulted in an additional increase in b-type cytochrome compared with lactate-sulfate-grown cells.

Odom, J M; Peck, H D

1981-01-01

350

Der einfluß der temperatur auf thermostabilität, isoenzym-muster und reaktionskinetik der laktat-dehydrogenase aus fischen  

Microsoft Academic Search

Methodological problems complicate investigations on thermostability of lactate dehydrogenase (LDH). It is difficult to demonstrate a correlation between adaptation-temperature (AT) and LDH thermostability. Heat-inactivation characteristics change completely if diluted or undiluted tissue extracts are heated. In purified LDH (purchased from Boehringer, Mannheim, FRG), additions such as casein, bovine-serum albumin, NADH and pyruvate — even in small concentrations — can alter

H. Kfinnemann

1973-01-01

351

Modifying the lactation curve to improve lactation milk and persistency.  

PubMed

Daily, stage and lactation estimated breeding values (EBV) and the shape of the lactation curve for each cow are controlled by a unique set of random (genetic) regression coefficients under a test day model, thus providing a basis for genetic improvement of these characteristics. Three selection procedures were developed for simultaneous improvement of total lactation milk and persistency: 1) index selection based on daily EBV, 2) index selection based on stage EBV, and 3) index selection based on random regression (RR) coefficients. A numerical example was given to demonstrate the computation of indexes based on stage EBV and based on RR coefficients. A conversion equation was derived to convert between genetic changes in EBV and RR coefficients. Index selection based on daily EBV would require the finding of 305 weighting factors for a lactation period of 305 d, making it impractical to determine the weighting factors on a daily basis. Alternatively, a lactation period was partitioned into a few stages to facilitate the construction of index selection based on stage EBV and index selection based on RR coefficients. These selection procedures make use of the annual genetic gains routinely computed in national genetic evaluations to restrict the genetic gains between different lactation stages to achieve the desired curve. When there is no prior knowledge of annual genetic gains, the proportional restriction of genetic gains between stages may be used. In summary, this study provides a simple means of modifying the lactation curve by manipulating genetic changes in different lactation stages at a pre-specified rate. PMID:12741575

Togashi, K; Lin, C Y

2003-04-01

352

Cold stress induces switchover of respiratory pathway to lactate glycolysis in psychrotrophic Rhizobium strains  

Microsoft Academic Search

Two psychrotrophic strains ofRhizobium, DDSS69, a non-cold acclimated strain, and ATRI, a cold acclimated strain, were subjected to cold stress. A 4-fold increase\\u000a in the specific activity of lactate dehydrogenase (LDH) was characteristic for cold stressed cells of DDSS69, whereas ATR1\\u000a showed a higher LDH activity in general, which increased 1.5-fold under cold stress. Cold sensitive mutants of DDSS69 which

N. Sardesai; C. R. Babu

2000-01-01

353

A europium luminescence assay of lactate and citrate in biological fluids  

Microsoft Academic Search

Lactate and citrate are essential oxy-anions in nature. Their determination is typically based on multi-component enzymatic methods of analysis, using lactic acid dehydrogenase (LDH) or citrate lyase, 1 most commonly linked to absorption spectropho- tometric analysis of NAD + at 340 nm. The limit of sensitivity of the spectrophotometric assay is about 0.1 mM; and therefore requires a relatively high

Robert Pal; David Parker; Leslie C. Costellob

2009-01-01

354

Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit, Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters  

PubMed Central

Background/Aims The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium. Methodology and Principal Findings Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization. Conclusions This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the observations made in the IVF studies in hamsters suggest that capacitation failures could be a plausible cause of unsuccessful fertilization encountered during human assisted reproductive technologies, like IVF and ICSI. Our studies indicate a role of sperm capacitation in the post-penetration events during fertilization.

Sailasree, Purnima; Singh, Durgesh K.; Kameshwari, Duvurri B.; Shivaji, Sisinthy

2014-01-01

355

Induced lactation in adoptive mothers.  

PubMed

The results of a pilot study of 6 women who undertook breast feeding their adopted infants are discussed, with a review of methods available for inducing lactation if this form of infant feeding is undertaken. It is concluded that whilst induced lactation can be successful for the mother and baby, further studies are necessary before conclusive advice can be given that this technique is effective and safe. PMID:6598379

Thearle, M J; Weissenberger, R

1984-11-01

356

Cellobiose Dehydrogenase, an Active Agent in Cellulose Depolymerization  

PubMed Central

The ability of cellobiose dehydrogenase purified from Phanerochaete chrysosporium to modify a Douglas fir kraft pulp was assessed. Although the addition of cellobiose dehydrogenase alone had little effect, supplementation with cellobiose and iron resulted in a substantial reduction in the degree of polymerization of the pulp cellulose. When the reaction was monitored over time, a progressive depolymerization of the cellulose was apparent with the concomitant production of cellobiono-1,5-lactone. Analysis of the reaction filtrates indicated that glucose and arabinose were the only neutral sugars generated. These sugars are derived from the degradation of the cellobiose rather than resulting from modifications of the pulp. These results suggest that the action of cellobiose dehydrogenase results in the generation of hydroxyl radicals via Fenton's chemistry which subsequently results in the depolymerization of cellulose. This appears to be the mechanism whereby a substantial reduction in the degree of polymerization of the cellulose can be achieved without a significant release of sugar.

Mansfield, S. D.; De Jong, E.; Saddler, J. N.

1997-01-01

357

Substrate-dependent proton load and recovery of stunned hearts during pyruvate dehydrogenase stimulation.  

