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1

The enzymatic reaction catalyzed by lactate dehydrogenase exhibits one dominant reaction path  

NASA Astrophysics Data System (ADS)

Enzymes are the most efficient chemical catalysts known, but the exact nature of chemical barrier crossing in enzymes is not fully understood. Application of transition state theory to enzymatic reactions indicates that the rates of all possible reaction paths, weighted by their relative probabilities, must be considered in order to achieve an accurate calculation of the overall rate. Previous studies in our group have shown a single mechanism for enzymatic barrier passage in human heart lactate dehydrogenase (LDH). To ensure that this result was not due to our methodology insufficiently sampling reactive phase space, we implement high-perturbation transition path sampling in both microcanonical and canonical regimes for the reaction catalyzed by human heart LDH. We find that, although multiple, distinct paths through reactive phase space are possible for this enzymatic reaction, one specific reaction path is dominant. Since the frequency of these paths in a canonical ensemble is inversely proportional to the free energy barriers separating them from other regions of phase space, we conclude that the rarer reaction paths are likely to have a negligible contribution. Furthermore, the non-dominate reaction paths correspond to altered reactive conformations and only occur after multiple steps of high perturbation, suggesting that these paths may be the result of non-biologically significant changes to the structure of the enzymatic active site.

Masterson, Jean E.; Schwartz, Steven D.

2014-10-01

2

D-lactate dehydrogenase of Desulfovibrio vulgaris.  

PubMed

D-Lactate dehydrogenase, the starting enzyme for carbon and energy metabolism in dissimilatory sulfate-reducing bacteria, has been purified 36-fold from the soluble fraction of the sonicate of Desulfovibrio vulgaris, Miyazaki. The enzyme is specific for D-lactate (Km = 0.8 mM) and DL-2-hydroxybutyrate (probably its D-isomer) as the electron donor substrate. It reduces, in the presence of lactate, various artificial electron acceptors such as 1-methoxyphenazinium methyl sulfate, ferricyanide, tetrazolium dyes, methylene blue, and 2,6-dichlorophenol-indophenol. When 2 mol of ferricyanide was reduced, 1 mol of pyruvate was produced during the reaction. Among natural electron carriers, only cytochrome c-553 isolated from the same organism can be reduced by the enzyme. The ferric complex of pyridine-2,6-dicarboxylate can act as an electron acceptor if cytochrome c-553 is present in the reaction system. NAD+, NADP+, FAD, FMN, cytochrome c3, high-molecular-weight cytochrome, eucaryotic cytochromes c (yeast and horse) and O2 could not be reduced. The enzyme does not have any diaphorase activity. The D-lactate dehydrogenase of D. vulgaris must therefore be named D-lactate:ferricytochrome c-553 oxidoreductase [EC subclass 1.1.2]. A similar enzyme exists in the formate dehydrogenase-less mutant of D. vulgaris, Miyazaki, and in D. vulgaris, Hildenborough. PMID:7275946

Ogata, M; Arihara, K; Yagi, T

1981-05-01

3

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 ...

4

Controlling the highest lactate dehydrogenase activity known in nature.  

PubMed

In the shipjack, Euthynnus pelamis, white muscle appears to possess a powerful anaerobic capacity as well as a significant carbohydrate based aerobic potential. Lactate dehydrogenase occurs at higher activities than found thus far anywhere else in nature and clearly functions in anaerobic glycolysis. Alpha-glycerophosphate dehydrogenase also occurs in unusually high activities and appears to play a role in aerobic glycolysis. Regulation of these two reactions is accomplished by temperature, pH, and creatine phosphate levels. High temperature, low pH, and low creatine phosphate levels all appear to favor lactate dehydrogenase over alpha-glycerophosphate dehydrogenase; low temperature, high pH, and high creatine-phosphate levels, all expected during the quiescent state in this species, and when metabolism in aerobic, all favor alpha-glycerophosphate dehydrogenase activity. PMID:24351

Guppy, M; Hochachka, P W

1978-03-01

5

21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.  

Code of Federal Regulations, 2012 CFR

...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...

2012-04-01

6

21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.  

Code of Federal Regulations, 2013 CFR

...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

7

21 CFR 862.1440 - Lactate dehydrogenase test system.  

Code of Federal Regulations, 2013 CFR

...ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1440 Lactate dehydrogenase test system. (a)...

2013-04-01

8

21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.  

...ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1445 Lactate dehydrogenase isoenzymes test system....

2014-04-01

9

21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.  

Code of Federal Regulations, 2011 CFR

...ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1445 Lactate dehydrogenase isoenzymes test system....

2011-04-01

10

21 CFR 862.1440 - Lactate dehydrogenase test system.  

...Lactate dehydrogenase measurements are used in the diagnosis and treatment of liver diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial infarction, and tumors of the...

2014-04-01

11

Pleiotropic effects of lactate dehydrogenase inactivation in Lactobacillus casei  

Microsoft Academic Search

In lactic acid bacteria, conversion of pyruvic to lactic acid through the activity of lactate dehydrogenase (Ldh) constitutes the final step of the homofermentative pathway. Lactobacillus casei has two characterized genes encoding Ldh activities. The ldhL gene codes for an L-Ldh, which specifically catalyzes the formation of l-lactate, whereas the hicD gene codes for a d-hydroxyisocaproate dehydrogenase (HicDH), which catalyzes

Rosa Viana; María Jesús Yebra; José Luis Galán; Vicente Monedero; Gaspar Pérez-Martínez

2005-01-01

12

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 PMID:4004236

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

1985-01-01

13

Lactate production yield from engineered yeasts is dependent from the host background, the lactate dehydrogenase source and the lactate export  

Microsoft Academic Search

BACKGROUND: Metabolic pathway manipulation for improving the properties and the productivity of microorganisms is becoming a well established concept. For the production of important metabolites, but also for a better understanding of the fundamentals of cell biology, detailed studies are required. In this work we analysed the lactate production from metabolic engineered Saccharomyces cerevisiae cells expressing a heterologous lactate dehydrogenase

Paola Branduardi; Michael Sauer; Luca De Gioia; Giuseppe Zampella; Minoska Valli; Diethard Mattanovich; Danilo Porro

2006-01-01

14

Interaction Between Lactate Dehydrogenase and Tween 80 in Aqueous Solution  

Microsoft Academic Search

Purpose. The weak aqueous interaction between the protein lactate dehydrogenase (LDH) and the nonionic surfactant Tween 80 has been investigated, because weak protein-amphiphile interactions are of significant importance in pharmaceutical formulations, but are experimentally hard to determine. The system LDH\\/sodium dodecyl sulphate (SDS) was used as reference because SDS, by its strong protein binding, denatures LDH completely.

Anna Hillgren; Hans Evertsson; Maggie Aldén

2002-01-01

15

Effects of lactate dehydrogenase suppression and glycerol-3-phosphate dehydrogenase overexpression on cellular metabolism  

Microsoft Academic Search

Abstact  In order to conduct a physiological functional study of lactate dehydrogenase (LDH) and glycerol-3-phosphate dehydrogenase\\u000a (GPDH), we engineered a CHO dhfr\\u000a ? cell, by overexpressing either the anti-sense LDH-A RNA (anti-LDH cells) or GPDH (GP3 cells), or both (GP3\\/anti-LDH cells).\\u000a LDH activity in the cell cytosol, and lactate content and pHe change in the growth media were found to decrease

Dae-won Jeong; Il Taeg Cho; Tae Soo Kim; Gun Won Bae; Ik-Hwan Kim; Ick Young Kim

2006-01-01

16

Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals.  

PubMed

Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes. PMID:25247702

Schueren, Fabian; Lingner, Thomas; George, Rosemol; Hofhuis, Julia; Dickel, Corinna; Gärtner, Jutta; Thoms, Sven

2014-01-01

17

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

18

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

PubMed Central

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.

2011-01-01

19

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. PMID:24898534

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

2014-01-01

20

NAD-dependent lactate dehydrogenase catalyses the first step in respiratory utilization of lactate by Lactococcus lactis?  

PubMed Central

Lactococcus lactis can undergo respiration when hemin is added to an aerobic culture. The most distinctive feature of lactococcal respiration is that lactate could be consumed in the stationary phase concomitantly with the rapid accumulation of diacetyl and acetoin. However, the enzyme responsible for lactate utilization in this process has not yet been identified. As genes for fermentative NAD-dependent l-lactate dehydrogenase (l-nLDH) and potential electron transport chain (ETC)-related NAD-independent l-LDH (l-iLDH) exist in L. lactis, the activities of these enzymes were measured in this study using crude cell extracts prepared from respiratory and fermentation cultures. Further studies were conducted with purified preparations of recombinant LDH homologous proteins. The results showed that l-iLDH activity was hardly detected in both crude cell extracts and purified l-iLDH homologous protein while l-nLDH activity was very significant. This suggested that l-iLDHs were inactive in lactate utilization. The results of kinetic analyses and the effects of activator, inhibitor, substrate and product concentrations on the reaction equilibrium showed that l-nLDH was much more prone to catalyze the pyruvate reduction reaction but could reverse its role provided that the concentrations of NADH and pyruvate were extremely low while NAD and lactate were abundant. Metabolite analysis in respiratory culture revealed that the cellular status in the stationary phase was beneficial for l-nLDH to catalyze lactate oxidation. The factors accounting for the respiration- and stationary phase-dependent lactate utilization in L. lactis are discussed here. PMID:24251099

Zhao, Rui; Zheng, Sui; Duan, Cuicui; Liu, Fei; Yang, Lijie; Huo, Guicheng

2013-01-01

21

Conversion of Lactobacillus pentosus D-lactate dehydrogenase to a D-hydroxyisocaproate dehydrogenase through a single amino acid replacement.  

PubMed

The single amino acid replacement of Tyr52 with Leu drastically increased the activity of Lactobacillus pentosus NAD-dependent D-lactate dehydrogenase toward larger aliphatic or aromatic 2-ketoacid substrates by 3 or 4 orders of magnitude and decreased the activity toward pyruvate by about 30-fold, converting the enzyme into a highly active D-2-hydroxyisocaproate dehydrogenase. PMID:12897026

Tokuda, Chizuka; Ishikura, Yoshiro; Shigematsu, Mayu; Mutoh, Hiroyuki; Tsuzuki, Shino; Nakahira, Yusaku; Tamura, Yusuke; Shinoda, Takeshi; Arai, Kazuhito; Takahashi, O; Taguchi, Hayao

2003-08-01

22

Conversion of Lactobacillus pentosus d-Lactate Dehydrogenase to a d-Hydroxyisocaproate Dehydrogenase through a Single Amino Acid Replacement  

PubMed Central

The single amino acid replacement of Tyr52 with Leu drastically increased the activity of Lactobacillus pentosus NAD-dependent d-lactate dehydrogenase toward larger aliphatic or aromatic 2-ketoacid substrates by 3 or 4 orders of magnitude and decreased the activity toward pyruvate by about 30-fold, converting the enzyme into a highly active d-2-hydroxyisocaproate dehydrogenase. PMID:12897026

Tokuda, Chizuka; Ishikura, Yoshiro; Shigematsu, Mayu; Mutoh, Hiroyuki; Tsuzuki, Shino; Nakahira, Yusaku; Tamura, Yusuke; Shinoda, Takeshi; Arai, Kazuhito; Takahashi, O; Taguchi, Hayao

2003-01-01

23

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. PMID:24466056

Shi, Yun; Pinto, B. Mario

2014-01-01

24

[Changes in lactate dehydrogenase isoforms in the process of oncogenesis].  

PubMed

Isoenzymes of lactate dehydrogenase were studied by disc-electrophoresis in polyacrylamide gel, and in the clinic--in 1% agar gel. Oncovirus A12 invasion of the culture of rat embryo fibroblasts (REF) was found to result in the increased percentage of the cathode fractions activity (LDG-4 and LD-5) and in the disappearance of LDG-1 yet during the first day of the experiment prior to hypoxia and enhanced proliferation, i. e. it is most likely to be primary. In the homogenates of cancerous tumor and large intestine polyps of man also a reliable increase of the cathode and a decrease or disappearance of the anode fractions accur. A correlation of the experimental and clinical data allowed a suggestion to be made that LDG isoenzymes changes are genetically conditioned and play an important role in the process of oncogenesis, providing conditions for the increased intensity of glycolysis and proliferation. PMID:636370

Ageenko, A I; Vitorgan, Iu E; Chernomordik, A E

1978-01-01

25

Enzymatic urea adaptation: lactate and malate dehydrogenase in elasmobranchs.  

PubMed

Lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) electrophoretic tissue patterns of two different orders of Elasmobranchii: Carchariniformes (Galeus melanostomus and Prionace glauca) and Squaliformes (Etmopterus spinax and Scymnorinus licha) were studied. The number of loci expressed for these enzymes was the same of other elasmobranch species. Differences in tissue distribution were noted in LDH from G. melanostomus due to the presence of an additional heterotetramer in the eye tissue. There were also differences in MDH. In fact, all the tissues of E. spinax and G. melanostomus showed two mitochondrial bands. Major differences were noted in the number of isozymes detected in the four compared elasmobranchs. The highest polymorphism was observed in E. spinax and G. melanostomus, two species that live in changeable environmental conditions. The resistance of isozymes after urea treatment was examined; the resulting patterns showed a quite good resistance of the enzymes, higher for LDH than MDH, also at urea concentration much greater than physiological one. These results indicated that the total isozyme resistance can be considered higher in urea accumulators (such as elasmobranchs) than in the non-accumulators (such as teleosts). PMID:16497106

Laganà, G; Bellocco, E; Mannucci, C; Leuzzi, U; Tellone, E; Kotyk, A; Galtieri, A

2006-01-01

26

D- and L-lactate dehydrogenases during invertebrate evolution  

PubMed Central

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 main isozymes of this gene family in chordates, but little is known about the evolution of L-Ldh in invertebrates. While most invertebrates preferentially oxidize L-lactic acid, several species of mollusks, a few arthropods and polychaetes were found to have exclusively D-LDH enzymatic activity. Therefore, it has been suggested that L-LDH and D-LDH are mutually exclusive. However, recent characterization of putative mammalian D-LDH with significant similarity to yeast proteins showing D-LDH activity suggests that at least mammals have the two naturally occurring forms of LDH specific to L- and D-lactate. This study describes the phylogenetic relationships of invertebrate L-LDH and D-LDH with special emphasis on crustaceans, and discusses gene duplication events during the evolution of L-Ldh. Results Our phylogenetic analyses of L-LDH in vertebrates are consistent with the general view that the main isozymes (LDH-A, LDH-B and LDH-C) evolved through a series of gene duplications after the vertebrates diverged from tunicates. We report several gene duplication events in the crustacean, Daphnia pulex, and the leech, Helobdella robusta. Several amino acid sequences with strong similarity to putative mammalian D-LDH and to yeast DLD1 with D-LDH activity were found in both vertebrates and invertebrates. Conclusion The presence of both L-Ldh and D-Ldh genes in several chordates and invertebrates suggests that the two enzymatic forms are not necessarily mutually exclusive. Although, the evolution of L-Ldh has been punctuated by multiple events of gene duplication in both vertebrates and invertebrates, a shared evolutionary history of this gene in the two groups is apparent. Moreover, the high degree of sequence similarity among D-LDH amino acid sequences suggests that they share a common evolutionary history. PMID:18828920

2008-01-01

27

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

Microsoft Academic Search

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

Osamu Kato; Jung-Won Youn; K Corinna Stansen; Daisuke Matsui; Tadao Oikawa; Volker F Wendisch

2010-01-01

28

Direct electrochemistry of lactate dehydrogenase immobilized on silica sol–gel modified gold electrode and its application  

Microsoft Academic Search

The direct electrochemistry of lactate dehydrogenase (LDH) immobilized in silica sol–gel film on gold electrode was investigated, and an obvious cathodic peak at about ?200mV (versus SCE) was found for the first time. The LDH-modified electrode showed a surface controlled irreversible electrode process involving a one electron transfer reaction with the charge-transfer coefficient (?) of 0.79 and the apparent heterogeneous

Junwei Di; Jiongjia Cheng; Quan Xu; Huie Zheng; Jingyue Zhuang; Yongbo Sun; Keyu Wang; Xiangyin Mo; Shuping Bi

2007-01-01

29

Sequence analysis of teleost retina-specific lactate dehydrogenase C: evolutionary implications for the vertebrate lactate dehydrogenase gene family.  

PubMed Central

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 spermatocytes of mammals and a single family of birds and in the eye or liver tissues of teleost fishes. Ldh-C might have arisen independently in these taxa as duplications of either Ldh-A or Ldh-B. Several authors have challenged this traditional hypothesis on the basis of amino acid sequence and immunological similarity of the three LDH isozymes. They suggest that the primordial LDH gene was duplicated to form Ldh-C and a locus that later gave rise to Ldh-A and Ldh-B. We have differentiated between these hypotheses by determining the cDNA sequence of the retina-specific LDH-C from a teleost, Fundulus heteroclitus. On the basis of amino acid sequence similarity, we conclude that the LDH-C isozymes in fish and mammals are not orthologous but derive from independent gene duplications. Furthermore, our phylogenetic analyses support previous hypotheses that teleost Ldh-C is derived from a duplication of the Ldh-B locus. PMID:8419929

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

1993-01-01

30

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

31

Enhanced activity of 3alpha-hydroxysteroid dehydrogenase by addition of the co-solvent 1-butyl-3-methylimidazolium (L)-lactate in aqueous phase of biphasic systems for reductive production of steroids.  