PubMed

Stimulation of pyruvate dehydrogenase (PDH) improves functional recovery of postischemic hearts. This study examined the potential for a mechanism mediated by substrate-dependent proton production and intracellular pH. After 20 min of ischemia, isolated rabbit hearts were reperfused with or without 5 mM dichloroacetate (DCA) in the presence of either 5 mM glucose, 5 mM glucose + 2.5 mM lactate, or 5 mM glucose + 2.5 mM pyruvate. DCA inhibits PDH kinase, increasing the proportion of dephosphorylated, active PDH. Unlike pyruvate or glucose alone, lactate + glucose did not support the effects of DCA on the recovery of rate-pressure product (RPP) (without DCA, RPP = 14,000 +/- 1,200, n = 6; with DCA, RPP = 13,700 +/- 1,800, n = 9). Intracellular pH, from (31)P nuclear magnetic resonance spectra, returned to normal within 2.1 min of reperfusion with all substrates except for lactate + glucose + DCA or lactate + DCA, which delayed pH recovery for up to 12 min (at 2.1 min pH = 6. 00 +/- 0.08, lactate + glucose + DCA; pH = 6.27 +/- 0.34, for lactate + DCA). Hearts were also reperfused after 10 min of ischemia with 0.5 mM palmitate + 5 mM DCA and either 2.5 mM pyruvate or 2.5 mM lactate. Again, intracellular pH recovery was delayed in the presence of lactate. PDH activation in the presence of lactate also decreased coupling of oxidative metabolism to mechanical work. These findings have implications for therapeutic use of stimulated carbohydrate oxidation in stunned hearts. PMID:10899076

Griffin, J L; White, L T; Lewandowski, E D

2000-07-01

358

Depletion of lactate by dichloroacetate reduces cardiac efficiency after hemorrhagic shock.  

PubMed

We have demonstrated previously that dichloroacetate (DCA) treatment in rodents ameliorates, via activation of the pyruvate dehydrogenase complex, the cardiovascular depression observed after hemorrhagic shock. To explore the mechanism of this effect, we administered DCA in a large animal model of hemorrhagic shock. Mongrel hounds were anesthetized with 1.5% isoflurane and were measured for hemodynamics, myocardial contractility, and myocardial substrate utilization. They were hemorrhaged to a mean arterial pressure of 35 mm Hg for 90 min or until arterial lactate levels reached 7.0 mM (1137 +/- 47 mL or 49 +/- 2% total blood volume). Animals were chosen at random to receive DCA dissolved in water or an equal volume of saline at the onset of resuscitation. Two-thirds of the shed blood volume was returned immediately after giving an equivalent volume of saline. Two hours after the onset of resuscitation, mean arterial pressure was not different between DCA and control groups (79 +/- 3 vs. 82 +/- 3 mm Hg, respectively). Arterial lactate levels were significantly reduced by DCA (0.5 +/- 0.06 vs. 2.0 +/- 0.2 mM). However, DCA treatment was associated with a decreased stroke volume index (0.56 +/- 0.06 vs. 0.82 +/- 0.08 mL/kg/beat) and a decreased myocardial efficiency (19 vs. 41 L x mm Hg/mL/100 g tissue). During resuscitation by DCA, myocardial lactate consumption was reduced (21.4 +/- 3.7 vs. 70.7 +/- 16.3 micromole/min/100 g tissue) despite a three-fold increase in myocardial pyruvate dehydrogenase activity, while free fatty acid levels actually began to rise. Although increased lactate oxidation should be beneficial during resuscitation, we propose that DCA treatment led to a deprivation of myocardial lactate supply, which reduced net myocardial lactate oxidation, thus compromising myocardial function during resuscitation from hemorrhagic shock. PMID:10947168

Barbee, R W; Kline, J A; Watts, J A

2000-08-01

359

Stereochemistry and mechanism of reactions catalyzed by tryptophanase Escherichia coli.  

PubMed

Several beta replacement and alpha,beta elimination reactions catalyzed by tryptophanase from Escherichia coli are shown to proceed stereospecifically with retention of configuration. These conversions include synthesis of tryptophan from (2S,3R)- and (2s,3s)-[3(-3H)]serine in the presence of indole, deamination of these serines in D2O to pyruvate and ammonia, and cleavage of (2S,3R)-and (2S,3S)-[3(-3H)]tryptophan in D2O to indole, pyruvate, and ammonia. A coupled reaction with lactate dehydrogenase was used to trap the stereospecifically labeled [3-H,2H,3H]pryuvates as lactate, which was oxidized to acetate for chirality analysis of the methyl group. During deamination of tryptophan there is significant intramolecular transfer of the alpha proton of the amino acid to C-3 of indole. To determine the exposed face of the cofactor.substrate complex on the enzyme surface and to analyze its conformational orientation, sodium boro[3H]hydride was used to reduce tryptophanase-bound alaninepyridoxal phosphate Schiff's base. Degradation of the resulting pyridoxylalanine to (2S)-[2(-3H)]alanine and (4'S)-[4'(-3H)]pyridoxamine demonstrates that reduction occurs from the exposed si face at C-4' of the complex and that the ketimine double bond is trans. PMID:353050

Vederas, J C; Schleicher, E; Tsai, M D; Floss, H G

1978-08-10

360

Suppression of NDA-type alternative mitochondrial NAD(P)H dehydrogenases in arabidopsis thaliana modifies growth and metabolism, but not high light stimulation of mitochondrial electron transport.  

PubMed

The plant respiratory chain contains several pathways which bypass the energy-conserving electron transport complexes I, III and IV. These energy bypasses, including type II NAD(P)H dehydrogenases and the alternative oxidase (AOX), may have a role in redox stabilization and regulation, but current evidence is inconclusive. Using RNA interference, we generated Arabidopsis thaliana plants simultaneously suppressing the type II NAD(P)H dehydrogenase genes NDA1 and NDA2. Leaf mitochondria contained substantially reduced levels of both proteins. In sterile culture in the light, the transgenic lines displayed a slow growth phenotype, which was more severe when the complex I inhibitor rotenone was present. Slower growth was also observed in soil. In rosette leaves, a higher NAD(P)H/NAD(P)? ratio and elevated levels of lactate relative to sugars and citric acid cycle metabolites were observed. However, photosynthetic performance was unaffected and microarray analyses indicated few transcriptional changes. A high light treatment increased AOX1a mRNA levels, in vivo AOX and cytochrome oxidase activities, and levels of citric acid cycle intermediates and hexoses in all genotypes. However, NDA-suppressing plants deviated from the wild type merely by having higher levels of several amino acids. These results suggest that NDA suppression restricts citric acid cycle reactions, inducing a shift towards increased levels of fermentation products, but do not support a direct association between photosynthesis and NDA proteins. PMID:24486764

Wallström, Sabá V; Florez-Sarasa, Igor; Araújo, Wagner L; Escobar, Matthew A; Geisler, Daniela A; Aidemark, Mari; Lager, Ida; Fernie, Alisdair R; Ribas-Carbó, Miquel; Rasmusson, Allan G

2014-05-01

361

Skeletal muscle PGC-1? controls whole-body lactate homeostasis through estrogen-related receptor ?-dependent activation of LDH B and repression of LDH A  

PubMed Central

The peroxisome proliferator-activated receptor-? coactivator 1? (PGC-1?) controls metabolic adaptations. We now show that PGC-1? in skeletal muscle drives the expression of lactate dehydrogenase (LDH) B in an estrogen-related receptor-?–dependent manner. Concomitantly, PGC-1? reduces the expression of LDH A and one of its regulators, the transcription factor myelocytomatosis oncogene. PGC-1? thereby coordinately alters the composition of the LDH complex and prevents the increase in blood lactate during exercise. Our results show how PGC-1? actively coordinates lactate homeostasis and provide a unique molecular explanation for PGC-1?–mediated muscle adaptations to training that ultimately enhance exercise performance and improve metabolic health.

Summermatter, Serge; Santos, Gesa; Perez-Schindler, Joaquin; Handschin, Christoph

2013-01-01

362

1 Biooxidation with PQQ- and FAD-Dependent Dehydrogenases  

Microsoft Academic Search

Among the obligate aerobic bacteria, acetic acid bacteria are well known for their powerful ability to oxidize alcohols, sugars, or sugar alcohols and to accumulate the corresponding oxidation products in the culture medium. These reactions are restricted to one-step incomplete oxidation (so-called oxidative fermentation) and are catalyzed by primary dehydrogenases located on the outer surface of the cytoplasmic membrane, the

Osao Adachi; Yoshitaka Ano; Hirohide Toyama; Kazunobu Matsushita

363

Enantioselective oxidation of aldehydes catalyzed by alcohol dehydrogenase.  

PubMed

Teaching old dogs new tricks: Alcohol dehydrogenases (ADHs) may be established redox biocatalysts but they still are good for a few surprises. ADHs can be used to oxidize aldehydes, and this was demonstrated by the oxidative dynamic kinetic resolution of profens. In the presence of a suitable cofactor regeneration system, this reaction can occur with high selectivity. PMID:22936647

Könst, Paul; Merkens, Hedda; Kara, Selin; Kochius, Svenja; Vogel, Andreas; Zuhse, Ralf; Holtmann, Dirk; Arends, Isabel W C E; Hollmann, Frank

2012-09-24

364

Fecal lactate and ulcerative colitis.  

PubMed

Impaired metabolism of short-chain fatty acids, as well as a modified fecal ionogram, have been reported in ulcerative colitis. Fecal water samples from 62 patients with ulcerative colitis were analyzed in the present investigation to evaluate changes in SCFAs and lactic acid in relation to activity and severity of disease. Short-chain fatty acid levels were high in quiescent and mild disease (162.6 +/- 63.6 and 147.8 +/- 63.2 mM/L, respectively), but significantly decreased in the severe form (64.7 +/- 46.9 mM/L). Lactate showed a progressive increase from mild colitis (3.0 +/- 1.8 mM/L) to severe colitis (21.4 +/- 18.6 mM/L). It thus appears that mild colitis displayed a fecal pattern characterized by normal pH and bicarbonate, slightly impaired electrolyte handling, high short-chain fatty acid values, and only moderately increased lactate. Severe colitis, on the other hand, was characterized by low fecal pH, bicarbonate, and potassium, high sodium and chloride, low short-chain fatty acid levels, and very high lactate levels. A critical lowering of intraluminal pH, which shifts bacterial metabolism from short-chain fatty acid to lactate production, may be responsible for the intraluminal pooling of lactate. PMID:3181680

Vernia, P; Caprilli, R; Latella, G; Barbetti, F; Magliocca, F M; Cittadini, M

1988-12-01

365

Antibacterial characteristics and activity of water-soluble chitosan derivatives prepared by the Maillard reaction.  

PubMed

The antibacterial activity of water-soluble chitosan derivatives prepared by Maillard reactions against Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Escherichia coli, Shigella dysenteriae, and Salmonella typhimurium was examined. Relatively high antibacterial activity against various microorganisms was noted for the chitosan-glucosamine derivative as compared to the acid-soluble chitosan. In addition, it was found that the susceptibility of the test organisms to the water-soluble chitosan derivative was higher in deionized water than in saline solution. Metal ions were also found to reduce the antibacterial activity of the water-soluble chitosan derivative on S. aureus. The marked increase in glucose level, protein content and lactate dehydrogenase (LDH) activity was observed in the cell supernatant of S. aureus exposed to the water-soluble chitosan derivative in deionized water. The results suggest that the water-soluble chitosan produced by Maillard reaction may be a promising commercial substitute for acid-soluble chitosan. PMID:21989311

Chung, Ying-Chien; Yeh, Jan-Ying; Tsai, Cheng-Fang

2011-01-01

366

Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase.  

PubMed

Metformin is considered to be one of the most effective therapeutics for treating type 2 diabetes because it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain or posing a risk of hypoglycaemia. For over half a century, this agent has been prescribed to patients with type 2 diabetes worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production, while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide knockdown of hepatic mitochondrial glycerophosphate dehydrogenase in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decreases in plasma glucose concentrations, and inhibition of endogenous glucose production. These findings were replicated in whole-body mitochondrial glycerophosphate dehydrogenase knockout mice. These results have significant implications for understanding the mechanism of metformin's blood glucose lowering effects and provide a new therapeutic target for type 2 diabetes. PMID:24847880

Madiraju, Anila K; Erion, Derek M; Rahimi, Yasmeen; Zhang, Xian-Man; Braddock, Demetrios T; Albright, Ronald A; Prigaro, Brett J; Wood, John L; Bhanot, Sanjay; MacDonald, Michael J; Jurczak, Michael J; Camporez, Joao-Paulo; Lee, Hui-Young; Cline, Gary W; Samuel, Varman T; Kibbey, Richard G; Shulman, Gerald I

2014-06-26

367

UDP-glucose dehydrogenase from bovine liver: primary structure and relationship to other dehydrogenases.  