PubMed

The enzyme activity of 3alpha-hydrosteroid dehydrogenase (HSDH) was enhanced by the addition of the co-solvent 1-butyl-3-methylimidazolium (L)-lactate ([Bmim][lactate]) to 50 mM Tris-HCl buffer. When utilizing [Bmim][lactate], the reaction velocity of HSDH increased. Also, reductive production of androsterone was investigated in an aqueous-organic solvent biphasic system containing 5% [Bmim][lactate] as the co-solvent of aqueous phase. In a coupled-enzyme system comprising HSDH and formate dehydrogenase (FDH), a two-fold increase in production rate of androsterone was obtained when utilizing [Bmim][lactate] with NADH regeneration. PMID:17092593

Okochi, Mina; Nakagawa, Izumi; Kobayashi, Takeshi; Hayashi, Shuhei; Furusaki, Shintaro; Honda, Hiroyuki

2007-02-01

32

Pyruvate dehydrogenase and the path of lactate degradation in Desulfovibrio vulgaris Miyazaki F.  

PubMed

Pyruvate dehydrogenase from Desulfovibrio vulgaris Miyazaki F was partially purified from the soluble fraction of the bacterial sonicate, and characterized. The enzyme catalyzes oxidative decarboxylation of pyruvate to produce acetyl-CoA, in contrast to statements in current review articles in which acetyl phosphate is indicated to be a direct decomposition product of pyruvate in sulfate-reducing bacteria. The established reaction stoichiometry is: pyruvate + CoA + FMN----acetyl-CoA + CO2 + FMNH2. The Km values are 2.9 mM for pyruvate, 32 microM for CoA and 6.7 mumol for FMN. Participation of thiamine diphosphate in the enzymic process was not proven. 2-Oxobutyrate, but not 2-oxoglutarate, can substitute for pyruvate. The three flavin compounds, FMN, FAD, and flavodoxin, as well as clostridial ferredoxin, serve as electron carriers for the enzyme. Thus the enzyme is a kind of pyruvate synthase [EC 1.2.7.1], but acts in the direction of pyruvate degradation in the growing cells. The rate of cytochrome C3 reduction is extremely low, but in the presence of flavodoxin as an electron mediator, the reduction rate of cytochrome C3 becomes faster than the reduction rate of flavodoxin alone. It seems that the physiological electron acceptor for this enzyme is flavodoxin, which might be complexed with cytochrome C3 to produce a very efficient electron transfer system in the cell. The soluble fraction of D. vulgaris cells has been proved to contain, in addition to the pyruvate dehydrogenase, lactate dehydrogenase (Ogata, M., Arihara, K., & Yagi, T. (1981) J. Biochem. 89, 1423-1431), phosphate acetyltransferase and acetate kinase, i.e., all the enzymes necessary to convert lactate to acetate, producing ATP by substrate level phosphorylation. PMID:3023304

Ogata, M; Yagi, T

1986-08-01

33

The effects of season and temperature on D-lactate dehydrogenase, pyruvate kinase and arginine kinase in the foot of Helix pomatia L.  

PubMed

The effects of pH, season, environmental and experimental temperatures on the activities and kinetic parameters of D-lactate dehydrogenase, pyruvate kinase and arginine kinase from the foot of the pulmonate snail Helix pomatia were analyzed. Both in phosphate and Tris buffers D-lactate dehydrogenase was the enzyme with the most acid maximum, arginine kinase that with the most alkaline, whilst pyruvate kinase occupied an intermediate position. Pyruvate kinase activity, measured at 20 degrees C, was positively correlated with the environmental temperature at the moment of collecting the animal, whereas neither arginine kinase nor D-lactate dehydrogenase showed such a relationship. A seasonal study based on approximately 100 specimens established that arginine kinase activity remained the same throughout the year. Pyruvate kinase activity was slightly lower, and D-lactate dehydrogenase activity significantly higher, in winter than in summer animals. Snails subjected in spring to a short warm-up period before enzyme extraction showed extreme variability and some extraordinarily high values of pyruvate kinase activity, suggesting that either season or elevated temperature may have an immediate effect on the activity of this enzyme. Individual variability of all three enzymes ranges from 300 to 400%. The activities of pyruvate kinase and D-lactate dehydrogenase are strongly correlated in summer, forming a "constant-proportion-group", whereas in winter, with D-lactate dehydrogenase activity increasing and pyruvate kinase activity decreasing these two enzymes become "uncoupled". The Km value of pyruvate kinase is independent of experimental temperature between 10 and 25 degrees C, whereas that of D-lactate dehydrogenase and arginine kinase increases about three-fold within this range. Thus the temperature relationship of a single enzymic reaction cannot be used as an arguemnt for or against the occurrence of temperature compensation of whole animal metabolism. The possibility of modulation of enzyme activity by environmental temperature is discussed. PMID:35457

Wieser, W; Wright, E

1979-04-01

34

Direct evidence of catalytic heterogeneity in lactate dehydrogenase by temperature jump infrared spectroscopy.  

PubMed

Protein conformational heterogeneity and dynamics are known to play an important role in enzyme catalysis, but their influence has been difficult to observe directly. We have studied the effects of heterogeneity in the catalytic reaction of pig heart lactate dehydrogenase using isotope edited infrared spectroscopy, laser-induced temperature jump relaxation, and kinetic modeling. The isotope edited infrared spectrum reveals the presence of multiple reactive conformations of pyruvate bound to the enzyme, with three major reactive populations having substrate C2 carbonyl stretches at 1686, 1679, and 1674 cm(-1), respectively. The temperature jump relaxation measurements and kinetic modeling indicate that these substates form a heterogeneous branched reaction pathway, and each substate catalyzes the conversion of pyruvate to lactate with a different rate. Furthermore, the rate of hydride transfer is inversely correlated with the frequency of the C2 carbonyl stretch (the rate increases as the frequency decreases), consistent with the relationship between the frequency of this mode and the polarization of the bond, which determines its reactivity toward hydride transfer. The enzyme does not appear to be optimized to use the fastest pathway preferentially but rather accesses multiple pathways in a search process that often selects slower ones. These results provide further support for a dynamic view of enzyme catalysis where the role of the enzyme is not just to bring reactants together but also to guide the conformational search for chemically competent interactions. PMID:25149276

Reddish, Michael J; Peng, Huo-Lei; Deng, Hua; Panwar, Kunal S; Callender, Robert; Dyer, R Brian

2014-09-18

35

Direct Evidence of Catalytic Heterogeneity in Lactate Dehydrogenase by Temperature Jump Infrared Spectroscopy  

PubMed Central

Protein conformational heterogeneity and dynamics are known to play an important role in enzyme catalysis, but their influence has been difficult to observe directly. We have studied the effects of heterogeneity in the catalytic reaction of pig heart lactate dehydrogenase using isotope edited infrared spectroscopy, laser-induced temperature jump relaxation, and kinetic modeling. The isotope edited infrared spectrum reveals the presence of multiple reactive conformations of pyruvate bound to the enzyme, with three major reactive populations having substrate C2 carbonyl stretches at 1686, 1679, and 1674 cm?1, respectively. The temperature jump relaxation measurements and kinetic modeling indicate that these substates form a heterogeneous branched reaction pathway, and each substate catalyzes the conversion of pyruvate to lactate with a different rate. Furthermore, the rate of hydride transfer is inversely correlated with the frequency of the C2 carbonyl stretch (the rate increases as the frequency decreases), consistent with the relationship between the frequency of this mode and the polarization of the bond, which determines its reactivity toward hydride transfer. The enzyme does not appear to be optimized to use the fastest pathway preferentially but rather accesses multiple pathways in a search process that often selects slower ones. These results provide further support for a dynamic view of enzyme catalysis where the role of the enzyme is not just to bring reactants together but also to guide the conformational search for chemically competent interactions. PMID:25149276

Reddish, Michael; Peng, Huo-Lei; Deng, Hua; Panwar, Kunal S.; Callender, Robert; Dyer, R. Brian

2014-01-01

36

The Core of Allosteric Motion in Thermus caldophilus l-Lactate Dehydrogenase.  

PubMed

For Thermus caldophilus l-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S0.5 value 10(3)-fold and increased the Vmax value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus l-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172-185) and MR2 (positions 211-221), form a compact core for allosteric motion, and His(179) of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased Vmax 4-fold but reduced pyruvate S0.5 only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase Vmax, but 10(2)-reduced pyruvate S0.5, and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule. PMID:25258319

Ikehara, Yoko; Arai, Kazuhito; Furukawa, Nayuta; Ohno, Tadashi; Miyake, Tatsuya; Fushinobu, Shinya; Nakajima, Masahiro; Miyanaga, Akimasa; Taguchi, Hayao

2014-11-01

37

Catabolism of mannitol in Lactococcus lactis MG1363 and a mutant defective in lactate dehydrogenase.  

PubMed

Mannitol metabolism in Lactococcus lactis MG1363 and in a derivative strain deficient in lactate dehydrogenase (LDH(d)) was characterized. Both strains had the ability to grow on mannitol as an energy source, although this polyol was a poorer substrate for growth than glucose. When compared to glucose, the metabolism of mannitol caused an NADH burden due to formation of an additional NADH molecule at the reaction catalysed by mannitol-1-phosphate dehydrogenase (Mtl1PDH). This resulted in a prominent accumulation of mannitol 1-phosphate (Mtl1P) both in growing and resting cells, suggesting the existence of a severe bottleneck at Mtl1PDH. Growth on mannitol induced the activity of Mtl1PDH in both the LDH(d) and MG1363 strains. The lower accumulation of Mtl1P in mannitol-grown cells when compared to glucose-grown LDH(d) cells, as monitored by in vivo (13)C-NMR, reflects this induction. A clear shift towards the production of ethanol was observed on mannitol, indicating pressure to regenerate NAD(+) when this substrate was used. A strategy to obtain a mannitol-overproducing strain is proposed. PMID:12427938

Neves, Ana Rute; Ramos, Ana; Shearman, Claire; Gasson, Michael J; Santos, Helena

2002-11-01

38

Incorporating expression data in metabolic modeling: a case study of lactate dehydrogenase  

E-print Network

Integrating biological information from different sources to understand cellular processes is an important problem in systems biology. We use data from mRNA expression arrays and chemical kinetics to formulate a metabolic model relevant to K562 erythroleukemia cells. MAP kinase pathway activation alters the expression of metabolic enzymes in K562 cells. Our array data show changes in expression of lactate dehydrogenase (LDH) isoforms after treatment with phorbol 12-myristate 13-acetate (PMA), which activates MAP kinase signaling. We model the change in lactate production which occurs when the MAP kinase pathway is activated, using a non-equilibrium, chemical-kinetic model of homolactic fermentation. In particular, we examine the role of LDH isoforms, which catalyze the conversion of pyruvate to lactate. Changes in the isoform ratio are not the primary determinant of the production of lactate. Rather, the total concentration of LDH controls the lactate concentration.

Joshua Downer; Joel R. Sevinsky; Natalie G. Ahn; Katheryn A. Resing; M. D. Betterton

2005-11-12

39

Nasopharyngeal Lactate Dehydrogenase Concentrations Predict Bronchiolitis Severity in a Prospective Multicenter Emergency Department Study  

PubMed Central

We re-examined the finding of an inverse relationship between values of nasopharyngeal lactate dehydrogenase (LDH), a marker of the innate immune response, and bronchiolitis severity. In a prospective, multicenter study of 258 children we found in a mutlivariable model that higher nasopharyngeal LDH values in young children with bronchiolitis were independently associated with a decreased risk of hospitalization. PMID:22517336

Mansbach, Jonathan M.; Piedra, Pedro A.; Laham, Federico R.; McAdam, Alexander J.; Clark, Sunday; Sullivan, Ashley F.; Camargo, Carlos A.

2012-01-01

40

Hypoxia and cobalt stimulate lactate dehydrogenase (LDH) activity in vascular smooth muscle cells  

Microsoft Academic Search

O2 plays a dominant role in the metabolism and viability of cells; changes in O2 supply lead to many physiological responses in the cell. Recent reports have shown that hypoxia induces the transcription of a number of genes, among them those for the glycolytic enzymes. We have investigated signalling events that may lead to enhanced activity of lactate dehydrogenase (LDH)

Hugo H. Marti; Hans H. Jung; Josef Pfeilschifter; Christian Bauer

1994-01-01

41

Histamine and lactate dehydrogenase (LDH) release in ischemic myocardium of the guinea-pig  

Microsoft Academic Search

Histamine has been proved to be released during myocardial infarction and ischemic arrhythmias in dogs. The aim of the present experiments was to evaluate if ischemia and reperfusion modify histamine and lactate dehydrogenase (LDH) release in isolated guinea-pig heart. The results obtained show a steady increase of LDH release both in the ischemic and reperfusion phases. The release of histamine

E. Masini; E. Giannella; S. Bianchi; P. F. Mannaioni

1987-01-01

42

Control of Lactate Dehydrogenase, Lactate Glycolysis, and ?-Amylase by O2 Deficit in Barley Aleurone Layers 1  

PubMed Central

After 4 days in an atmosphere of N2, aleurone layers of barley (Hordeum vulgare L. cv Himalaya) remained viable as judged by their ability to produce near normal amounts of ?-amylases when incubated with gibberellic acid (GA3) in air. However, layers did not produce ?-amylase when GA3 was supplied under N2, apparently because ?-amylase mRNA failed to accumulate. When an 8-hour pulse of [U-14C]glucose was supplied under N2 to freshly prepared aleurone layers, both [14C]lactate and [14C]ethanol accumulated; the [14C]lactate/[14C]ethanol ratio was about 0.3. Prior incubation of layers for 1 day under N2 changed this ratio to about 0.8, indicating an increase in the relative importance of the lactate branch of glycolysis. l(+)Lactate dehydrogenase (LDH) activity was low in freshly prepared aleurone layers and increased 10-fold during 2 days under N2, whereas alcohol dehydrogenase activity (ADH) was high initially and rose by 60%. The responses of LDH and ADH activities to O2 tension were dissimilar; when layers were incubated in various O2/N2 mixtures, LDH activity peaked at 2 to 5% O2 whereas ADH activity was highest at 0% O2. The LDH activity was resolved into several enzymically active bands by native polyacrylamide gel electrophoresis. We conclude that barley aleurone layers are highly adapted to O2 deficiency, that they possess an inducible LDH system as well as an ADH system, and we infer that the LDH and ADH systems are independently regulated. Images Fig. 2 Fig. 5 PMID:16663667

Hanson, Andrew D.; Jacobsen, John V.

1984-01-01

43

Design and synthesis of novel lactate dehydrogenase A inhibitors by fragment-based lead generation.  

PubMed

Lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate, utilizing NADH as a cofactor. It has been identified as a potential therapeutic target in the area of cancer metabolism. In this manuscript we report our progress using fragment-based lead generation (FBLG), assisted by X-ray crystallography to develop small molecule LDHA inhibitors. Fragment hits were identified through NMR and SPR screening and optimized into lead compounds with nanomolar binding affinities via fragment linking. Also reported is their modification into cellular active compounds suitable for target validation work. PMID:22417091

Ward, Richard A; Brassington, Claire; Breeze, Alexander L; Caputo, Alessandro; Critchlow, Susan; Davies, Gareth; Goodwin, Louise; Hassall, Giles; Greenwood, Ryan; Holdgate, Geoffrey A; Mrosek, Michael; Norman, Richard A; Pearson, Stuart; Tart, Jonathan; Tucker, Julie A; Vogtherr, Martin; Whittaker, David; Wingfield, Jonathan; Winter, Jon; Hudson, Kevin

2012-04-12

44

Efficacy of DNA vaccines carrying Eimeria acervulina lactate dehydrogenase antigen gene against coccidiosis.  

PubMed

The efficacies of DNA vaccines encoding either Eimeria acervulina lactate dehydrogenase (LDH) antigen or a combination of LDH antigen and chicken IL-2 or IFN-gamma were evaluated against chicken coccidiosis. Three vaccine plasmids pVAX-LDH, pVAX-LDH-IFN-gamma and pVAX-LDH-IL-2 were constructed using the eukaryotic expression vector pVAX1. Expressions of proteins encoded by plasmids DNA in vivo were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay. Average body weight gain, oocyst output, survival rate and lesion scores were measured to evaluate the protective effects of vaccination on challenge infection. The results showed that DNA vaccines could obviously alleviate body weight loss, duodenal lesions, oocyst output and enhance oocyst decrease ratio. Anti-coccidial indexes (ACIs) of pVAX-LDH-IFN-gamma and pVAX-LDH-IL-2 groups were higher than that of other groups. Flow cytometric analysis of T lymphocytes in spleen and cecal tonsil demonstrated that DNA vaccines had significantly increased percentages of CD3(+) T cells compared with pVAX1 alone or TE buffer. The results provided the first proof that DNA vaccine carrying E. acervulina LDH antigen gene induced protective immunity against homologous infection and its effect could be enhanced by co-expression of chicken IL-2 or IFN-gamma. PMID:20566413

Song, Hongyan; Yan, Ruofeng; Xu, Lixin; Song, Xiaokai; Shah, Muhammad Ali A; Zhu, Huili; Li, Xiangrui

2010-10-01

45

Liquid-liquid extraction of lactate dehydrogenase from muscle using polymer-bound triazine dyes.  