PubMed Central

The primary structure of bovine liver UDP-glucose dehydrogenase (UDPGDH), a hexameric, NAD(+)-linked enzyme, has been determined at the protein level. The 52-kDa subunits are composed of 468 amino acid residues, with a free N-terminus and a Ser/Asn microhetergeneity at one position. The sequence shares 29.6% positional identity with GDP-mannose dehydrogenase from Pseudomonas, confirming a similarity earlier noted between active site peptides. This degree of similarity is comparable to the 31.1% identity vs. the UDPGDH from type A Streptococcus. Database searching also revealed similarities to a hypothetical sequence from Salmonella typhimurium and to "UDP-N-acetyl-mannosaminuronic acid dehydrogenase" from Escherichia coli. Pairwise identities between bovine UDPGDH and each of these sequences were all in the range of approximately 26-34%. Multiple alignment of all 5 sequences indicates common ancestry for these 4-electron-transferring enzymes. There are 27 strictly conserved residues, including a cysteine residue at position 275, earlier identified by chemical modification as the expected catalytic residue of the second half-reaction (conversion of UDP-aldehydoglucose to UDP-glucuronic acid), and 2 lysine residues, at positions 219 and 338, one of which may be the expected catalytic residue for the first half-reaction (conversion of UDP-glucose to UDP-aldehydoglucose). A GXGXXG pattern characteristic of the coenzyme-binding fold is found at positions 11-16, close to the N-terminus as with "short-chain" alcohol dehydrogenases.(ABSTRACT TRUNCATED AT 250 WORDS)

Hempel, J.; Perozich, J.; Romovacek, H.; Hinich, A.; Kuo, I.; Feingold, D. S.

1994-01-01

368

Enzymatic in situ determination of stereospecificity of NAD-dependent dehydrogenases  

SciTech Connect

Amino acid racemases inherently catalyze the exchange of alpha-hydrogen of amino acids with deuterium during racemization in /sup 2/H/sub 2/O. When the reactions catalyzed by alanine racemase and L-alanine dehydrogenase (EC 1.4.1.1), which is pro-R specific for the C-4 hydrogen transfer of NADH, are coupled in /sup 2/H/sub 2/O, (4R-2H)NADH is exclusively produced. Similarly, (4S-2H)NADH is made in /sup 2/H/sub 2/O with amino-acid racemase with low substrate specificity and L-leucine dehydrogenase, which is pro-S specific. We have established a simple procedure for the in situ analysis of stereospecificity of C-4 hydrogen transfer of NADH by an NAD-dependent dehydrogenase by combination with either of the above two couples of enzymes in the same reaction mixture. When the C-4 hydrogen of NAD+ is fully retained after sufficient incubation, the stereospecificity of hydrogen transfer by a dehydrogenase is the same as that of alanine dehydrogenase or leucine dehydrogenase. However, when the C-4 hydrogen of NAD+ is exchanged with deuterium, the enzyme to be examined shows the different stereospecificity from alanine dehydrogenase or leucine dehydrogenase. Thus, we can readily determine the stereospecificity by /sup 1/H NMR measurement without isolation of the coenzymes and products.

Esaki, N.; Shimoi, H.; Nakajima, N.; Ohshima, T.; Tanaka, H.; Soda, K.

1989-06-15

369

Cell-cell and intracellular lactate shuttles  

PubMed Central

Once thought to be the consequence of oxygen lack in contracting skeletal muscle, the glycolytic product lactate is formed and utilized continuously in diverse cells under fully aerobic conditions. ‘Cell–cell’ and ‘intracellular lactate shuttle’ concepts describe the roles of lactate in delivery of oxidative and gluconeogenic substrates as well as in cell signalling. Examples of the cell–cell shuttles include lactate exchanges between between white-glycolytic and red-oxidative fibres within a working muscle bed, and between working skeletal muscle and heart, brain, liver and kidneys. Examples of intracellular lactate shuttles include lactate uptake by mitochondria and pyruvate for lactate exchange in peroxisomes. Lactate for pyruvate exchanges affect cell redox state, and by itself lactate is a ROS generator. In vivo, lactate is a preferred substrate and high blood lactate levels down-regulate the use of glucose and free fatty acids (FFA). As well, lactate binding may affect metabolic regulation, for instance binding to G-protein receptors in adipocytes inhibiting lipolysis, and thus decreasing plasma FFA availability. In vitro lactate accumulation upregulates expression of MCT1 and genes coding for other components of the mitochondrial reticulum in skeletal muscle. The mitochondrial reticulum in muscle and mitochondrial networks in other aerobic tissues function to establish concentration and proton gradients necessary for cells with high mitochondrial densities to oxidize lactate. The presence of lactate shuttles gives rise to the realization that glycolytic and oxidative pathways should be viewed as linked, as opposed to alternative, processes, because lactate, the product of one pathway, is the substrate for the other.

Brooks, George A

2009-01-01

370

Crystal structure of Arabidopsis thaliana cytokinin dehydrogenase  

SciTech Connect

Since first discovered in Zea mays, cytokinin dehydrogenase (CKX) genes have been identified in many plants including rice and Arabidopsis thaliana, which possesses CKX homologues (AtCKX1-AtCKX7). So far, the three-dimensional structure of only Z. mays CKX (ZmCKX1) has been determined. The crystal structures of ZmCKX1 have been solved in the native state and in complex with reaction products and a slowly reacting substrate. The structures revealed four glycosylated asparagine residues and a histidine residue covalently linked to FAD. Combined with the structural information, recent biochemical analyses of ZmCKX1 concluded that the final products of the reaction, adenine and a side chain aldehyde, are formed by nonenzymatic hydrolytic cleavage of cytokinin imine products resulting directly from CKX catalysis. Here, we report the crystal structure of AtCKX7 (gene locus At5g21482.1, UniProt code Q9FUJ1).