PubMed

An extract from porcine muscle containing soluble enzymes has been partitioned between the two liquid phases of an aqueous, biphasic system consisting of dextran, polyethylene glycol, and water. The influence of polymer-bound triazine dyes (Cibacron blue F3G-A and Procion yellow HE-3G) on the partition of lactate dehydrogenase and total protein was studied. The effects of pH and concentrations of polymers and buffer on this so-called affinity partitioning were also determined. The two-phase systems were used in extraction procedures for purification of lactate dehydrogenase to a specific activity of 456-494 U (7.6-8.4 mukat) per mg protein. The use of these systems for extraction of enzymes in technical scale is discussed. PMID:3752985

Johansson, G; Joelsson, M

1986-08-01

46

A Role for Lactate Dehydrogenases in the Survival of Neisseria gonorrhoeae in Human Polymorphonuclear Leukocytes and Cervical Epithelial Cells.  

PubMed

Lactate is an abundant metabolite, produced by host tissues and commensal organisms, and it represents an important potential carbon source for bacterial pathogens. In the case of Neisseria spp., the importance of the lactate permease in colonization of the host has been demonstrated, but there have been few studies of lactate metabolism in pathogenic Neisseria in the postgenomic era. We describe herein the characterization of genome-annotated, respiratory, and substrate-level lactate dehydrogenases (LDHs) from the obligate human pathogen Neisseria gonorrhoeae. Biochemical assays using N. gonorrhoeae 1291 wild type and isogenic mutant strains showed that cytoplasmic LdhA (NAD(+)-dependent D-lactate dehydrogenase) and the membrane-bound respiratory enzymes, LdhD (D-lactate dehydrogenase) and LldD (L-lactate dehydrogenase) are correctly annotated. Mutants lacking LdhA and LdhD showed greatly reduced survival in neutrophils compared with wild type cells, highlighting the importance of D-lactate metabolism in gonococcal survival. Furthermore, an assay of host colonization using the well-established human primary cervical epithelial cell model revealed that the two respiratory enzymes make a significant contribution to colonization of and survival within the microaerobic environment of the host. Taken together, these data suggest that host-derived lactate is critical for the growth and survival of N. gonorrhoeae in human cells. PMID:24737798

Atack, John M; Ibranovic, Ines; Ong, Cheryl-Lynn Y; Djoko, Karrera Y; Chen, Nathan H; Vanden Hoven, Rachel; Jennings, Michael P; Edwards, Jennifer L; McEwan, Alastair G

2014-10-15

47

Computational analyses of mammalian lactate dehydrogenases: Human, mouse, opossum and platypus LDHs  

Microsoft Academic Search

Computational methods were used to predict the amino acid sequences and gene locations for mammalian lactate dehydrogenase (LDH) genes and proteins using genome sequence databanks. Human LDHA, LDHC and LDH6A genes were located in tandem on chromosome 11, while LDH6B and LDH6C genes were on chromosomes 15 and 12, respectively. Opossum LDHC and LDH6B genes were located in tandem with

Roger S. Holmes; Erwin Goldberg

2009-01-01

48

Physical and Functional Association of Lactate Dehydrogenase (LDH) with Skeletal Muscle Mitochondria*  

PubMed Central

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.

2013-01-01

49

Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target  

PubMed Central

Babesia microti is an emerging zoonotic protozoan organism that causes “malaria-like” symptoms that can be fatal in immunocompromised people. Owing to lack of specific therapeutic regiment against the disease, we cloned and characterized B. microti lactate dehydrogenase (BmLDH) as a potential molecular drug receptor. The in vitro kinetic properties of BmLDH enzyme was evaluated using nicotinamide adenine dinucleotide (NAD+) as a co-factor and lactate as a substrate. Inhibitory assay was also done using gossypol as BmLDH inhibitor to determine the inhibitory concentration 50 (IC50). The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite. In vitro enzyme kinetic studies further revealed that BmLDH is an active enzyme with a high catalytic efficiency at optimal pH of 10.2. The Km values of NAD+ and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively. The IC50 value for gossypol was 0.345 ?M, while at 2.5 ?M, gossypol caused 100% inhibition of BmLDH catalytic activity. These findings, therefore, provide initial evidence that BmLDH could be a potential drug target, although further in vivo studies are needed to validate the practical application of lactate dehydrogenase inhibitors against B. microti infection. PMID:25125971

Vudriko, Patrick; Masatani, Tatsunori; Cao, Shinuo; Terkawi, Mohamad Alla; Kamyingkird, Ketsarin; Mousa, Ahmed A; Adjou Moumouni, Paul F; Nishikawa, Yoshifumi; Xuan, Xuenan

2014-01-01

50

Lysine-5 Acetylation Negatively Regulates Lactate Dehydrogenase A and Is Decreased in Pancreatic Cancer  

PubMed Central

SUMMARY Tumor cells commonly have increased glucose uptake and lactate accumulation. Lactate is produced from pyruvate by lactate dehydrogenase A (LDH-A), which is frequently overexpressed in tumor cells and is important for cell growth. Elevated transcription by c-Myc or HIF1? may contribute to increased LDH-A in some cancer types. Here, we show that LDH-A is acetylated at lysine 5 (K5) and that this acetylation inhibits LDH-A activity. Furthermore, the K5-acetylated LDH-A is recognized by the HSC70 chaperone and delivered to lysosomes for degradation. Replacement of endogenous LDH-A with an acetylation mimetic mutant decreases cell proliferation and migration. Importantly, K5 acetylation of LDH-A is reduced in human pancreatic cancers. Our study reveals a mechanism of LDH-A upregulation in pancreatic cancers. PMID:23523103

Zhao, Di; Zou, Shao-Wu; Liu, Ying; Zhou, Xin; Mo, Yan; Wang, Ping; Xu, Yan-Hui; Dong, Bo; Xiong, Yue; Lei, Qun-Ying; Guan, Kun-Liang

2013-01-01

51

Lactate dehydrogenase ontogeny, paternal gene activation, and tetramer assembly in embryos of brook trout, lake trout, and their hybrids  

Microsoft Academic Search

Measurement of lactate dehydrogenase in reciprocal hybrids of trout during development revealed that a maternal effect was involved in the regulation of enzyme levels until resorption of the yolk sac was completed. Malate dehydrogenase specific activities were the same in these embryos and larvae. The more negatively charged B subunits of LDH predominated during early stages of embryogenesis in lake

Erwin Goldberg; J. P. Cuerrier; J. C. Ward

1969-01-01

52

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

53

Lactate dehydrogenase is the key enzyme for pneumococcal pyruvate metabolism and pneumococcal survival in blood.  

PubMed

Streptococcus pneumoniae is a fermentative microorganism and causes serious diseases in humans, including otitis media, bacteremia, meningitis, and pneumonia. However, the mechanisms enabling pneumococcal survival in the host and causing disease in different tissues are incompletely understood. The available evidence indicates a strong link between the central metabolism and pneumococcal virulence. To further our knowledge on pneumococcal virulence, we investigated the role of lactate dehydrogenase (LDH), which converts pyruvate to lactate and is an essential enzyme for redox balance, in the pneumococcal central metabolism and virulence using an isogenic ldh mutant. Loss of LDH led to a dramatic reduction of the growth rate, pinpointing the key role of this enzyme in fermentative metabolism. The pattern of end products was altered, and lactate production was totally blocked. The fermentation profile was confirmed by in vivo nuclear magnetic resonance (NMR) measurements of glucose metabolism in nongrowing cell suspensions of the ldh mutant. In this strain, a bottleneck in the fermentative steps is evident from the accumulation of pyruvate, revealing LDH as the most efficient enzyme in pyruvate conversion. An increase in ethanol production was also observed, indicating that in the absence of LDH the redox balance is maintained through alcohol dehydrogenase activity. We also found that the absence of LDH renders the pneumococci avirulent after intravenous infection and leads to a significant reduction in virulence in a model of pneumonia that develops after intranasal infection, likely due to a decrease in energy generation and virulence gene expression. PMID:25245810

Gaspar, Paula; Al-Bayati, Firas A Y; Andrew, Peter W; Neves, Ana Rute; Yesilkaya, Hasan

2014-12-01

54

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. PMID:4340864

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

1972-01-01

55

Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo 13C-NMR.  

PubMed

The metabolism of glucose by nongrowing cells of Lactococcus lactis strain FI7851, constructed from the wild-type L. lactis strain MG1363 by disruption of the lactate dehydrogenase (ldh) gene [Gasson, M.J., Benson, K., Swindel, S. & Griffin, H. (1996) Lait 76, 33-40] was studied in a noninvasive manner by 13C-NMR. The kinetics of the build-up and consumption of the pools of intracellular intermediates mannitol 1-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate, and phosphoenolpyruvate as well as the utilization of [1-13C]glucose and formation of products (lactate, acetate, mannitol, ethanol, acetoin, 2,3-butanediol) were monitored in vivo with a time resolution of 30 s. The metabolism of glucose by the parental wild-type strain was also examined for comparison. A clear shift from typical homolactic fermentation (parental strain) to a mixed acid fermentation (lactate dehdydrogenase deficient; LDHd strain) was observed. Furthermore, high levels of mannitol were transiently produced and metabolized once glucose was depleted. Mannitol 1-phosphate accumulated intracellularly up to 76 mM concentration. Mannitol was formed from fructose 6-phosphate by the combined action of mannitol-1-phosphate dehydrogenase and phosphatase. The results show that the formation of mannitol 1-phosphate by the LDHd strain during glucose catabolism is a consequence of impairment in NADH oxidation caused by a highly reduced LDH activity, the transient production of mannitol 1-phosphate serving as a regeneration pathway for NAD+ regeneration. Oxygen availability caused a drastic change in the pattern of intermediates and end-products, reinforcing the key-role of the fulfilment of the redox balance. The flux control coefficients for the step catalysed by mannitol-1-phosphate dehydrogenase were calculated and the implications in the design of metabolic engineering strategies are discussed. PMID:10849005

Neves, A R; Ramos, A; Shearman, C; Gasson, M J; Almeida, J S; Santos, H

2000-06-01

56

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. PMID:21159175

2010-01-01

57

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. PMID:24992632

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

2014-01-01

58

Kinetic resolution of 2-hydroxybutanoate racemic mixtures by NAD-independent l-lactate dehydrogenase  

Microsoft Academic Search

Optically active d-2-hydroxybutanoate is an important building block intermediate for medicines and biodegradable poly(2-hydroxybutanoate). Kinetic resolution of racemic 2-hydroxybutanoate may be a green and desirable alternative for d-2-hydroxybutanoate production. In this work, d-2-hydroxybutanoate at a high concentration (0.197M) and a high enantiomeric excess (99.1%) was produced by an NAD-independent l-lactate dehydrogenase (l-iLDH) containing biocatalyst. 2-Oxobutanoate, another important intermediate, was co-produced

Chao Gao; Wen Zhang; Cuiqing Ma; Peng Liu; Ping Xu

2011-01-01

59

Resting oxygen consumption varies among lactate dehydrogenase genotypes in the sow bug, Porcellio scaber  

PubMed Central

Laboratory studies of respiration in the sow bug, Porcellio scaber, reveal that respiration rates are related to genetic variation at the lactate dehydrogenase (Ldh) locus. In population samples taken from Burlington, North Carolina and Pacific Grove, California, respiration rates differed among Ldh genotypes, but not among genotypes at the other enzyme polymorphisms. In both population samples, the respiration rate of the common Ldh homozygote exceeded the respiration rate of the heterozygote by more than 50 per cent. The differences in respiration rates are consistent with previously reported viability differentials at the Ldh polymorphism.

Mitton, J. B.; Carter, P. A.; DiGiacomo, A.

1997-01-01

60

Diverse allosteric and catalytic functions of tetrameric d-lactate dehydrogenases from three Gram-negative bacteria  

PubMed Central

NAD-dependent d-lactate dehydrogenases (d-LDHs) reduce pyruvate into d-lactate with oxidation of NADH into NAD+. Although non-allosteric d-LDHs from Lactobacilli have been extensively studied, the catalytic properties of allosteric d-LDHs from Gram-negative bacteria except for Escherichia coli remain unknown. We characterized the catalytic properties of d-LDHs from three Gram-negative bacteria, Fusobacterium nucleatum (FNLDH), Pseudomonas aeruginosa (PALDH), and E. coli (ECLDH) to gain an insight into allosteric mechanism of d-LDHs. While PALDH and ECLDH exhibited narrow substrate specificities toward pyruvate like usual d-LDHs, FNLDH exhibited a broad substrate specificity toward hydrophobic 2-ketoacids such as 2-ketobutyrate and 2-ketovalerate, the former of which gave a 2-fold higher kcat/S0.5 value than pyruvate. Whereas the three enzymes consistently showed hyperbolic shaped pyruvate saturation curves below pH 6.5, FNLDH and ECLDH, and PALDH showed marked positive and negative cooperativity, respectively, in the pyruvate saturation curves above pH 7.5. Oxamate inhibited the catalytic reactions of FNLDH competitively with pyruvate, and the PALDH reaction in a mixed manner at pH 7.0, but markedly enhanced the reactions of the two enzymes at low concentration through canceling of the apparent homotropic cooperativity at pH 8.0, although it constantly inhibited the ECLDH reaction. Fructose 1,6-bisphosphate and certain divalent metal ions such as Mg2+ also markedly enhanced the reactions of FNLDH and PALDH, but none of them enhanced the reaction of ECLDH. Thus, our study demonstrates that bacterial d-LDHs have highly divergent allosteric and catalytic properties.

2014-01-01

61

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

62

Catabolism of circulating enzymes: plasma clearance, endocytosis, and breakdown of lactate dehydrogenase-1 in rabbits  

SciTech Connect

Lactate dehydrogenase-1, intravenously injected into rabbits, was cleared with first-order kinetics (half-life 27 min), until at least 80% of the injected activity had disappeared from plasma. Radioactivity from injected SVI-labeled enzyme disappeared at this same rate. Trichloroacetic-acid-soluble breakdown products started to appear in the circulation shortly after injection of the labeled enzyme. Body scans of the rabbits for 80 min after injection of T I-labeled enzyme revealed rapid accumulation of label in the liver, peaking 10-20 min after injection. Subsequently, activity in the liver declined and radioactivity (probably labeled breakdown products of low molecular mass) steadily accumulated in the bladder. Tissue fractionation of liver, 19 min after injection of labeled enzyme, indicated that the radioactivity was present both in endosomes and in lysosomes, suggesting uptake by endocytosis, followed by breakdown in the lysosomes. Measurements of radioactivity in liver and plasma suggest that the liver is responsible for the breakdown of at least 75% of the injected enzyme. Radioautography of tissue sections of liver and spleen showed accumulated radioactivity in sinusoidal liver cells and red pulpa, respectively. These results are very similar to those for lactate dehydrogenase-5, creatine kinase MM, and several other enzymes that we have previously studied in rats.

Smit, M.J.; Beekhuis, H.; Duursma, A.M.; Bouma, J.M.; Gruber, M.

1988-12-01

63

Integration of aqueous two-phase extraction and affinity precipitation for the purification of lactate dehydrogenase.  

PubMed

Integration of extraction in aqueous two-phase system and affinity precipitation was investigated as a technique for purification of lactate dehydrogenase (LDH) from porcine muscle extract. An enteric coating polymer, Eudragit S 100, which can be made reversibly soluble and insoluble by change in pH was used as the ligand carrier. The ligand used was Cibacron blue 3GA. The polymer is nearly totally partitioned to the top phase (> 98%) in PEG-dextran aqueous two-phase system. The enzyme, lactate dehydrogenase, was first spontaneously partitioned to the bottom phase in a 6% (w/w) PEG 8000-8% (w/w) dextran T250 phase system. New PEG phase and Eudragit-dye were then added to the bottom phase, which helped in extraction of LDH to the top phase. After a washing step with a fresh bottom phase, Eudragit-dye-target protein affinity complex was precipitated out from the top phase by lowering the pH to 5.1. The enzyme was recovered by treatment of the complex with 0.5 M NaCl with a yield of 54% and a specific activity of 245 units/mg. The purification of LDH by this procedure was better than that obtained by a single step of affinity partitioning. PMID:7516243

Guoqiang, D; Kaul, R; Mattiasson, B

1994-05-01

64

[Myoglobinuria due to enzyme abnormalities in glycolytic pathway--especially lactate dehydrogenase M subunit deficiency].  