Bae, Euiyoung; Bingman, Craig A.; Bitto, Eduard; Aceti, David J.; Phillips, Jr., George N. (UW)

2008-08-13

371

Parity, lactation and hip fracture  

Microsoft Academic Search

The relationship between parity, lactation and the occurrence of hip fracture was investigated in a case-control study of white women. The cases were patients (n=174) aged 45 years and over with a radiologically confirmed first hip fracture sampled from among admissions to 30 hospitals in New York and Philadelphia between September 1987 and July 1989. Controls (n=174) were selected from

S. Hoffman; J. A. Grisso; J. L. Kelsey; M. D. Gammon; L. A. O'Brien

1993-01-01

372

R-lipoic acid inhibits mammalian pyruvate dehydrogenase kinase.  

PubMed

The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1>PDK4 approximately PDK2>PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects. PMID:15512796

Korotchkina, Lioubov G; Sidhu, Sukhdeep; Patel, Mulchand S

2004-10-01

373

Alcohol Dehydrogenase and Ethanol in the Stems of Trees 1  

PubMed Central

Anaerobic fermentation in plants is usually thought to be a transient phenomenon, brought about by environmental limitations to oxygen availability, or by structural constraints to oxygen transport. The vascular cambium of trees is separated from the air by the outer bark and secondary phloem, and we hypothesized that the cambium may experience sufficient hypoxia to induce anaerobic fermentation. We found high alcohol dehydrogenase activity in the cambium of several tree species. Mean activity of alcohol dehydrogenase in Populus deltoides was 165 micromoles NADH oxidized per minute per gram fresh weight in May. Pyruvate decarboxylase activity was also present in the cambium of P. deltoides, with mean activity of 26 micromoles NADH oxidized per minute per gram fresh weight in May. Lactate dehydrogenase activity was not present in any tree species we examined. Contrary to our expectation, alcohol dehydrogenase activity was inversely related to bark thickness in Acer saccharum and unrelated to bark thickness in two Populus species. Bark thickness may be less important in limiting oxygen availability to the cambium than is oxygen consumption by rapidly respiring phloem and cambium in actively growing trees. Ethanol was present in the vascular cambium of all species examined, with mean concentrations of 35 to 143 nanomoles per gram fresh weight, depending on species. Ethanol was also present in xylem sap and may have been released from the cambium into the transpiration stream. The presence in the cambium of the enzymes necessary for fermentation as well as the products of fermentation is evidence that respiration in the vascular cambium of trees may be oxygen-limited, but other biosynthetic origins of ethanol have not been ruled out.

Kimmerer, Thomas W.; Stringer, Mary A.

1988-01-01

374

The role of an iron-sulfur cluster in an enzymatic methylation reaction. Methylation of CO dehydrogenase/acetyl-CoA synthase by the methylated corrinoid iron-sulfur protein.  

PubMed

This paper focuses on how a methyl group is transferred from a methyl-cobalt(III) species on one protein (the corrinoid iron-sulfur protein (CFeSP)) to a nickel iron-sulfur cluster on another protein (carbon monoxide dehydrogenase/acetyl-CoA synthase). This is an essential step in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation. The results described here strongly indicate that transfer of methyl group to carbon monoxide dehydrogenase/acetyl-CoA synthase occurs by an SN2 pathway. They also provide convincing evidence that oxidative inactivation of Co(I) competes with methylation. Under the conditions of our anaerobic assay, Co(I) escapes from the catalytic cycle one in every 100 turnover cycles. Reductive activation of the CFeSP is required to regenerate Co(I) and recruit the protein back into the catalytic cycle. Our results strongly indicate that the [4Fe-4S] cluster of the CFeSP is required for reductive activation. They support the hypothesis that the [4Fe-4S] cluster of the CFeSP does not participate directly in the methyl transfer step but provides a conduit for electron flow from physiological reductants to the cobalt center. PMID:10206956

Menon, S; Ragsdale, S W

1999-04-23

375

Semi-quantitative RT-PCR analysis of fat metabolism genes in mammary tissue of lactating and non-lactating water buffalo (Bubalus bubalis).  

PubMed

Understanding the mechanism of milk fat synthesis and secretion is important for dairy industry, as the nature of the cream fraction influences the manufacturing properties and organoleptic qualities of milk and dairy products. So, there is a need to understand the mechanism of milk fat synthesis and to elucidate the key genes regulating milk fat synthesis by studying the expression of genes involved in milk fat synthesis. Present manuscript reports the expression of genes involved in milk fat synthesis and metabolism in buffalo mammary tissue. The expression of lipogenic genes was studied in lactating and non-lactating mammary tissue of water buffalo by semi-quantitative reverse transcription PCR expression analysis. The genes studied were acetyl-CoA carboxylase (ACACA), stearoyl-CoA desaturase (SCD), 3 hydroxybutyrate dehydrogenase (BDH), LIPIN, lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma (PPARG), and sterol regulatory element binding protein (SREBF). The expression of ACACA, BDH, LIPIN, PPARG, LPL, and SREBF was higher in lactating as compared to non-lactating buffalo whereas no difference was found in the expression of SCD between both the stages. PMID:21965031

Yadav, Poonam; Mukesh, Manishi; Kataria, Ranjit Singh; Yadav, Anita; Mohanty, Ashok Kumar; Mishra, Bishnu Prasad

2012-04-01

376

Instrumentation Applicable to Artificial Heart Development and Utilization.  

National Technical Information Service (NTIS)

A measurement of the lactate in blood has been developed using electrochemical monitoring of an enzyme reaction. The reaction is the oxidation of lactate by ferricyanide, as catalyzed by a yeast cytochrome b2 preparation containing lactate dehydrogenase. ...