PubMed

Glycolysis is an important energy productive system. Enzyme abnormalities the in glycolytic pathway, which cause myoglobinuria, are deficiencies of phosphofructokinase, phosphoglycerate kinase, phosphoglycerate mutase, and lactate dehydrogenase (LDH). Common symptoms of these enzyme abnormalities are muscle cramp, muscle pain, and rhabdomyolysis after strenuous exercise. Acute renal failure owing to myoglobinuria is the most noteworthy symptom. In daily life, symptoms are rarely observed and prognosis is usually good. Correct and fast diagnosis of such latent symptomatic disorders is important to prevent a turn for the worse of these symptoms. LDH M subunit deficiency was first discovered by urinary discoloration and a discrepancy of laboratory data. Since then, only four cases have been reported in the Japanese population. The response to ischemic forearm work is characteristic (an increase of venous lactate concentration after ischemic work is not observed and a marked increase of venous pyruvate is found). The increase of pyruvate concentration is specific in LDH-M subunit deficiency, and is not observed in other abnormalities of the glycolytic pathway. Glycolysis was markedly retarded in the patient's muscle in the glyceraldehyde 3-phosphate dehydrogenase (GA3PD) step, possibly due to the impaired reoxidation of NADH produced by GA3PD activity. Then, the excess NADH is reoxidized by alpha-glycerophosphate dehydrogenase and triose phosphates are drained to alpha-glycerophosphate and glycerol. Therefore ATP production is significantly impaired and muscle tissue is damaged. A genetical study revealed a deletion of 20 base-pairs in exon 6 in LDH-M subunit deficiency. This mutation results in a frame-shift translation and premature termination. PMID:1828277

Maekawa, M; Kanno, T; Sudo, K

1991-02-01

65

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

PubMed Central

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 Vmax, Km and I50 values between control and frozen LDH. However, control and dehydrated LDH differed significantly in Km values for pyruvate, lactate, and NAD, I50 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

2013-01-01

66

The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates.  

PubMed Central

Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is suggested that, under anaerobic conditions, muscles of marine invertebrates form lactate and/or octopine or succinate (or similar end product) according to the activities of the enzymes present in the muscles (see above). The muscles investigated possess low activities of cytosolic glycerol 3-phosphate dehydrogenase, which indicates that glycerol phosphate formation is quantitatively unimportant under anaerobic conditions, and low activities of mitochondrial glycerol phosphate dehydrogenase, which indicates that the glycerol phosphate cycle is unimportant in the re-oxidation of glycolytically produced NADH in these muscles under aerobic conditions. Conversely, high activities of glutamate-oxaloacetate transaminase are present in some muscles, which indicates that the malate-aspartate cycle may be important in oxidation of glycolytically produced NADH under aerobic conditions. 3. High activities of nucleoside diphosphate kinase were found in muscles that function for prolonged periods under anaerobic conditions (e.g... PMID:13783

Zammit, V A; Newsholme, E A

1976-01-01

67

Quinoline 3-sulfonamides inhibit lactate dehydrogenase A and reverse aerobic glycolysis in cancer cells  

PubMed Central

Background Most normal cells in the presence of oxygen utilize glucose for mitochondrial oxidative phosphorylation. In contrast, many cancer cells rapidly convert glucose to lactate in the cytosol, a process termed aerobic glycolysis. This glycolytic phenotype is enabled by lactate dehydrogenase (LDH), which catalyzes the inter-conversion of pyruvate and lactate. The purpose of this study was to identify and characterize potent and selective inhibitors of LDHA. Methods High throughput screening and lead optimization were used to generate inhibitors of LDHA enzymatic activity. Effects of these inhibitors on metabolism were evaluated using cell-based lactate production, oxygen consumption, and 13C NMR spectroscopy assays. Changes in comprehensive metabolic profile, cell proliferation, and apoptosis were assessed upon compound treatment. Results 3-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl) amino) benzoic acid was identified as an NADH-competitive LDHA inhibitor. Lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 nM and 10 to 80-fold selectivity over LDHB. Molecules in this family rapidly and profoundly inhibited lactate production rates in multiple cancer cell lines including hepatocellular and breast carcinomas. Consistent with selective inhibition of LDHA, the most sensitive breast cancer cell lines to lactate inhibition in hypoxic conditions were cells with low expression of LDHB. Our inhibitors increased rates of oxygen consumption in hepatocellular carcinoma cells at doses up to 3 microM, while higher concentrations directly inhibited mitochondrial function. Analysis of more than 500 metabolites upon LDHA inhibition in Snu398 cells revealed that intracellular concentrations of glycolysis and citric acid cycle intermediates were increased, consistent with enhanced Krebs cycle activity and blockage of cytosolic glycolysis. Treatment with these compounds also potentiated PKM2 activity and promoted apoptosis in Snu398 cells. Conclusions Rapid chemical inhibition of LDHA by these quinoline 3-sulfonamids led to profound metabolic alterations and impaired cell survival in carcinoma cells making it a compelling strategy for treating solid tumors that rely on aerobic glycolysis for survival. PMID:24280423

2013-01-01

68

THE EFFECT OF EXERCISE ON PLASMA ACTIVITIES OF LACTATE DEHYDROGENASE AND CREATINE KINASE IN RED-TAILED HAWKS (Buteo jamaicensis)  

Microsoft Academic Search

Plasma activities of lactate dehydrogenase (LD) and creatine kinase (CK) have been used as diagnostic indicators of muscle fitness and damage, respectively, in mammals. Activities of these enzymes were measured in three groups of red-tailed hawks (Buteojamaicensis) differing in flight capability (trained, untrained, and disabled) to determine whether their plasma enzyme activities were indicative of muscle fitness and flight training

SHANNON T. KNUTH; SUSAN B. CHAPLIN

69

Redox Balance via Lactate Dehydrogenase Is Important for Multiple Stress Resistance and Virulence in Enterococcus faecalis  

PubMed Central

Enterococcus faecalis is a highly stress resistant opportunistic pathogen. The intrinsic ruggedness of this bacterium is supposed to be the basis of its capacity to colonize the hostile environments of hospitals and to cause several kinds of infections. We show in this work that general resistance to very different environmental stresses depends on the ability of E. faecalis to maintain redox balance via lactate dehydrogenase (LDH). Furthermore, LDH-deficient mutants are less successful than the wild type at colonizing host organs in a murine model of systemic infection. Taken together, our results, as well as those previously published for Staphylococcus aureus (A. R. Richardson, S. J. Libby, and F. C. Fang, Science 319:1672–1676, 2008), identify LDH as an attractive drug target. These drugs may have additional applications, as in the fight against glycopeptide antibiotic-resistant bacteria and even cancer. PMID:23649090

Rana, Nosheen Fatima; Sauvageot, Nicolas; Laplace, Jean-Marie; Bao, YinYin; Nes, Ingolf; Rince, Alain; Posteraro, Brunella; Sanguinetti, Maurizio

2013-01-01

70

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. PMID:24885701

2014-01-01

71

The Approach to the Michaelis Complex in Lactate Dehydrogenase: The Substrate Binding Pathway  

PubMed Central

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 His195 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 Kd values (the ratios of the microscopic rate constants for each unimolecular step) are not far from one. Either substantial, ?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

2005-01-01

72

In situ Regeneration of NADH via Lipoamide Dehydrogenase-catalyzed Electron Transfer Reaction Evidenced by Spectroelectrochemistry  

SciTech Connect

NAD/NADH is a coenzyme found in all living cells, carrying electrons from one reaction to another. We report on characterizations of in situ regeneration of NADH via lipoamide dehydrogenase (LD)-catalyzed electron transfer reaction to regenerate NADH using UV-vis spectroelectrochemistry. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of NADH regeneration were measured as 0.80 {+-} 0.15 mM and 1.91 {+-} 0.09 {micro}M s-1 in a 1-mm thin-layer spectroelectrochemical cell using gold gauze as the working electrode at the applied potential -0.75 V (vs. Ag/AgCl). The electrocatalytic reduction of the NAD system was further coupled with the enzymatic conversion of pyruvate to lactate by lactate dehydrogenase to examine the coenzymatic activity of the regenerated NADH. Although the reproducible electrocatalytic reduction of NAD into NADH is known to be difficult compared to the electrocatalytic oxidation of NADH, our spectroelectrochemical results indicate that the in situ regeneration of NADH via LD-catalyzed electron transfer reaction is fast and sustainable and can be potentially applied to many NAD/NADH-dependent enzyme systems.

Tam, Tsz Kin; Chen, Baowei; Lei, Chenghong; Liu, Jun

2012-08-01

73

Evolutionary implications of the cDNA sequence of the single lactate dehydrogenase of a lamprey.  

PubMed Central

All vertebrates other than lampreys exhibit multiple loci encoding lactate dehydrogenase +ADL-LDH; (S)-lactate:NAD+ oxidoreductase, EC 1.1.1.27+BD. Of these loci, Ldh-A is expressed predominantly in muscle, Ldh-B is expressed predominantly in heart, and Ldh-C (where present) exhibits different tissue-restricted patterns of expression depending on the taxon. To examine the relationship of the single LDH of lampreys to other vertebrate LDHs, we have determined the cDNA sequence of the LDH of the sea lamprey Petromyzon marinus and compared it to previously published sequences from bacteria, plants, and vertebrates. The lamprey sequence exhibits a mixture of features of both LDH-A and LDH-B at the amino acid level that may account for its intermediate kinetic properties. Both distance and maximum parsimony analyses strongly reject a relationship of lamprey LDH with mammalian LDH-C but do not significantly distinguish among remaining alternative phylogenetic hypotheses. Evolutionary parsimony analyses suggest that the lamprey LDH is related to Ldh-A and that the single locus condition has arisen as a result of the loss of Ldh-B (prior to the appearance of Ldh-C). The collection of LDH sequences for further studies of the evolution of the vertebrate LDH gene family will be facilitated by the PCR approach that we have used to obtain the lamprey sequence. PMID:1542673

Stock, D W; Whitt, G S

1992-01-01

74

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. PMID:24816116

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

2014-01-01

75

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

76

Assessment of Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activities in Cow’s Milk as an Indicator of Subclinical Mastitis  

Microsoft Academic Search

This study examined the activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase\\u000a (AST) in the milk of lactating Holstein cows in association with subclinical mastitis (SCM). A total of 94 milk samples were\\u000a collected from 58 lactating dairy cows representing stages of lactation from the second to the tenth week after calving. Those\\u000a which were classified as

H. Babaei; L. Mansouri-Najand; M. M. Molaei; A. Kheradmand; M. Sharifan

2007-01-01

77

Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop, Pecten maximus (L).  

PubMed

cDNA for octopine dehydrogenase (ODH) from the adductor muscle of the great scallop, Pecten maximus, was cloned using 5'- and 3'-RACE. The cDNA comprises an ORF of 1197 nucleotides and the deduced amino acid sequence encodes a protein of 399 amino acids. ODH was heterologously expressed in Escherichia coli with a C-terminal penta His-tag. ODH-5His was purified to homogeneity using metal-chelate affinity chromatography and Sephadex G-100 gel filtration. Recombinant ODH had kinetic properties similar to those of wild-type ODH isolated from the scallop's adductor muscle. Site-directed mutagenesis was used to elucidate the involvement of several amino acid residues for the reaction catalyzed by ODH. Cys148, which is conserved in all opine dehydrogenases known to date, was converted to serine or alanine, showing that this residue is not intrinsically important for catalysis. His212, Arg324 and Asp329, which are also conserved in all known opine dehydrogenase sequences, were subjected to site-directed mutagenesis. Modification of these residues revealed their importance for the catalytic activity of the enzyme. Conversion of each of these residues to alanine resulted in strong increases in K(m) and decreases in k(cat) values for pyruvate and L-arginine, but had little effect on the K(m) and k(cat) values for NADH. Assuming a similar structure for ODH compared with the only available structure of a bacterial opine dehydrogenase, these three amino acids may function as a catalytic triad in ODH similar to that found in lactate dehydrogenase or malate dehydrogenase. The carboxyl group of pyruvate is then stabilized by Arg324. In addition to orienting the substrate, His212 will act as an acid-base catalyst by donating a proton to the carbonyl group of pyruvate. The acidity of this histidine is further increased by the proximity of Asp329. PMID:18028427

Müller, Andre; Janssen, Frank; Grieshaber, Manfred K

2007-12-01

78

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

79

The promotion effect of titania nanoparticles on the direct electrochemistry of lactate dehydrogenase sol–gel modified gold electrode  

Microsoft Academic Search

The promotion effect of titania nanoparticles (nano-TiO2) on the direct electron transfer between lactate dehydrogenase (LDH) and the silica sol–gel modified gold electrode was investigated by adding nano-TiO2 (50nm) in the modification process. This nano-TiO2–LDH electrode showed a pair of quasi-reversible cyclic voltammetry peaks with the formal potential of 70mV (vs. SCE). Compared to the previous result of LDH modified

Jiongjia Cheng; Junwei Di; Jianhui Hong; Kaian Yao; Yongbo Sun; Jingyue Zhuang; Quan Xu; Huie Zheng; Shuping Bi

2008-01-01

80

Nuclear magnetic resonance and molecular genetic studies of the membrane-bound D-lactate dehydrogenase of Escherichia coli.  

PubMed

In this study we demonstrate the potential of combining fluorine-19 nuclear magnetic resonance (NMR) spectroscopy with molecular genetics. We are using the membrane-bound enzyme D-lactate dehydrogenase of Escherichia coli as a model system to characterize interactions between proteins and lipids. We have labeled D-lactate dehydrogenase with 4-, 5-, and 6-fluorotryptophans and obtained high-resolution fluorine-19 NMR spectra showing five resonances, in agreement with the five tryptophan residues expected from the DNA sequence. The five 19F resonances in the spectra have been assigned to the specific tryptophan residues in the primary sequence of D-lactate dehydrogenase by site-directed oligonucleotide mutagenesis of the cloned gene. We observe large differences in the relative fluorine-19 chemical shifts of each tryptophan residue when labeled by different isomers of fluorotryptophan. We have determined by NMR methods that two tryptophans are exposed to the solvent and that none of the tryptophan residues are within 10 A of the lipid phase. On the basis of 19F NMR spectroscopy of the labeled tryptophan residues, the conformation of D-lactate dehydrogenase is similar in aqueous solution and in the presence of a variety of lipids and detergents. This result indicates that the presence of lipids or detergents is not required to maintain the tertiary structure of this membrane-bound enzyme. In contrast, Triton X-100 induces a change to an abnormal conformation of the enzyme as judged from both NMR spectroscopy and the effect of temperature on the maximal velocity of the enzyme in the presence of this detergent. PMID:3548821

Rule, G S; Pratt, E A; Simplaceanu, V; Ho, C

1987-01-27

81

Lactate dehydrogenase activity in bovine and porcine muscle as influenced by electrical stimulation, aging, freezing, thawing and heating  

E-print Network

, semitendinosus, biceps femoris, rectus femoris and adductor muscles were dissected from nine fresh hams. Each muscle was analyzed for lactate dehydrogenase (LDH) activity after receiving one of eighteen treatments: fresh, untreated held at 4 C for 4 d; aged...). The semimembranosus (SM), semitendinosus (ST), biceps femoris (BF) and rectus femoris (RF) muscles were dissected and closely trimmed of all connective tissue membranes and seam and intermuscular fat. The hams were treated as follows: Ham Pl-Fresh. The four...

Collins, Sharen Sue

2012-06-07

82

Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.  

PubMed

Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles. Around 3,000 clones were screened to obtain 152 mutants. Only heterozygous mutants were identified. An attempt to knockout the remaining wild-type allele from one such heterozygote yielded only two mutants after screening 567 clones. One had an extra valine. Another evidenced a duplication event, possessing at lease one wild-type and two different frameshifted alleles. Both mutants still retained LDH activity. Together, our data strongly suggest that a complete knockout of LDHA is lethal in CHO cells, despite simultaneous down-regulation of PDHK1, 2, and 3. PMID:24841241

Yip, Shirley S M; Zhou, Meixia; Joly, John; Snedecor, Bradley; Shen, Amy; Crawford, Yongping

2014-09-01

83

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. PMID:21968246

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

2011-01-01

84

Antibody-mediated rejection: importance of lactate dehydrogenase and neutrophilia in early diagnosis.  

PubMed

We report the importance of elevated serum lactate dehydrogenase (LDH) and neutrophilia (NT) in two renal transplant recipients who developed renal impairment in the early post-operative period. One of our recipients developed oliguria and increased serum creatinine with unexplained elevation of LDH and NT. The biopsy was C4d positive with platelet and fibrin thrombi in the glomerular capillaries and arterioles and interpreted as acute vasculitis or thrombotic form of antibody-mediated rejection (VAMR) with positive donor-specific antibodies (DSA). Despite intensive treatment, this graft was lost. When another patient developed a similar picture, prompt immunoadsorption was started without waiting for a confirmatory biopsy or DSA, and both were later reported as positive. Improvement in renal function was associated with decreasing levels of LDH and NT. Neither of these was elevated in cases of acute cellular rejection (ACR) or antibody mediated rejection (AMR) with isolated tubular injury (TAMR). It may therefore be reasonable to assume that LDH and NT are potential diagnostic and prognostic markers of VAMR. PMID:21566312

Khan, Taqi Toufeeq; Mirza, Anzar Baig; Zahid, Rafat; Haleem, Abdul; Al Hussaini, Hussa; Al Sulaiman, Mohammad; Mousa, Dujana

2011-05-01

85

Lactate Dehydrogenase Activity in Gingival Crevicular Fluid as a Marker in Orthodontic Tooth Movement  

PubMed Central

Objectives: This study aims at analyzing the changes in gingival crevicular fluid (GCF) lactate dehydrogenase (LDH) activity during orthodontic movement. Methods: Twenty patients all requiring first premolar extractions were selected and treated with conventional straight wire mechanotherapy. Canine retraction was done using 125 g Nitinol closed coil springs. The maxillary canine on one side served as the experimental site while the contralateral canine served as the control. GCF was collected from the canines before initiation of retraction, then 1 hour after initiating canine retraction, followed by 1 day, 7 days, 14 days and 21 days. GCF LDH levels were estimated and compared with the control site. Results The results revealed significantly higher LDH levels on the 7th, 14th and 21st day at the sites where orthodontic force had been applied. The levels also showed a significant increase from 0 hour to the 21st day. Peak levels were seen on 14th and 21st day following initiation of retraction. Conclusions: The study showed that LDH could be successfully estimated in the GCF and its increased levels could indicate active tooth movement, which could aid the clinician in monitoring active orthodontic tooth movement. PMID:21760863

Alfaqeeh, Sarah A; Anil, Sukumaran

2011-01-01

86

INACTIVATION OF LACTATE DEHYDROGENASE BY SEVERAL CHEMICALS: IMPLICATIONS FOR IN VITRO TOXICOLOGY STUDIES  

PubMed Central

Lactate dehydrogenase (LDH) release is frequently used as an end-point for cytotoxicity studies. We have been unable to measure LDH release during studies using para-aminophenol (PAP) in LLC-PK1 cells. When LLC-PK1 cells were incubated with either PAP (0–10 mM) or menadione (0–1000 ?M), viability was markedly reduced when assessed by alamar Blue or total LDH activity but not by release of LDH into the incubation medium. In addition, we incubated cells with PAP or menadione and compared LDH activity using two different assays. Both assays confirmed our observation of decreased LDH activity in cell lysates without corresponding increases in LDH activity in incubation media. Using purified LDH and 10 mM PAP, we that PAP produced loss of LDH activity that was inversely proportional to the amount of LDH initially added. In additional experiments, we incubated 0.5 units of LDH for 1 h with varying concentrations of PAP, menadione, hydrogen peroxide (H2O2) or cisplatin. All four chemicals produced concentration-dependent decreases in LDH activity. In previous experiments, inclusion of antioxidants such as reduced glutathione (GSH) and ascorbate protected cells from PAP toxicity. GSH (1 mM) preserved LDH activity in the presence of toxicants while ascorbate (1 mM) only prevented LDH loss induced by PAP. These studies suggest that LDH that is released into the incubation medium is susceptible to degradation when reactive chemicals are present. PMID:17079110

Kendig, Derek M.; Tarloff, Joan B.

2007-01-01

87

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. PMID:22077414

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

2011-01-01

88

Establishment of permanent chimerism in a lactate dehydrogenase-deficient mouse mutant with hemolytic anemia  

SciTech Connect

Pluripotent hemopoietic stem cell function was investigated in the homozygous muscle type lactate dehydrogenase (LDH-A) mutant mouse using bone marrow transplantation experiments. Hemopoietic tissues of LDH-A mutants showed a marked decreased in enzyme activity that was associated with severe hemolytic anemia. This condition proved to be transplantable into wild type mice (+/+) through total body irradiation (TBI) at a lethal dose of 8.0 Gy followed by engraftment of mutant bone marrow cells. Since the mutants are extremely radiosensitive (lethal dose50/30 4.4 Gy vs 7.3 Gy in +/+ mice), 8.0-Gy TBI followed by injection of even high numbers of normal bone marrow cells did not prevent death within 5-6 days. After a nonlethal dose of 4.0 Gy and grafting of normal bone marrow cells, a transient chimerism showing peripheral blood characteristics of the wild type was produced that returned to the mutant condition within 12 weeks. The transfusion of wild type red blood cells prior to and following 8.0-Gy TBI and reconstitution with wild type bone marrow cells prevented the early death of the mutants and permanent chimerism was achieved. The chimeras showed all hematological parameters of wild type mice, and radiosensitivity returned to normal. It is concluded that the mutant pluripotent stem cells are functionally comparable to normal stem cells, emphasizing the significance of this mouse model for studies of stem cell regulation.

Datta, T.; Doermer, P.

1987-12-01

89

Overexpression of lactate dehydrogenase-A in human intrahepatic cholangiocarcinoma: its implication for treatment  

PubMed Central

Background Previous studies have shown that lactate dehydrogenase-A (LDH-A) is strongly expressed in several malignancies, that LDH-A expression is associated with poor prognosis, and that LDH-A inhibition severely diminishes tumorigenicity. However, little is known about the implications of LDH-A expression in intrahepatic cholangiocarcinoma. The purpose of this study was to investigate the expression of LDH-A and to clarify its effect on intrahepatic cholangiocarcinoma. Methods We studied the expression of LDH-A in tissue samples from patients with intrahepatic cholangiocarcinoma (n?=?54) using the ultrasensitive surfactant protein (S-P) immunohistochemical method. We then inhibited LDH-A using small hairpin RNA (shRNA) in the cholangiocarcinoma cell line HuCCT-1 in vitro to study the role it plays in promoting growth and escaping apoptosis. Results We report that LDH-A was overexpressed in 52 of 54 (96%) paraffin-embedded cancer tissue samples and 0 of 54 para-carcinoma tissue samples. Reduction of LDH-A by RNA interference (RNAi) inhibited cell growth and induced apoptosis in HuCCT-1 cells. This result correlated with the elevation of cytoplasmic reactive oxygen species (ROS) levels. Conclusions LDH-A expression is closely correlated with histopathological variables of intrahepatic cholangiocarcinoma, indicating that LDH-A may serve as a new treatment target. PMID:24679073

2014-01-01

90

Improved partitioning in aqueous two-phase system of tyrosine-tagged recombinant lactate dehydrogenase.  

PubMed

The partitioning of Bacillus stearothermophilus lactate dehydrogenase (LDH) in an aqueous two-phase system was studied. Particularly, the influence of tyrosine tags on the partitioning was evaluated. The hydrophobic effect, caused by the addition of tyrosine residues, was determined in a system based on dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer (EO30PO70). Five different LDH variants were constructed with N-terminal tags containing tyrosines (Y3 and Y6), tyrosines and prolines (Y3P2 and Y6P2), and only prolines (P2). LDH fused with tags containing tyrosines increased the partitioning coefficient, and the more tyrosines added to the protein, the larger improvement in partitioning. When prolines were added between the tyrosine-rich tag and the protein, a further increased partitioning was obtained. The enhanced partitioning was attributed to the rigid structure of the proline, which in turn led to an increase in the exposure of the tag to the surroundings. The best tyrosine tag, Y6P2, increased the partition coefficient four times and additionally, a higher thermostability was observed. PMID:12135559

Fexby, Sara; Bülow, Leif

2002-07-01

91

Stable high surface area lactate dehydrogenase particles produced by spray freezing into liquid nitrogen.  

PubMed

Enzyme activities were determined for lactate dehydrogenase (LDH) powder produced by lyophilization, and two fast freezing processes, spray freeze-drying (SFD) and spray freezing into liquid (SFL) nitrogen. The 0.25 mg/mL LDH aqueous feed solutions included either 30 or 100 mg/mL trehalose. The SFL process produced powders with very high enzyme activities upon reconstitution, similar to lyophilization. However, the specific surface area of 13 m(2)/g for SFL was an order of magnitude larger than for lyophilization. In SFD activities were reduced in the spraying step by the long exposure to the gas-liquid interface for 0.1-1s, versus only 2 ms in SFL. The ability to produce stable high surface area submicron particles of fragile proteins such as LDH by SFL is of practical interest in protein storage and in various applications in controlled release including encapsulation into bioerodible polymers. The SFL process has been scaled down for solution volumes <1 mL to facilitate studies of therapeutic proteins. PMID:17027245

Engstrom, Josh D; Simpson, Dale T; Cloonan, Carrie; Lai, Edwina S; Williams, Robert O; Barrie Kitto, G; Johnston, Keith P

2007-02-01

92

An atomic-resolution view of neofunctionalization in the evolution of apicomplexan lactate dehydrogenases  

PubMed Central

Malate and lactate dehydrogenases (MDH and LDH) are homologous, core metabolic enzymes that share a fold and catalytic mechanism yet possess strict specificity for their substrates. In the Apicomplexa, convergent evolution of an unusual LDH from MDH produced a difference in specificity exceeding 12 orders of magnitude. The mechanisms responsible for this extraordinary functional shift are currently unknown. Using ancestral protein resurrection, we find that specificity evolved in apicomplexan LDHs by classic neofunctionalization characterized by long-range epistasis, a promiscuous intermediate, and few gain-of-function mutations of large effect. In canonical MDHs and LDHs, a single residue in the active-site loop governs substrate specificity: Arg102 in MDHs and Gln102 in LDHs. During the evolution of the apicomplexan LDH, however, specificity switched via an insertion that shifted the position and identity of this ‘specificity residue’ to Trp107f. Residues far from the active site also determine specificity, as shown by the crystal structures of three ancestral proteins bracketing the key duplication event. This work provides an unprecedented atomic-resolution view of evolutionary trajectories creating a nascent enzymatic function. DOI: http://dx.doi.org/10.7554/eLife.02304.001 PMID:24966208

Boucher, Jeffrey I; Jacobowitz, Joseph R; Beckett, Brian C; Classen, Scott; Theobald, Douglas L

2014-01-01

93

Computational analyses of mammalian lactate dehydrogenases: human, mouse, opossum and platypus LDHs.  

PubMed

Computational methods were used to predict the amino acid sequences and gene locations for mammalian lactate dehydrogenase (LDH) genes and proteins using genome sequence databanks. Human LDHA, LDHC and LDH6A genes were located in tandem on chromosome 11, while LDH6B and LDH6C genes were on chromosomes 15 and 12, respectively. Opossum LDHC and LDH6B genes were located in tandem with the opossum LDHA gene on chromosome 5 and contained 7 (LDHA and LDHC) or 8 (LDH6B) exons. An amino acid sequence prediction for the opossum LDH6B subunit gave an extended N-terminal sequence, similar to the human and mouse LDH6B sequences, which may support the export of this enzyme into mitochondria. The platypus genome contained at least 3 LDH genes encoding LDHA, LDHB and LDH6B subunits. Phylogenetic studies and sequence analyses indicated that LDHA, LDHB and LDH6B genes are present in all mammalian genomes examined, including a monotreme species (platypus), whereas the LDHC gene may have arisen more recently in marsupial mammals. PMID:19679512

Holmes, Roger S; Goldberg, Erwin

2009-10-01

94

Kinetic study of heavy metal salt effects on the activity of L-lactate dehydrogenase in solution or immobilized on an oxygen electrode  

Microsoft Academic Search

A sensitive and convenient biosensor for detection of heavy metal salts has been developed. The method is based on the effects of heavy metal salts on the catalytic activity of L-lactate dehydrogenase (LDH) in solution or coimmobilized with L-lactate oxidase (LOD) on an oxygen electrode. At metal concentrations below 100 ?M, the kinetic behavior, with the LDH substrate NADH, showed

S. Fennouh; V. Casimiri; A. Geloso-Meyer; C. Burstein

1998-01-01

95

Impact of probiotic-supplemented diet on the expression level of lactate dehydrogenase in the leukocytes of rabbits.  

PubMed

Probiotics are known as living, nonpathogenic microorganisms that colonize the intestine and provide benefit to the host. The present study aims to measure one important energy metabolism-related enzyme activity in blood of rabbits fed on probiotics of recommended concentration. In addition, it also aims for the evaluation of the expression level of lactate dehydrogenase (LDH) enzyme using reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Two groups of rabbits are used: control group receiving normal standardized diet and the other probiotic-supplemented group receiving the same diet containing probiotic, namely, Mega acidophilus (200 million cfu/kg body weight/day) for 4 weeks. The obtained results revealed that the rabbits supplemented with probiotics showed a significant decrease in the levels of serum total cholesterol (TC), triacylglycerol, high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) when compared with control group. Risk factors detected by measuring TC/HDL-c and LDL-c/HDL-c ratios showed statistically significant decrease in probiotic-supplemented rabbits when compared with control group. In addition, blood glucose and total LDH activity were elevated in probiotic-supplemented rabbits when compared with control group. RT-PCR products of LDH-M gene produced two specific amplicons. One amplicon has the expected size of 243 bp from all samples of rabbits as revealed by GelPro software. The level of LDH-M expression was found to be increased in the probiotic-supplemented group. However, unexpected amplicons are produced at 586 bp in all the samples, which may be a dimeric form of the amplified region. It was concluded that this probiotic blend is beneficiary for the metabolic reactions of lipids in the body. Moreover, LDH expression level can be considered as a biomarker for the effect of probiotic and hence monitoring the metabolic changes as reflected from its administration. PMID:22865283

Ghoneim, Magdy A E; Moselhy, Said S

2014-04-01

96

Lactate dehydrogenase regulation in aged skeletal muscle: Regulation by anabolic steroids and functional overload.  

PubMed

Aging alters the skeletal muscle response to overload-induced growth. The onset of functional overload is characterized by increased myoblast proliferation and an altered muscle metabolic profile. The onset of functional overload is associated with increased energy demands that are met through the interconversion of lactate and pyruvate via the activity of lactate dehydrogenase (LDH). Testosterone targets many of the processes activated at the onset of functional overload. However, the effect of aging on this metabolic plasticity at the onset of functional overload and how anabolic steroid administration modulates this response is not well understood. The purpose of this study was to determine if aging would alter overload-induced LDH activity and expression at the onset of functional overload and whether anabolic steroid administration would modulate this response. Five-month and 25-month male Fischer 344xF1 BRN were given nandrolone decanoate (ND) or sham injections for 14days and then the plantaris was functionally overloaded (OV) for 3days by synergist ablation. Aging reduced muscle LDH-A & LDH-B activity 70% (p<0.05). Aging also reduced LDH-A mRNA abundance, however there was no age effect on LDH-B mRNA abundance. In 5-month muscle, both ND and OV decreased LDH-A and LDH-B activity. However, there was no synergistic or additive effect. In 5-month muscle, ND and OV decreased LDH-A mRNA expression with no change in LDH-B expression. In 25-month muscle, ND and OV increased LDH-A and LDH-B activity. LDH-A mRNA expression was not altered by ND or OV in aged muscle. However, there was a main effect of OV to decrease LDH-B mRNA expression. There was also an age-induced LDH isoform shift. ND and OV treatment increased the "fast" LDH isoforms in aged muscle, whereas ND and OV increased the "slow" isoforms in young muscle. Our study provides evidence that aging alters aspects of skeletal muscle metabolic plasticity normally induced by overload and anabolic steroid administration. PMID:24835193

Washington, Tyrone A; Healey, Julie M; Thompson, Raymond W; Lowe, Larry L; Carson, James A

2014-09-01

97

Plasmodium falciparum and Plasmodium vivax specific lactate dehydrogenase: genetic polymorphism study from Indian isolates.  