D. L. Williams A. R. Doig

1969-01-01

377

Aluminum lactate – An attractive precursor for sol–gel synthesis of alumina-based glasses  

Microsoft Academic Search

A novel aluminum precursor, aluminum lactate (Al(lact)3), was used in the sol–gel synthesis of alumina-based systems. The peculiar chelation properties of aluminum lactate in aqueous solution make this precursor quite attractive for the sol–gel synthesis of alumina-containing materials. Based on the reaction of aluminum lactate with different precursors such as phosphorus, boron, fluorine and others, (Na2O–)Al2O3–P2O5, (Na2O–)Al2O3–B2O3, (Na2O–)Al2O3–B2O3–P2O5, (Na–)Al–P–O–F, amorphous

Long Zhang; Carla C. de Araujo; Hellmut Eckert

2007-01-01

378

A model for the spatial location of pyruvate dehydrogenase phosphatase in mammalian pyruvate dehydrogenase complex.  

PubMed

Recent experimental findings on the structural--functional features of pyruvate dehydrogenase phosphatase (PDP) isolated from various sources are compared. Two alternative mechanisms (a and b) of dephosphorylation of the E1 component in the pyruvate dehydrogenase complex (PDC) are discussed: a) the reaction occurs as a result of stochastic collisions of PDP and PDC, and the generation of an enzyme--substrate complex (PDP--E1--PDC) and dephosphorylation of the E1 component occur independently at different PDP binding sites on the PDC core; b) the dephosphorylation is performed simultaneously by a certain number of PDP molecules symmetrically bound on the PDC core. The second mechanism is suggested by the self-assembly theory of multicomponent enzyme systems and can be proved by kinetic experiments. Based on self-assembly principles and data on feasible binding sites of peripheral components of the PDC, the stoichiometry and mutual location of PDP, pyruvate dehydrogenase kinase, and the E1 component on the core of mammalian PDC are postulated to provide optimal functioning of the PDC. Structural mechanisms of stimulation of PDP activity by Ca2+ and polyamines are also discussed. PMID:10205302

Ermakov, G L; Goldstein, B N

1999-03-01

379

Nutrient deprivation induces the Warburg effect through ROS/AMPK-dependent activation of pyruvate dehydrogenase kinase.  

PubMed

The Warburg effect is known to be crucial for cancer cells to acquire energy. Nutrient deficiencies are an important phenomenon in solid tumors, but the effect on cancer cell metabolism is not yet clear. In this study, we demonstrate that starvation of HeLa cells by incubation with Hank's buffered salt solution (HBSS) induced cell apoptosis, which was accompanied by the induction of reactive oxygen species (ROS) production and AMP-activated protein kinase (AMPK) phosphorylation. Notably, HBSS starvation increased lactate production, cytoplasmic pyruvate content and decreased oxygen consumption, but failed to change the lactate dehydrogenase (LDH) activity or the glucose uptake. We found that HBSS starvation rapidly induced pyruvate dehydrogenase kinase (PDK) activation and pyruvate dehydrogenase (PDH) phosphorylation, both of which were inhibited by compound C (an AMPK inhibitor), NAC (a ROS scavenger), and the dominant negative mutant of AMPK. Our data further revealed the involvement of ROS production in AMPK activation. Moreover, DCA (a PDK inhibitor), NAC, and compound C all significantly decreased HBSS starvation-induced lactate production accompanied by enhancement of HBSS starvation-induced cell apoptosis. Not only in HeLa cells, HBSS-induced lactate production and PDH phosphorylation were also observed in CL1.5, A431 and human umbilical vein endothelial cells. Taken together, we for the first time demonstrated that a low-nutrient condition drives cancer cells to utilize glycolysis to produce ATP, and this increases the Warburg effect through a novel mechanism involving ROS/AMPK-dependent activation of PDK. Such an event contributes to protecting cells from apoptosis upon nutrient deprivation. PMID:23376776

Wu, Ching-An; Chao, Yee; Shiah, Shine-Gwo; Lin, Wan-Wan

2013-05-01

380

Analysis of quaternary structure of a [LDH-like] malate dehydrogenase of Plasmodium falciparum with oligomeric mutants  

Microsoft Academic Search

l-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to l-lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archae and other prokaryotes.\\u000a Initial sequence analysis and identification of critical amino acid residues involved in inter-subunit salt-bridge interactions\\u000a predict tetrameric structure for PfMDH. The catalytically

Anupam Pradhan; Prasenjit Mukherjee; Abhai K. Tripathi; Mitchell A. Avery; Larry A. Walker; Babu L. Tekwani

2009-01-01

381

Metabolic Analysis of Corynebacterium glutamicum during Lactate and Succinate Productions under Oxygen Deprivation Conditions  

Microsoft Academic Search

Lactate and succinate were produced from glucose by Corynebacterium glutamicum under oxygen deprivation conditions without growth. Addition of bicarbonate to the reaction mixture led not only to a 3.6-fold increase in succinate production rate, but also to a 2.3- and 2.5-fold increase, respectively, of the rates of lactate production and glucose consumption, compared to the control. Furthermore, when small amounts

Masayuki Inui; Shikiko Murakami; Shohei Okino; Hideo Kawaguchi; Alain A. Vertès; Hideaki Yukawa

2004-01-01

382

Etiology and therapeutic approach to elevated lactate  

PubMed Central

Lactate levels are commonly evaluated in acutely ill patients. Although most commonly used in the context of evaluating shock, lactate can be elevated for many reasons. While tissue hypoperfusion is probably the most common cause of elevation, many other etiologies or contributing factors exist. Clinicians need to be aware of the many potential causes of lactate elevation as the clinical and prognostic importance of an elevated lactate varies widely by disease state. Moreover, specific therapy may need to be tailored to the underlying cause of elevation. The current review is based on a comprehensive PubMed search and contains an overview of the pathophysiology of lactate elevation followed by an in-depth look at the varied etiologies, including medication-related causes. The strengths and weaknesses of lactate as a diagnostic/prognostic tool and its potential use as a clinical endpoint of resuscitation will be discussed. The review ends with some general recommendations on management of patients with elevated lactate.

Andersen, Lars W.; Mackenhauer, Julie; Roberts, Jonathan C.; Berg, Katherine M.; Cocchi, Michael N.; Donnino, Michael W.

2014-01-01

383

Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?  

PubMed Central

The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of ?,?-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated.