PubMed

Control and eradication of malaria is hindered by the acquisition of drug resistance by Plasmodium species. This has necessitated a persistent search for novel drugs and more efficient targets. Plasmodium species specific lactate dehydrogenase is one of the potential therapeutic and diagnostic targets, because of its indispensable role in endoerythrocytic stage of the parasite. A target molecule that is highly conserved in the parasite population can be more effectively used in diagnostics and therapeutics, hence, in the present study polymorphism in PfLDH (Plasmodiumfalciparum specific LDH) and PvLDH (Plasmodiumvivax specific LDH) genes was analyzed using PCR-single strand confirmation polymorphism (PCR-SSCP) and sequencing. Forty-six P. falciparum and thirty-five P. vivax samples were screened from different states of India. Our findings have revealed presence of a single PfLDH genotype and six PvLDH genotypes among the studied samples. Interestingly, along with synonymous substitutions, nonsynonymous substitutions were reported to be present for the first time in the PvLDH genotypes. Further, through amino acid sequence alignment and homology modeling studies we observed that the catalytic residues were conserved in all PvLDH genotypes and the nonsynonymous substitutions have not altered the enzyme structure significantly. Evolutionary genetics studies have confirmed that PfLDH and PvLDH loci are under strong purifying selection. Phylogenetic analysis of the pLDH gene sequences revealed that P. falciparum compared to P. vivax, has recent origin. The study therefore supports PfLDH and PvLDH as suitable therapeutic and diagnostic targets as well as phylogenetic markers to understand the genealogy of malaria species. PMID:24953504

Keluskar, Priyadarshan; Singh, Vineeta; Gupta, Purva; Ingle, Sanjay

2014-08-01

98

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. PMID:21779323

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

2011-01-01

99

Lactate Dehydrogenase B: A Metabolic Marker of Response to Neoadjuvant Chemotherapy in Breast Cancer  

PubMed Central

Purpose Although breast cancers are known to be molecularly heterogeneous, their metabolic phenotype is less well understood and may predict response to chemotherapy. This study aimed to evaluate metabolic genes as individual predictive biomarkers in breast cancer. Methods mRNA microarray data from breast cancer cell lines were used to identify bimodal genes – those with highest potential for robust high/low classification in clinical assays. Metabolic function was evaluated in vitro for the highest scoring metabolic gene, lactate dehydrogenase B (LDHB). Its expression was associated with neoadjuvant chemotherapy response and relapse within clinical and PAM50-derived subtypes. Results LDHB was highly expressed in cell lines with glycolytic, basal-like phenotypes. Stable knockdown of LDHB in cell lines reduced glycolytic dependence, linking LDHB expression directly to metabolic function. Using patient datasets, LDHB was highly expressed in basal-like cancers and could predict basal-like subtype within clinical groups (odds ratio = 21 for hormone-receptor (HR)-positive/HER2-negative; odds ratio = 10 for triple-negative). Furthermore, high LDHB predicted pathological complete response (pCR) to neoadjuvant chemotherapy for both HR-positive/HER2-negative (odds ratio = 4.1, P < .001) and triple-negative (odds ratio = 3.0, P = .003) cancers. For triple-negative tumors without pCR, high LDHB post-treatment also identified proliferative tumors with increased risk of recurrence (hazard ratio = 2.2, P = .006). Conclusions Expression of LDHB predicted response to neoadjuvant chemotherapy within clinical subtypes independently of standard prognostic markers and PAM50-subtyping. These observations support prospective clinical evaluation of LDHB as a predictive marker of response for breast cancer patients receiving neoadjuvant chemotherapy. PMID:23697991

Dennison, Jennifer B.; Molina, Jennifer R.; Mitra, Shreya; Gonzalez-Angulo, Ana M.; Balko, Justin M.; Kuba, Maria G.; Sanders, Melinda E.; Pinto, Joseph A.; Gomez, Henry L.; Arteaga, Carlos L.; Brown, Robert E.; Mills, Gordon B.

2013-01-01

100

Effect of water activity on inactivation of Listeria monocytogenes and lactate dehydrogenase during high pressure processing.  

PubMed

The aim of this study was to investigate the effect of water activity (aw) on the inactivation of Listeria monocytogenes and lactate dehydrogenase (LDH) during high pressure processing (HPP). For microbial inactivation lyophilized cells of L. monocytogenes 19,115 were left dry or were suspended in 10 ml of 0.1% peptone water, 10 ml of glycerol, or mixtures of glycerol and peptone water. All samples of various aws were high pressure (HP) processed at ambient temperature at 600 MPa for 300 s. Following HPP, samples were serially diluted in 0.1% peptone and spread-plated on Tryptic Soy agar supplemented with Yeast Extract. For enzyme inactivation, 4.2 mg of lyophilized LDH was suspended in 2 ml of 100 mM phosphate buffer (pH 7.4), 2 ml of peptone water or glycerol, or in 2 ml mixtures of glycerol and peptone water. A lyophilized sample with no added liquid was also included. All enzyme samples were subjected to HPP as described above. After HPP, LDH was diluted to 0.28 microg/ml in 100 mM phosphate buffer (pH 7.4). LDH activity was assessed by measuring the change in concentration of beta-NADH as a function of time. Dynamic light scattering analysis (DLS) was performed to examine the size distribution, polydispersity, and hydrodynamic radius of LDH before and after HPP. No significant difference in CFU/g was observed between lyophilized cells not subjected to HPP and lyophilized cells subjected to 600 MPa for 300 s (P<0.05). However, lyophilized cells that were suspended in 100% to 60% peptone water showed a approximately 7.5-log(10) reduction when subjected to HPP. Survival of L. monocytogenes following HPP significantly increased (P<0.05) when the peptone water concentration was decreased below 60% (aw approximately 0.8). DLS results revealed that LDH suspended in buffer underwent aggregation following HPP (600 MPa, 300 s). Inactivation rate constants obtained using a first-order kinetic model indicated that untreated and HP processed lyophilized LDH had similar activities. When LDH was subject to HPP in solutions containing glycerol, enzyme activity decreased as the water content increased (r2=0.95). Lyophilization completely protected L. monocytogenes and LDH from inactivation by high pressure. Furthermore, enzyme activity and cell survival increased as water activity was decreased. We postulate low aw results in protein stabilization, which prevents protein denaturation and cell death during HPP. PMID:18403036

Hayman, Melinda M; Kouassi, Gilles K; Anantheswaran, Ramaswamy C; Floros, John D; Knabel, Stephen J

2008-05-10

101

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. PMID:24708595

2014-01-01

102

The reaction of choline dehydrogenase with some electron acceptors.  

PubMed Central

1. The choline dehydrogenase (EC 1.1.99.1) WAS SOLUBILIZED FROM ACETONE-DRIED POWDERS OF RAT LIVER MITOCHONDRIA BY TREATMENT WITH Naja naja venom. 2. The kinetics of the reaction of enzyme with phenazine methosulphate and ubiquinone-2 as electron acceptors were investigated. 3. With both electron acceptors the reaction mechanism appears to involve a free, modified-enzyme intermediate. 4. With some electron acceptors the maximum velocity of the reaction is independent of the nature of the acceptor. With phenazine methosulphate and ubiquinone-2 as acceptors the Km value for choline is also independent of the nature of the acceptor molecule. 5. The mechanism of the Triton X-100-solubilized enzyme is apparently the smae as that for the snake venom solubilized enzyme. PMID:1218095

Barrett, M C; Dawson, A P

1975-01-01

103

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. PMID:21969607

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

104

Efficient Production of (R)-2-Hydroxy-4-Phenylbutyric Acid by Using a Coupled Reconstructed d-Lactate Dehydrogenase and Formate Dehydrogenase System  

PubMed Central

Background (R)-2-Hydroxy-4-phenylbutyric acid [(R)-HPBA] is a key precursor for the production of angiotensin-converting enzyme inhibitors. However, the product yield and concentration of reported (R)-HPBA synthetic processes remain unsatisfactory. Methodology/Principal Findings The Y52L/F299Y mutant of NAD-dependent d-lactate dehydrogenase (d-nLDH) in Lactobacillus bulgaricus ATCC 11842 was found to have high bio-reduction activity toward 2-oxo-4-phenylbutyric acid (OPBA). The mutant d-nLDHY52L/F299Y was then coexpressed with formate dehydrogenase in Escherichia coli BL21 (DE3) to construct a novel biocatalyst E. coli DF. Thus, a novel bio-reduction process utilizing whole cells of E. coli DF as the biocatalyst and formate as the co-substrate for cofactor regeneration was developed for the production of (R)-HPBA from OPBA. The biocatalysis conditions were then optimized. Conclusions/Significance Under the optimum conditions, 73.4 mM OPBA was reduced to 71.8 mM (R)-HPBA in 90 min. Given its high product enantiomeric excess (>99%) and productivity (47.9 mM h?1), the constructed coupling biocatalysis system is a promising alternative for (R)-HPBA production. PMID:25089519

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

2014-01-01

105

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

106

Effects of the suppression of lactate dehydrogenase A on the growth and invasion of human gastric cancer cells.  

PubMed

Lactate dehydrogenase A (LDH-A), which regulates glycolytic flux by catalyzing pyruvate to lactate in the cytoplasm, is believed to be one of the highly attractive therapeutic targets for cancers. Firstly, we detected the expression of LDH-A in gastric cancer (GC) cells. LDH-A inhibitor oxamate was then used to suppress the LDH-A activity in GC cells. Cell proliferation, lactic acid production, Transwell migration assay and apoptosis were assessed, respectively. The results showed that inhibition of LDH-A by oxamate decreased the lactate production. In the presence of glucose, oxamate inhibited cell proliferation in a dose-dependent manner. Flow cytometry assay further confirmed a pro-apoptotic effect of oxamate, and this was likely through increased expression of Bax, activated caspase-3, and decreased expression of Bcl-2. Therefore, we believe that oxamate inhibits cell growth, suppresses tumor invasion, and induces apoptosis in GC cells. LDH-A may be a potential therapeutic target for GC. PMID:25394466

Liu, Xiaojun; Yang, Zhongxia; Chen, Zhaofeng; Chen, Rui; Zhao, Da; Zhou, Yongning; Qiao, Liang

2015-01-01

107

Mitochondrial Lactate Dehydrogenase Is Involved in Oxidative-Energy Metabolism in Human Astrocytoma  

E-print Network

in muscles [8]. The involvement of a lactate shuttle in peroxisomes confers this organelle with the ability to metabolize fatty acids. The presence of this enzyme was recently confirmed in liver peroxisomes [9

Appanna, Vasu

108

Cyclic AMP and AKAP-mediated targeting of protein kinase A regulates lactate dehydrogenase subunit A mRNA stability.  

PubMed

Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA subunits and AKAP 95 from RSW extracts by immunoprecipitation resulted in a marked loss of mRNA stabilization activity indicating that the presence of the PKA regulatory and catalytic subunits as well as AKAP 95 in the CSR-protein complexes was absolutely necessary to achieve LDH-A mRNA stabilization. PMID:15878851

Jungmann, Richard A; Kiryukhina, Olga

2005-07-01

109

Importance of lactate dehydrogenase for the regulation of glycolytic flux and insulin secretion in insulin-producing cells.  

PubMed

The role of lactate dehydrogenase (LDH) in the generation of the metabolic signal for insulin secretion was studied after stable overexpression in INS-1 and RINm5F insulin-producing cells. INS-1 cells with a 25-fold overexpression of LDH-A, the highest level achieved, showed a 20-30% decrease in the glucose oxidation rate at glucose concentrations above 5 mM when compared with control cells, whereas values were unchanged at lower glucose concentrations. Lactate release increased in parallel with a decrease in the glucose oxidation rate. However, the INS-1 cell glucose-induced insulin secretory response, together with the rate of glucose utilization, were not significantly affected by LDH-A overexpression. Despite 3-fold overexpression of LDH-A in glucose-unresponsive RINm5F cells, there was no change in insulin secretion, glucose metabolism or lactate production in these cells. Exogenously added pyruvate and lactate potentiated glucose-stimulated insulin secretion in INS-1 cells, an effect that was abolished after LDH-A overexpression. Both compounds significantly decreased glucose oxidation rates in control cells. After overexpression of LDH-A in INS-1 cells, the effects of pyruvate and lactate on glucose oxidation were diminished. On the other hand, after LDH-A overexpression, both glycolytic metabolites decreased the glucose utilization rate at 5 mM glucose. The present data suggest that the level of LDH expression in insulin-secreting cells is critical for correct channelling of pyruvate towards mitochondrial metabolism. Interestingly, glucokinase-mediated glycolytic flux was decreased after LDH-A overexpression. Thus preferential channelling of glucose towards aerobic metabolism by glucokinase may be determined, at least in part, by the low level of constitutive expression of LDH-A in pancreatic beta-cells. In conclusion, the level of LDH expression in insulin-secreting cells is an important determinant of the physiological insulin-secretory capacity, and also determines how pyruvate and lactate affect insulin secretion. PMID:11085930

Alcazar, O; Tiedge, M; Lenzen, S

2000-12-01

110

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

111

Decreased Hematocrit-To-Viscosity Ratio and Increased Lactate Dehydrogenase Level in Patients with Sickle Cell  

E-print Network

with Sickle Cell Anemia and Recurrent Leg Ulcers Philippe Connes1,2,3* , Yann Lamarre1,2 , Marie-à-Pitre, Guadeloupe Abstract Leg ulcer is a disabling complication in patients with sickle cell anemia (SCA Dehydrogenase Level in Patients with Sickle Cell Anemia and Recurrent Leg Ulcers. PLoS ONE 8(11): e79680. doi:10

Paris-Sud XI, Université de

112

Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations  

SciTech Connect

Background: The model bacterium Clostridium cellulolyticum efficiently hydrolyzes crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels. Therefore genetic engineering will likely be required to improve the ethanol yield. Random mutagenesis, plasmid transformation, and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism. Results: The first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh) and L-malate dehydrogenase (Ccel_0137; mdh) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products (by molarity), corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four-times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant s TCA pathway. Conclusions: The efficient intron-based gene inactivation system produced the first gene-targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in C. cellulolyticum and rapid genetic engineering to significantly alter the mixture of fermentation products. The initial application of this system successfully engineered a strain with high ethanol productivity from complex biomass substrates.

Li, Yongchao [ORNL; Tschaplinski, Timothy J [ORNL; Engle, Nancy L [ORNL; Hamilton, Choo Yieng [ORNL; Rodriguez, Jr., Miguel [ORNL; Liao, James C [ORNL; Schadt, Christopher Warren [ORNL; Guss, Adam M [ORNL; Yang, Yunfeng [ORNL; Graham, David E [ORNL

2012-01-01

113

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. PMID:20604468

1989-01-01

114

Enzymes Related to Lactate Metabolism in Green Algae and Lower Land Plants 1  

PubMed Central

Cell-free extracts of Chlorella pyrenoidosa contained two enzymes capable of oxidizing d-lactate; these were glycolate dehydrogenase and NAD+-dependent d-lactate dehydrogenase. The two enzymes could be distinguished by differential centrifugation, glycolate dehydrogenase being largely particulate and NAD+-d-lactate dehydrogenase being soluble. The reduction of pyruvate by NADH proceeded more rapidly than the reverse reaction, and the apparent Michaelis constants for pyruvate and NADH were lower than for d-lactate and NAD+. These data indicated that under physiological conditions, the NAD+-linked d-lactate dehydrogenase probably functions to produce d-lactate from pyruvate. Lactate dehydrogenase activity dependent on NAD+ was found in a number of other green algae and in the green tissues of a few lower land plants. When present in species which contain glycolate oxidase rather than glycolate dehydrogenase, the enzyme was specific for l-lactate rather than d-lactate. A cyclic system revolving around the production and utilization of d-lactate in some species and l-lactate in certain others is proposed. PMID:16658670

Gruber, Peter J.; Frederick, Sue Ellen; Tolbert, N. E.

1974-01-01

115

Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations  

PubMed Central

Background The model bacterium Clostridium cellulolyticum efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering. Results The first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh) and L-malate dehydrogenase (Ccel_0137; mdh) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain, resulting in a substantial shift in fermentation toward ethanol production. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products, corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant's tricarboxylic acid pathway. Conclusions The efficient intron-based gene inactivation system produced the first non-random, targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in C. cellulolyticum and rapid genetic engineering to significantly alter the mixture of fermentation products. The initial application of this system successfully engineered a strain with high ethanol productivity from cellobiose, cellulose and switchgrass. PMID:22214220

2012-01-01

116

Clinicopathological Significance and Prognostic Value of Lactate Dehydrogenase A Expression in Gastric Cancer Patients  

PubMed Central

Introduction LDH-A, the enzyme responsible for transforming pyruvate into lactate, has been demonstrated to be up-regulated in many types of cancer and to give rise to more aggressive behavior by regulating proliferation and anti-apoptosis. However, its expression in gastric cancer (GC) has not been characterized thoroughly. The purpose of this study was to clarify the expression and potential impact of LDH-A in GC. Methods We examined LDH-A expression by immunohistochemistry on GC tissue microarray (TMA) and using Western blot on fresh GC tissues and cell lines. Prognostic value and correlation with other clinicopathologic factors were evaluated. We transfected siRNA into GC cells against LDH-A. LDH-A was analyzed by Western blotting and real-time RT-PCR. Cell growth was evaluated in vitro and in vivo. Lactate and ATP production by cells were determined. Results There was significantly higher LDH-A expression in carcinoma than in non-neoplastic mucosa (NNM). There was a positive correlation of LDH-A expression with age, histological type and Lymph node metastases. Survival analysis demonstrated that high expression of LDH-A in GC was associated with lower overall survival (OS). When stratified by Lauren grade and histological classification, significance appeared in diffuse type and undifferentiated type GC. In multivariate analysis, the LDH-A expression in GC was an independent prognostic risk factor for OS (hazard ratio?=?1.829, 95%CI 1.375–2.433,P<0.0001). Specific siRNA against LDH-A in GC cell line retarded cell growth both in vitro and in mouse models. LDH-A knockdown also reduced lactate and ATP production in GC cells. Conclusions Our study indicated the oncogenic role of LDH-A in GC. LDH-A expression is an independent prognostic risk factor in GC patients and up-regulated expression of LDH-A could be predictive of poor outcomes in diffuse type and undifferentiated type GC. Our results suggested that LDH-A might be a potential therapeutic target in gastric cancer. PMID:24608789

Sun, Xuren; Sun, Zhe; Zhu, Zhi; Guan, Haixia; Zhang, Junyan; Zhang, Yining; Xu, Huimian; Sun, Mingjun

2014-01-01

117

The blood counts and lactate dehydrogenase levels in thrombotic thrombocytopenic purpura (TTP).  

PubMed

The blood counts and lactic dehydrogenase values of eight patients with thrombotic thrombocytopenic purpura (TTP) were reviewed in relation to the clinical course. Three of the eight patients died. In these patients, the hemoglobin was significantly lower and the LDH higher at the time of presentation than that of the patients responding to treatment. The height of the absolute reticulocyte count and platelet count did not correlate as well with outcome as did the degree of anemia and LDH elevation. Microangiopathic changes were noted in all eight patients. A differential count showed that the total microangiopathic changes varied from 0.8 to 54%. The more severe microangiopathic changes occurred in the fatal cases. The observations indicate that the degree of anemia, elevation of LDH, and severity of microangiopathic changes at the time of presentation correlate with the outcome in TTP and provide useful parameters in the assessment of response to therapy. PMID:6685430

Crowley, J P; Metzger, J B; L'Europa, R A

1983-11-01

118

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

Microsoft Academic Search

Clostridium acetobutylicum strain P262 utilized lactate at a rapid rate [600 nmol min?1 (mg protein)?1], but lactate could not serve as the sole energy source. When acetate was provided as a co-substrate, the growth rate was\\u000a 0.05h?1. Butyrate, carbon dioxide and hydrogen were the end products of lactate and acetate utilization, and the stoichiometry was\\u000a 1 lactate + 0.4 acetate

Francisco Diez-Gonzalez; James B. Russell; Jean B. Hunter

1995-01-01

119

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

120

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. PMID:24764735

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

2014-01-01

121

Purification of a recombinant histidine-tagged lactate dehydrogenase from the malaria parasite, Plasmodium vivax, and characterization of its properties.  

PubMed

Lactate dehydrogenase (LDH) of the malaria parasite, Plasmodium vivax (Pv), serves as a drug target and immunodiagnostic marker. The LDH cDNA generated from total RNA of a clinical isolate of the parasite was cloned into pRSETA plasmid. Recombinant his-tagged PvLDH was over-expressed in E. coli Rosetta2DE3pLysS and purified using Ni(2+)-NTA resin giving a yield of 25-30 mg/litre bacterial culture. The recombinant protein was enzymatically active and its catalytic efficiency for pyruvate was 5.4 × 10(8) min(-1) M(-1), 14.5 fold higher than a low yield preparation reported earlier to obtain PvLDH crystal structure. The enzyme activity was inhibited by gossypol and sodium oxamate. The recombinant PvLDH was reactive in lateral flow immunochromatographic assays detecting pan- and vivax-specific LDH. The soluble recombinant PvLDH purified using heterologous expression system can facilitate the generation of vivax LDH-specific monoclonals and the screening of chemical compound libraries for PvLDH inhibitors. PMID:25048245

Sundaram, Balamurugan; Varadarajan, Nandan Mysore; Subramani, Pradeep Annamalai; Ghosh, Susanta Kumar; Nagaraj, Viswanathan Arun

2014-12-01

122

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of D-lactate dehydrogenase from Lactobacillus jensenii.  

PubMed

The thermostable D-lactate dehydrogenase from Lactobacillus jensenii (LjD-LDH) is a key enzyme for the production of the D-form of lactic acid from pyruvate concomitant with the oxidation of NADH to NAD(+). The polymers of lactic acid are used as biodegradable bioplastics. The LjD-LDH protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 28%(w/v) polyethylene glycol 400, 100?mM Tris-HCl pH 9, 200?mM magnesium sulfate at 295?K. X-ray diffraction data were collected to a maximum resolution of 2.1?Å. The crystal belonged to space group P3121, with unit-cell parameters a = b = 90.5, c = 157.8?Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.58?Å(3)?Da(-1), which corresponds to a solvent content of approximately 52.3%. The structure was solved by single-wavelength anomalous dispersion using a selenomethionine derivative. PMID:25084378

Kim, Sangwoo; Kim, Yong Hwan; Kim, Kyung-Jin

2014-08-01

123

Evaluation of Creatine Kinase, Lactate Dehydrogenase, and Amylase Concentrations in Umbilical Blood of Preterm Infants after Long-Term Tocolysis  

PubMed Central

Creatine kinase (CK), lactate dehydrogenase (LDH), and amylase levels of preterm infants following long-term tocolysis in pregnant women are limited. The objective of this study was to determine if the tocolytic therapy affects CK, LDH, and amylase levels in the umbilical blood. This study included 215 preterm infants born to women treated with and without ritodrine hydrochloride. CK, LDH, and amylase levels in the umbilical blood at delivery were determined. Infants were divided according to the ritodrine tocolysis, as follows: Group A (n = 91), not exposed to ritodrine; Group B (n = 44), IV ritodrine for <1 week; Group C (n = 80), IV ritodrine for ?1 week. The CK concentration in cord blood of Group C (198.8 ± 14.2?IU/L) was significantly higher in comparison with Group A (155.0 ± 7.3?IU/L, P < 0.05). There was no significant difference in LDH and amylase levels in the three groups. The CK significantly correlated with gestational age (r = 0.42, P < 0.01) and birth weight (r = 0.38, P < 0.01). LDH and amylase levels did not change with gestational age nor birth weight. In conclusion, long-term ritodrine tocolysis leads to increased umbilical blood CK level. PMID:24693289

Nakajima, Yoshiyuki; Masaoka, Naoki

2014-01-01

124

Recognition and separation of isoenzymes by metal chelates. Immobilized metal ion affinity partitioning of lactate dehydrogenase isoenzymes.  

PubMed

Poly(ethylene glycol) (PEG)-bound chelated metal ions partition preferentially into the top, PEG-rich, phase of a PEG-salt or PEG-dextran aqueous two-phase system. Extraction by this soluble affinity ligand of proteins is due to a selective interaction of the chelated metal ion with accessible histidine residues on the protein surface. Using Cu-iminodiacetate-PEG (Cu-IDA-PEG) the surface of lactate dehydrogenase (LDH) isoenzymes from different species was probed for the presence of metal chelate binding sites. It was demonstrated that the homotetramers (LDH-1)(H4) from rabbit, bovine and pig displayed weak binding to chelated copper whereas the M4-type isoenzymes (LDH-5) bound strongly to this ligand. The binding of the different heterotetramers increases as the number of M-type subunits increases. In contrast, the human isoenzymes are bound to chelated copper in a reversed sequence. The comparison of the affinity partitioning effect of Cu-IDA-PEG in PEG-salt and PEG-dextran systems revealed that the discriminatory effect of copper is promoted by high salt concentrations. Resolution of isoenzymes by multiple extraction using counter-current distribution provides valuable data on the partitioning of enzymes relative to that of the bulk proteins. The efficacy of metal chelate affinity partitioning for the purification of LDH from tissue samples by batchwise extraction was also demonstrated. PMID:7690357

Otto, A; Birkenmeier, G

1993-07-30

125

An alternative allosteric regulation mechanism of an acidophilic l-lactate dehydrogenase from Enterococcus mundtii 15-1A  

PubMed Central

A plant-derived Enterococcus mundtii 15-1A, that has been previously isolated from Brassica rapa L. subsp. nipposinica (L.H. Bailey) Hanelt var. linearifolia by our group, possesses two kinds of l-lactate dehydrogenase (l-LDH): LDH-1 and LDH-2. LDH-1 was activated under low concentration of fluctose-1,6-bisphosphate (FBP) at both pH 5.5 and 7.5. Although LDH-2 was also activated under the low concentration of FBP at pH 5.5, a high concentration of FBP is necessary to activate it at pH 7.5. The present study shows the crystal structures of the acidophilic LDH-2 in a complex with and without FBP and NADH. Although the tertiary structure of the ligands-bound LDH-2 is similar to that of the active form of other bacterial l-LDHs, the structure without the ligands is different from that of any other previously determined l-LDHs. Major structural alterations between the two structures of LDH-2 were observed at two regions in one subunit. At the N-terminal parts of the two regions, the ligands-bound form takes an ?-helical structure, while the form without ligands displays more disordered and extended structures. A vacuum-ultraviolet circular dichroism analysis showed that the ?-helix content of LDH-2 in solution is approximately 30% at pH 7.5, which is close to that in the crystal structure of the form without ligands. A D241N mutant of LDH-2, which was created by us to easily form an ?-helix at one of the two parts, exhibited catalytic activity even in the absence of FBP at both pH 5.5 and 7.5. PMID:25379380

Matoba, Yasuyuki; Miyasako, Masashi; Matsuo, Koichi; Oda, Kosuke; Noda, Masafumi; Higashikawa, Fumiko; Kumagai, Takanori; Sugiyama, Masanori

2014-01-01

126

Elevation of serum lactate dehydrogenase at posterior reversible encephalopathy syndrome onset in chemotherapy-treated cancer patients.  

PubMed

The pathophysiology of posterior reversible encephalopathy syndrome (PRES) is incompletely understood; however, an underlying state of immune dysregulation and endothelial dysfunction has been proposed. We examined alterations of serum lactate dehydrogenase (LDH), a marker of endothelial dysfunction, relative to the development of PRES in patients receiving chemotherapy. A retrospective Institutional Review Board approved database of 88 PRES patients was examined. PRES diagnosis was confirmed by congruent clinical diagnosis and MRI. Clinical features at presentation were recorded. Serum LDH values were collected at three time points: prior to, at the time of, and following PRES diagnosis. Student's t-test was employed. LDH values were available during the course of treatment in 12 patients (nine women; mean age 57.8 years [range 33-75 years]). Chemotherapy-associated PRES patients were more likely to be normotensive (25%) versus the non-chemotherapy group (9%). LDH levels at the time of PRES diagnosis were higher than those before and after (p=0.0263), with a mean difference of 114.8 international units/L. Mean time intervals between LDH measurement prior to and following PRES diagnosis were 44.8 days and 51.4 days, respectively. Mean elapsed time between last chemotherapy administration and PRES onset was 11.1days. In conclusion, serum LDH, a marker of endothelial dysfunction, shows statistically significant elevation at the onset of PRES toxicity in cancer patients receiving chemotherapy. Our findings support a systemic process characterized by endothelial injury/dysfunction as a factor, if not the prime event, in the pathophysiology of PRES. PMID:24780237

Fitzgerald, Ryan T; Wright, Steven M; Samant, Rohan S; Kumar, Manoj; Ramakrishnaiah, Raghu H; Van Hemert, Rudy; Brown, Aliza T; Angtuaco, Edgardo J

2014-09-01

127

Inhibition of lactate dehydrogenase A by microRNA?34a resensitizes colon cancer cells to 5?fluorouracil.  

PubMed

5?Fluorouracil (5?FU) chemotherapy is widely used in the treatment of advanced colon cancer. However, the development of resistance to 5?FU is a significant obstacle to successful treatment. MicroRNA?34a (miR?34a) has been reported to be downregulated in a number of tumor types and has also been shown to act as a tumor suppressor. However, the mechanisms underlying the biological effects of miR?34a in chemoresistance remain unclear. The present study showed that the expression of miR?34a is downregulated in 5?FU?resistant colon cancer cells. In addition, 5?FU?resistant colon cancer cells exhibited upregulation of lactate dehydrogenase A (LDHA) expression and activity compared with parental cells. Furthermore, LDHA was shown to be a direct target of miR?34a. Overexpression of miR?34a reduced the expression of LDHA, probably through binding to the 3' untranslated region, leading to the re?sensitization of 5?FU?resistant cancer cells to 5?FU. Additionally, overexpression of LDHA rendered colon cancer cells resistant to 5?FU, suggesting that the miR?34a?induced sensitization to 5?FU is mediated through the inhibition of LDHA. In conclusion, the current study showed that miR?34a is involved in sensitivity to 5?FU in part through its effects on LDHA expression. This indicates that miR?34a?mediated inhibition of glucose metabolism may be a therapeutic target in patients with chemoresistant colon cancer. PMID:25333573

Li, Xiangyong; Zhao, Haibin; Zhou, Xijian; Song, Lei

2015-01-01

128

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

129

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

130

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

131

A novel polyclonal antibody-based sandwich ELISA for detection of Plasmodium vivax developed from two lactate dehydrogenase protein segments  

PubMed Central

Background Immunoassays for Plasmodium detection are, presently, most frequently based on monoclonal antibodies (MAbs); Polyclonal antibodies (PAbs), which are cheaper to develop and manufacture, are much less frequently used. In the present study we describe a sandwich ELISA assay which is capable of detecting P. vivax Lactate Dehydrogenase (LDH) in clinical blood samples, without cross reacting with those infected with P. falciparum. Methods Two recombinant proteins were produced from different regions of the P. vivax LDH gene. Two sandwich ELISA assay were then designed: One which uses mouse anti-LDH 1-43aa PAbs as primary antibodies (“Test 1”) and another which uses anti-LDH 35-305aa PAbs (“Test 2”) as the primary antibodies. Rabbit anti-LDH 1-43aa PAbs were used as capture antibodies in both ELISA assays. Blood samples taken from P. vivax and P. falciparum infected patients (confirmed by light microscopy) were analysed using both tests. Results “Test 2” performed better at detecting microscopy-positive blood samples when compared to “Test 1”, identifying 131 of 154 positive samples (85%); 85 positives (55%) were identified using “test 1”. “Test 1” produced one false positive sample (from the 20 malaria-free control) blood samples; “test 2” produced none. Kappa coefficient analysis of the results produced a value of 0.267 when microscope-positive blood smears were compared with “test 1”, but 0.734 when microscope-positive blood smears were compared with the results from “test 2”. Positive predictive value (PPV) and negative predictive value (NPV) were observed to be 98% and 22% respectively, for “Test 1”, and 99% and 45%, for “test 2”. No cross reactivity was detected with P. falciparum positive blood samples (n?=?15) with either test assay. Conclusion Both tests detected P. vivax infected blood and showed no evidence of cross-reacting with P. falciparum. Further studies will need to be conducted to establish the full potential of this technique for malaria diagnostics. As well as representing a promising new cost-effective novel technique for P. vivax diagnosis and research, the method for developing this assay also highlights the potential for PAb-based strategies for diagnostics in general. PMID:24475751

2014-01-01

132

Protective effects of caffeic acid on lactate dehydrogenase isoenzymes, electrocardiogram, adenosine triphosphatases, and hematology on isoproterenol-induced myocardial infarcted rats.  

PubMed

The present study aims to evaluate the protective effects of caffeic acid on isoproterenol-treated myocardial infarction. Male albino Wistar rats were pretreated with caffeic acid (15 mg/kg) daily for 10 days. After the pretreatment, rats were injected with isoproterenol (100 mg/kg) at an interval of 24 h for 2 days to induce myocardial infarction. Isoproterenol-treated rats showed increased intensity of lactate dehydrogenase-1 and 2 isoenzyme bands and elevated ST segments. The activity of the heart sodium potassium adenosine triphosphatase was decreased, and the activities of the heart magnesium adenosine triphosphatase and calcium adenosine triphosphatase were increased in isoproterenol-treated rats. Isoproterenol-treated rats also showed a significant increase in the concentration of heart calcium. Furthermore, it significantly increased the counts of red blood cells, hemoglobin, white blood cells, and neutrophils and decreased significantly the concentration of erythrocyte sedimentation rate and the counts of lymphocytes and eosinophils. Pretreatment with caffeic acid showed protective effects on all the biochemical parameters, hematology and minimized alterations in lactate dehydrogenase isoenzymes and electrocardiogram. In vitro study confirmed the free radical scavenging potential of caffeic acid. The observed effects might be due to the free radical scavenging and membrane-stabilizing property of caffeic acid. PMID:21472895

Senthil Kumaran, K; Stanely Mainzen Prince, P

2011-01-01

133

Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity.  

PubMed

Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43?mol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate. PMID:24412354

Jiang, Ting; Xu, Yanbing; Sun, Xiucheng; Zheng, Zhaojuan; Ouyang, Jia

2014-03-01

134

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

PubMed

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, of LDH in tumours has never been investigated in the past. In the present study, we established an immunohistochemical method to evaluate the LDH-5 overexpression in tumours, using two novel antibodies raised against the rat muscle LDH-5 and the human LDH-5 (Abcam, UK). The subcellular patterns of expression in cancer cells were mixed nuclear and cytoplasmic. In direct contrast to cancer cells, stromal fibroblasts were reactive for LDH-5 only in a minority of cases. Serum LDH, although positively correlated with, does not reliably reflect the intratumoral LDH-5 status. Lactate dehydrogenase-5 overexpression was directly related to HIF1alpha and 2alpha, but not with the carbonic anhydrase 9 expression. Patients with tumours bearing high LDH-5 expression had a poor prognosis. Tumours with simultaneous LDH-5 and HIF1alpha (or HIF2alpha) overexpression, indicative of a functional HIF pathway, had a particularly aggressive behaviour. It is concluded that overexpression of LDH-5 is a common event in non-small-cell lung cancer, can be easily assessed in paraffin-embedded material and provides important prognostic information, particularly when combined with other endogenous markers of hypoxia and acidity. PMID:12942121

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

2003-09-01

135

Identification of Lactaldehyde Dehydrogenase in Methanocaldococcus jannaschii and Its Involvement in Production of Lactate for F420 Biosynthesis  

PubMed Central

One of the early steps in the biosynthesis of coenzyme F420 in Methanocaldococcus jannaschii requires generation of 2-phospho-l-lactate, which is formed by the phosphorylation of l-lactate. Preliminary studies had shown that l-lactate in M. jannaschii is not derived from pyruvate, and thus an alternate pathway(s) for its formation was examined. Here we report that l-lactate is formed by the NAD+-dependent oxidation of l-lactaldehyde by the MJ1411 gene product. The lactaldehyde, in turn, was found to be generated either by the NAD(P)H reduction of methylglyoxal or by the aldol cleavage of fuculose-1-phosphate by fuculose-1-phosphate aldolase, the MJ1418 gene product. PMID:16585745

Grochowski, Laura L.; Xu, Huimin; White, Robert H.