2014-01-01

384

Relationship between the lactation curve and udder disease incidence in different lactation stages in first-lactation Holstein cows.  

PubMed

We examined the relationships between the shape of the first parity lactation curve and udder disease incidence at different stages of lactation in 538 Holstein cows. Data used were first-parity daily milk yields and treatment records. Each cow was classified according to whether or not it had had udder disease at least once over the whole lactation period or in one of three stages within the lactation period. We then examined the differences in the shapes of the lactation curves between the disease incidence and non-incidence group in each stage. Cows that had high rates of increase in milk yield and high milk yields in early lactation were predisposed to udder disease afterwards. Cows with high milk production over a long period but with low lactation persistency were predisposed to udder disease after the peak of lactation. There was no difference in total milk yield between incidence and non-incidence groups in all stages, suggesting that, for a comparable level of lactation, cows without udder diseases have flatter lactation curves. PMID:20163652

Yamazaki, Takeshi; Takeda, Hisato; Nishiura, Akiko; Togashi, Kenji

2009-12-01

385

Metabolism changes during aging in the hippocampus and striatum of glud1 (glutamate dehydrogenase 1) transgenic mice.  

PubMed

The decline in neuronal function during aging may result from increases in extracellular glutamate (Glu), Glu-induced neurotoxicity, and altered mitochondrial metabolism. To study metabolic responses to persistently high levels of Glu at synapses during aging, we used transgenic (Tg) mice that over-express the enzyme Glu dehydrogenase (GDH) in brain neurons and release excess Glu in synapses. Mitochondrial GDH is important in amino acid and carbohydrate metabolism and in anaplerotic reactions. We monitored changes in nineteen neurochemicals in the hippocampus and striatum of adult, middle aged, and aged Tg and wild type (wt) mice, in vivo, using proton ((1)H) magnetic resonance spectroscopy. Significant differences between adult Tg and wt were higher Glu, N-acetyl aspartate (NAA), and NAA + NAA-Glu (NAAG) levels, and lower lactate in the Tg hippocampus and striatum than those of wt. During aging, consistent changes in Tg and wt hippocampus and striatum included increases in myo-inositol and NAAG. The levels of glutamine (Gln), a key neurochemical in the Gln-Glu cycle between neurons and astroglia, increased during aging in both the striatum and hippocampus of Tg mice, but only in the striatum of the wt mice. Age-related increases of Glu were observed only in the striatum of the Tg mice. PMID:24442550

Choi, In-Young; Lee, Phil; Wang, Wen-Tung; Hui, Dongwei; Wang, Xinkun; Brooks, William M; Michaelis, Elias K

2014-03-01

386

Mammalian alpha-Keto Acid Dehydrogenase Complexes.  

National Technical Information Service (NTIS)

Both complexes (pyruvate dehydrogenase complex and 2-oxoglutarate dehydrogenase complex) were isolated from pig heart muscle in highly purified states as soluble multienzyme units with high molecular weights by a convenient procedure. The results suggest ...

M. Koike

1968-01-01

387

Genetics Home Reference: Pyruvate dehydrogenase deficiency  

MedlinePLUS

... component of a group of proteins called the pyruvate dehydrogenase complex. This complex plays an important role in the ... into a form that cells can use. The pyruvate dehydrogenase complex converts a molecule called pyruvate, which is formed ...

388

Contrasting effects of selenite and tellurite on lipoamide dehydrogenase activity suggest a different biological behaviour of the two chalcogens.  

PubMed

The effects of selenite and tellurite on the mammalian enzyme lipoamide dehydrogenase were compared. Selenite acts as a substrate of lipoamide dehydrogenase in a process requiring the presence of lipoamide. In contrast, tellurite is a potent inhibitor, effective in the low micromolar range. The inhibitory effect of tellurite on lipoamide dehydrogenase is partially reverted by dithiothreitol indicating the participation of the thiol groups of the enzyme. Tellurite, but not selenite, stimulates the diaphorase activity of lipoamide dehydrogenase. In a mitochondrial matrix protein preparation, which contains lipoamide dehydrogenase, an inhibitory action similar to that observed on the purified enzyme was also elicited by tellurite. Human embryonic kidney cells (HEK 293 T) treated with tellurite show a partial inhibition of lipoamide dehydrogenase. In addition to the toxicological implications of tellurium compounds, the reported results suggest that tellurite and its derivatives can be used as potential tools for studying biochemical reactions. PMID:22100759

Folda, Alessandra; Citta, Anna; Scutari, Guido; Bindoli, Alberto; Rigobello, Maria Pia

2012-01-01

389

Ubiquinone-mediated coupling of NADH dehydrogenase to active transport in membrane vesicles from Escherichia coli.  

PubMed Central

Addition of ubiquinone-1 to E. coli ML 308-225 membrane vesicles dramatically increases coupling between NADH oxidation and active transport such that initial rates and steady-state levels of lactose and amino-acid accumulation are comparable to those observed during D-lactate oxidation. Similar but less dramatic effects are observed with the quinone and succinate or L-lactate. In the presence of NADH and ubiquinone-1, the vesicles also generate a membrane potential (interior negative) that is similar in magnitude to that observed in the presence of D-lactate. Stimulation of NADH-dependent transport by ubiquinone-1 cannot be accounted for by increased rates of oxidation of NADH, and the effect of the quinone on NADH-dependent lactose transport is not observed in vesicles depleted of NADH dehydrogenase activity. Thus, it is apparent that ubiquinone-1 shunts electrons from NADH dehydrogenase [NADH:(acceptor)oxidoreductase; EC 1.6.99.3] to the portion of the respiratory chain containing the energy-coupling site. The findings demonstrate unequivocally that inefficient coupling of NADH oxidation to active transport cannot be due to the presence of inverted vesicles. In addition, they provide further support for specific localization of the energy-coupling site.

Stroobant, P; Kaback, H R

1975-01-01

390

Dose-response effects of lactate infusions on gluconeogenesis from lactate in normal man.  

PubMed

Lactate is the predominant gluconeogenic precursor in man. To determine the dose-response relationships between plasma lactate concentration and rates of lactate incorporation in plasma glucose (lactate gluconeogenesis, LGN), we infused 17 normal volunteers with sodium lactate for 180 min at rates ranging from 6 to 40 mumol kg-1 min-1 and measured [U-14C]lactate incorporation into plasma glucose, as well as rates of lactate and glucose appearance in plasma. With the highest lactate infusions, plasma lactate increased up to 7 mM (compared to 1.1 +/- 0.13 mM during control sodium bicarbonate infusions, n = 10) and LGN averaged 4.73 +/- 0.23 mumol kg-1 min-1 (compared to 1.57 +/- 0.26 mumol kg-1 min-1 in bicarbonate control experiments, P < 0.001). The data relating plasma lactate concentration to LGN best fit a sigmoidal curve which plateaued at plasma lactate concentrations of approximately 6 mM and yielded an ED50 of 2.04 +/- 0.20 (SD) mM and a Vmax (6.25 +/- 1.2) (SD) (mumol kg-1 min-1). The sum of the basal rate of lactate appearance and the rate of lactate infusion was not significantly different from the overall rates of lactate appearance during the lactate infusions (35.8 +/- 2.2 vs. 34.8 +/- 2.9 mumol kg-1 min-1, P = 0.23). Thus, our results support the view that infusion of exogenous lactate does not suppress endogenous lactate appearance in plasma. PMID:8404995

Jenssen, T; Nurjhan, N; Consoli, A; Gerich, J E

1993-08-01

391

Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius  

Microsoft Academic Search

Allysine ethylene acetal [(S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (2)] was prepared from the corresponding keto acid by reductive amination using phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius ATCC 33205. Glutamate, alanine, and leucine dehydrogenases, and PDH from Sporosarcina species (listed in order of increasing effectiveness) also gave the desired amino acid but were less effective. The reaction requires ammonia and NADH. NAD produced during

Ronald L Hanson; Jeffrey M Howell; Thomas L LaPorte; Mary Jo Donovan; Dana L Cazzulino; Valerie Zannella; Michael A Montana; Venkata B Nanduri; Steven R Schwarz; Ronald F Eiring; Susan C Durand; John M Wasylyk; William L Parker; Mark S Liu; Francis J Okuniewicz; Bang-Chi Chen; John C Harris; Kenneth J Natalie; Keith Ramig; Shankar Swaminathan; Victor W Rosso; Shawn K Pack; Bruce T Lotz; Peter J Bernot; Andrew Rusowicz; David A Lust; Kai S Tse; John J Venit; Laszlo J Szarka; Ramesh N Patel

2000-01-01

392

Comparison of properties of malate dehydrogenase isoenzymes from hare and rabbit hearts  

Microsoft Academic Search

The hare heart mitochondrial malate dehydrogenase (mMDH) was established to have a much higher electrophoretic mobility than\\u000a the corresponding enzyme from the rabbit heart. Differences of kinetic properties of both mMDH and cytoplasmic malate dehydrogenase\\u000a (cMDH) from these two sources were shown. The hare heart mMDH and cMDH isoenzymes have a higher affinity to malate (in direct\\u000a reaction) and oxaloacetate

S. Strumilo; A. Owsieniuk; A. Radecka; A. Tylicki

2006-01-01

393

Succinate dehydrogenase deficiency in human  

Microsoft Academic Search

Mitochondrial succinate dehydrogenase (SDH) consists merely of four nuclearly encoded subunits. It participates in the electron transfer in the respiratory chain and in succinate catabolism in the Krebs cycle. Mutations in the four genes, SDHA, B, C and D, have been reported, resulting in strikingly diverse clinical presentations. So far, SDHA mutations have been reported to cause an encephalomyopathy in

J.-J. Brière; J. Favier; V. El. Ghouzzi; F. Djouadi; P. Bénit; A.-P. Gimenez; P. Rustin

2005-01-01

394

Unilateral periventricular leukomalacia in association with pyruvate dehydrogenase deficiency.  

PubMed

Pyruvate dehydrogenase (PDH) deficiency is a major cause of primary lactic acidosis and neurological dysfunction in infancy and early childhood. A deficiency of PDH E1 alpha, a subunit of the PDH complex, is a prominent cause of congenital lactic acidosis. We describe a female infant born at term and delivered by emergency Caesarean section because of fetal distress. There was no parental consanguinity. She presented at 5 months of age with failure to thrive, microcephaly, hypertonia, and developmental impairment. Her plasma and cerebrospinal fluid lactate were raised. She had raised plasma pyruvate with a normal lactate-pyruvate ratio. Magnetic resonance imaging of the brain showed a focal dilatation of the right lateral ventricle with unilateral periventricular leukomalacia (PVL) with subependymal cyst. Skin fibroblast culture assay revealed PDH deficiency, confirmed by mutation analysis of the E1 alpha subunit. At 18 months of age, she has hypertonia and global impairment and is making slow progress. Denver II assessment showed delay in gross motor, fine motor, adaptive, personal, social, and language categories. She has been treated with dichloroacetate and a ketogenic diet since the age of 10 and 13 months respectively, without any side effects. To our knowledge, unilateral PVL as a neuroradiological feature has not been described in children with PDH deficiency. PDH deficiency should be considered as a differential diagnosis if PVL is unilateral and if the perinatal history is not typical of PVL. PMID:21895644

Sharma, Ruchi; Sharrard, Mark J; Connolly, Daniel J; Mordekar, Santosh R

2012-05-01

395

Glutamate dehydrogenase of the unicellular green alga Scenedesmus acutus  

Microsoft Academic Search

The coenzyme-non-specific glutamate dehydrogenase (EC 1.4.1.3) from Scenedesmus acutus in inhibited by p-hydroxymercuribenzoate only in the deamination reaction. From this result and from its stability in the presence of urea it is concluded that this enzyme exhibits and equilibrium between three conformations: aminating and deaminating conformations induced by NADH-2-oxoglutarate and NAD+-glutamate, respectively, and the “native” conformation in the absence of

Valery R. Shatilov; Horst Sund

1983-01-01