2006-01-01

136

Effects of Gram-negative Bacteria, E.coli and Cold Exposure on Free Radicals Production, Lactate Dehydrogenase and Glutathione Peroxidase Activity in the Lungs of Rats, Rattus norvigicus  

Microsoft Academic Search

The purpose of this study was to explore the effects of LPS-gram negative bacteria and low ambient temperature on free radicals (FR) production, the activities of lactate dehydrogenase (LDH) and glutathione peroxidase (GPx) in the lungs of rats, Rattus norvigisu. Twenty four male rats, matched with age and weigh, were divided randomly into four groups namely control (C), Bacteria (B),

Al-Said A. Haffor

137

Effects of sustained swimming on rainbow trout muscle structure, blood oxygen transport, and lactate dehydrogenase isozymes: evidence for increased aerobic capacity of white muscle.  

PubMed

Groups of rainbow trout (Salmo gairdneri, Richardson) were continuously swum at 20 cm s-1 (1.0 body lengths s-1) for 0, 3, 30, and 200 days. No significant changes in fish condition factor, combined red and white muscle mass, muscle fibre size or fibre size distribution were observed. After 200 days of swimming there was a significant 2.2 fold increase in red muscle mass. Number of capillaries per red muscle fibre increased significantly in each group by a maximum of 27% after 200 days exercise. Number of capillaries per white muscle fibre increased significantly by 95% after 200 days exercise. Blood lactate, haemoglobin (Hb) concentration haematocrit, erythrocyte adenosine triphosphate, and whole blood oxygen affinity P50 were unchanged by swimming. After 30 and 200 days swimming there was a shift in expression of white muscle lactate dehydrogenase (LDH) isozymes from LDH-A to LDH-B. Within the duplicated LDH-B isozyme complex, there was a shift in expression from LDH-B to LDH-B' subunits. These results suggest that sustained swimming at 1(-1) bl s-1 increased the aerobic capacity of red and particularly white (fast) muscle of rainbow trout but did not alter the gas transport characteristics of the blood. PMID:3950562

Davie, P S; Wells, R M; Tetens, V

1986-02-01

138

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

139

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

140

Lactate production and measurement in critically ill horses.  

PubMed

Blood lactate concentration can be easily measured by practitioners using inexpensive point-of-care meters. Anaerobic tissue metabolism resulting from inadequate oxygen delivery (DO2) is the most important cause of an increase in blood lactate concentration in equine patients. However,hyperlactatemia also occurs under conditions of apparently adequate DO2, usually in association with sepsis and an intense inflammatory reaction. Numerous mechanisms have been proposed for aerobic hyperlactatemia, including increased Na+/K+-ATPase activity in response to inflammatory mediators; inhibition of pyruvate dehydrogenase, a key enzyme in glucose metabolism; and increased lactate production by activated inflammatory cells. The liver is responsible for most lactate metabolism, and liver disease might contribute to an increase in blood lactate concentration in some patients. Skeletal muscle is usually considered the most important source of lactate during sepsis. The roles of the lungs and the gastrointestinal tract in lactate production have been investigated but remain uncertain. PMID:22180135

Tennent-Brown, Brent S

2011-12-01

141

Direct production of allitol from D-fructose by a coupling reaction using D-tagatose 3-epimerase, ribitol dehydrogenase and formate dehydrogenase.  

PubMed

Allitol was produced from D-fructose via a new NADH-regenerating enzymatic reaction system using D-tagatose 3-epimerase (D-TE), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH). D-fructose was epimerized to D-psicose by the D-TE of Pseudomonas cichorii ST-24 and the D-psicose was subsequently reduced to allitol by the RDH of an RDH-constitutive mutant, X-22, derived from Klebsiella pneumoniae IFO 3321. NADH regeneration for the reduction of D-psicose by the RDH was achieved by the irreversible formate dehydrogenase reaction, which allowed the D-psicose produced from d-fructose to be successively transformed to allitol with a production yield from D-fructose of almost 100%. The reactions progressed without any by-product formation. After separation of the product from the reaction mixture by a simple procedure, a crystal of allitol was obtained in a yield exceeding 90%. This crystal was characterized and determined to be allitol by HPLC analysis, its IR and NMR spectra, its melting point, and optical rotation measurement. PMID:16232907

Takeshita, K; Ishida, Y; Takada, G; Izumori, K

2000-01-01

142

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. PMID:762012

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

1979-01-01

143

Catalytic reaction of cytokinin dehydrogenase: preference for quinones as electron acceptors  

Microsoft Academic Search

The catalytic reaction of cytokinin oxidase\\/dehydrogenase (EC 1.5.99.12) was studied in detail using the recombinant flavoenzyme from maize. Determination of the redox potential of the covalently linked flavin cofactor revealed a relatively high potential dictating the type of electron acceptor that can be used by the enzyme. Using 2,6-dichlorophenol indophenol, 2,3-dimethoxy-5-methyl-1,4-benzoquinone or 1,4-naphthoquinone as electron acceptor, turnover rates with N6-(2-isopentenyl)adenine

James T. English; Kristin D. Bilyeu; Ond?ej Novák; Pavel Pe?; Marek Šebela; Petr Galuszka; Marco W. Fraaije; Ivo Frébort; Jan Hrbá?; Jitka Frébortová

2004-01-01

144

Overexpression of Pyruvate Dehydrogenase Kinase 1 and Lactate Dehydrogenase A in Nerve Cells Confers Resistance to Amyloid ? and Other Toxins by Decreasing Mitochondrial Respiration and Reactive Oxygen Species Production*  

PubMed Central

We previously demonstrated that nerve cell lines selected for resistance to amyloid ? (A?) peptide exhibit elevated aerobic glycolysis in part due to increased expression of pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase A (LDHA). Here, we show that overexpression of either PDK1 or LDHA in a rat CNS cell line (B12) confers resistance to A? and other neurotoxins. Treatment of A?-sensitive cells with various toxins resulted in mitochondrial hyperpolarization, immediately followed by rapid depolarization and cell death, events accompanied by increased production of cellular reactive oxygen species (ROS). In contrast, cells expressing either PDK1 or LDHA maintained a lower mitochondrial membrane potential and decreased ROS production with or without exposure to toxins. Additionally, PDK1- and LDHA-overexpressing cells exhibited decreased oxygen consumption but maintained levels of ATP under both normal culture conditions and following A? treatment. Interestingly, immunoblot analysis of wild type mouse primary cortical neurons treated with A? or cortical tissue extracts from 12-month-old APPswe/PS1dE9 transgenic mice showed decreased expression of LDHA and PDK1 when compared with controls. Additionally, post-mortem brain extracts from patients with Alzheimer disease exhibited a decrease in PDK1 expression compared with nondemented patients. Collectively, these findings indicate that key Warburg effect enzymes play a central role in mediating neuronal resistance to ?? or other neurotoxins by decreasing mitochondrial activity and subsequent ROS production. Maintenance of PDK1 or LDHA expression in certain regions of the brain may explain why some individuals tolerate high levels of A? deposition without developing Alzheimer disease. PMID:22948140

Newington, Jordan T.; Rappon, Tim; Albers, Shawn; Wong, Daisy Y.; Rylett, R. Jane; Cumming, Robert C.

2012-01-01

145

Engineering Lactococcus lactis for production of mannitol: high yields from food-grade strains deficient in lactate dehydrogenase and the mannitol transport system.  

PubMed

Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTS(Mtl)). Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L. lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (Delta ldh Delta mtlA and Delta ldh Delta mtlF) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo (13)C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTS(Mtl). Disruption of this system completely blocked mannitol transport in L. lactis, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed. PMID:15006767

Gaspar, Paula; Neves, Ana Rute; Ramos, Ana; Gasson, Michael J; Shearman, Claire A; Santos, Helena

2004-03-01

146

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 Central

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

2009-01-01

147

Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: similarities with the erythropoietin 3' enhancer.  

PubMed Central

Production of the glycoprotein hormone erythropoietin (Epo) in response to hypoxic stimuli is almost entirely restricted to particular cells within liver and kidney, yet the transcriptional enhancer lying 3' to the Epo gene shows activity inducible by hypoxia after transfection into a wide variety of cultured cells. The implication of this finding is that many cells which do not produce Epo contain a similar, if not identical, oxygen-regulated control system, suggesting that the same system is involved in the regulation of other genes. We report that the human phosphoglycerate kinase 1 and mouse lactate dehydrogenase A genes are induced by hypoxia with characteristics which resemble induction of the Epo gene. In each case expression is induced by cobalt, but not by cyanide, and hypoxic induction is blocked by the protein-synthesis inhibitor cycloheximide. We show that the relevant cis-acting control sequences are located in the 5' flanking regions of the two genes, and we define an 18-bp element in the 5' flanking sequence of the phosphoglycerate kinase 1 gene which is both necessary and sufficient for the hypoxic response, and which has sequence and protein-binding similarities to the hypoxia-inducible factor 1 binding site within the Epo 3' enhancer. Images PMID:8022811

Firth, J D; Ebert, B L; Pugh, C W; Ratcliffe, P J

1994-01-01

148

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. PMID:25049573

Guha, Anirban; Gera, Sandeep; Sharma, Anshu

2012-01-01

149

Decreased Hematocrit-To-Viscosity Ratio and Increased Lactate Dehydrogenase Level in Patients with Sickle Cell Anemia and Recurrent Leg Ulcers  

PubMed Central

Leg ulcer is a disabling complication in patients with sickle cell anemia (SCA) but the exact pathophysiological mechanisms are unknown. The aim of this study was to identify the hematological and hemorheological alterations associated with recurrent leg ulcers. Sixty-two SCA patients who never experienced leg ulcers (ULC-) and 13 SCA patients with a positive history of recurrent leg ulcers (ULC+) - but with no leg ulcers at the time of the study – were recruited. All patients were in steady state condition. Blood was sampled to perform hematological, biochemical (hemolytic markers) and hemorheological analyses (blood viscosity, red blood cell deformability and aggregation properties). The hematocrit-to-viscosity ratio (HVR), which reflects the red blood cell oxygen transport efficiency, was calculated for each subject. Patients from the ULC+ group were older than patients from the ULC- group. Anemia (red blood cell count, hematocrit and hemoglobin levels) was more pronounced in the ULC+ group. Lactate dehydrogenase level was higher in the ULC+ group than in the ULC- group. Neither blood viscosity, nor RBC aggregation properties differed between the two groups. HVR was lower and RBC deformability tended to be reduced in the ULC+ group. Our study confirmed increased hemolytic rate and anemia in SCA patients with leg ulcers recurrence. Furthermore, our data suggest that although systemic blood viscosity is not a major factor involved in the pathophysiology of this complication, decreased red blood cell oxygen transport efficiency (i.e., low hematocrit/viscosity ratio) may play a role. PMID:24223994

Connes, Philippe; Lamarre, Yann; Hardy-Dessources, Marie-Dominique; Lemonne, Nathalie; Waltz, Xavier; Mougenel, Danièle; Mukisi-Mukaza, Martin; Lalanne-Mistrih, Marie-Laure; Tarer, Vanessa; Tressières, Benoit; Etienne-Julan, Maryse; Romana, Marc

2013-01-01

150

Decreased hematocrit-to-viscosity ratio and increased lactate dehydrogenase level in patients with sickle cell anemia and recurrent leg ulcers.  

PubMed

Leg ulcer is a disabling complication in patients with sickle cell anemia (SCA) but the exact pathophysiological mechanisms are unknown. The aim of this study was to identify the hematological and hemorheological alterations associated with recurrent leg ulcers. Sixty-two SCA patients who never experienced leg ulcers (ULC-) and 13 SCA patients with a positive history of recurrent leg ulcers (ULC+)--with no leg ulcers at the time of the study--were recruited. All patients were in steady state condition. Blood was sampled to perform hematological, biochemical (hemolytic markers) and hemorheological analyses (blood viscosity, red blood cell deformability and aggregation properties). The hematocrit-to-viscosity ratio (HVR), which reflects the red blood cell oxygen transport efficiency, was calculated for each subject. Patients from the ULC+ group were older than patients from the ULC- group. Anemia (red blood cell count, hematocrit and hemoglobin levels) was more pronounced in the ULC+ group. Lactate dehydrogenase level was higher in the ULC+ group than in the ULC- group. Neither blood viscosity, nor RBC aggregation properties differed between the two groups. HVR was lower and RBC deformability tended to be reduced in the ULC+ group. Our study confirmed increased hemolytic rate and anemia in SCA patients with leg ulcers recurrence. Furthermore, our data suggest that although systemic blood viscosity is not a major factor involved in the pathophysiology of this complication, decreased red blood cell oxygen transport efficiency (i.e., low hematocrit/viscosity ratio) may play a role. PMID:24223994

Connes, Philippe; Lamarre, Yann; Hardy-Dessources, Marie-Dominique; Lemonne, Nathalie; Waltz, Xavier; Mougenel, Danièle; Mukisi-Mukaza, Martin; Lalanne-Mistrih, Marie-Laure; Tarer, Vanessa; Tressières, Benoit; Etienne-Julan, Maryse; Romana, Marc

2013-01-01

151

Immune evasion mediated by tumor-derived lactate dehydrogenase induction of NKG2D ligands on myeloid cells in glioblastoma patients.  

PubMed

Myeloid cells are key regulators of the tumor microenvironment, governing local immune responses. Here we report that tumor-infiltrating myeloid cells and circulating monocytes in patients with glioblastoma multiforme (GBM) express ligands for activating the Natural killer group 2, member D (NKG2D) receptor, which cause down-regulation of NKG2D on natural killer (NK) cells. Tumor-infiltrating NK cells isolated from GBM patients fail to lyse NKG2D ligand-expressing tumor cells. We demonstrate that lactate dehydrogenase (LDH) isoform 5 secreted by glioblastoma cells induces NKG2D ligands on monocytes isolated from healthy individuals. Furthermore, sera from GBM patients contain elevated amounts of LDH, which correlate with expression of NKG2D ligands on their autologous circulating monocytes. NKG2D ligands also are present on circulating monocytes isolated from patients with breast, prostate, and hepatitis C virus-induced hepatocellular carcinomas. Together, these findings reveal a previously unidentified immune evasion strategy whereby tumors produce soluble factors that induce NKG2D ligands on myeloid cells, subverting antitumor immune responses. PMID:25136121

Crane, Courtney A; Austgen, Kathryn; Haberthur, Kristen; Hofmann, Carly; Moyes, Kara White; Avanesyan, Lia; Fong, Lawrence; Campbell, Michael J; Cooper, Stewart; Oakes, Scott A; Parsa, Andrew T; Lanier, Lewis L

2014-09-01

152

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

153

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

154

Rationally re-designed mutation of NAD-independent l-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli  

PubMed Central

Background NAD-independent l-lactate dehydrogenase (l-iLDH) from Pseudomonas stutzeri SDM can potentially be used for the kinetic resolution of small aliphatic 2-hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2-hydroxycarboxylic acids. Results Val-108 of l-iLDH was changed to Ala by rationally site-directed mutagenesis. The l-iLDH mutant exhibited much higher activity than wide-type l-iLDH towards l-mandelate, an aromatic 2-hydroxycarboxylic acid. Using the engineered Escherichia coli expressing the mutant l-iLDH as a biocatalyst, 40 g·L-1 of dl-mandelic acid was converted to 20.1 g·L-1 of d-mandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g·L-1 of benzoylformic acid. Conclusions A new biocatalyst with high catalytic efficiency toward an unnatural substrate was constructed by rationally re-design mutagenesis. Two building block intermediates (optically pure d-mandelic acid and benzoylformic acid) were efficiently produced by the one-pot biotransformation system. PMID:23176608

2012-01-01

155

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. PMID:640378

Sassaman, Clay

1978-01-01

156

Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.  

PubMed Central

The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon. Images PMID:8478320

Llanos, R M; Harris, C J; Hillier, A J; Davidson, B E

1993-01-01

157

N-terminal tagged lactate dehydrogenase proteins: evaluation of relative hydrophobicity by hydrophobic interaction chromatography and aqueous two-phase system partition.  

PubMed

The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS). The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein. The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli. HIC and ATPS behaviour of the proteins indicated significant differences in protein hydrophobicity, even though the modifications caused only 1% increase in protein molecular weight and 2% variation in isoelectric points. HIC and ATPS results correlated well (R(2) = 0.89). Protein expression was clearly affected by N-terminal modification, but there was no evidence that the modification affected protein activity. A GluAsnAlaAspVal modification resulted in increased protein expression. In most cases, HIC and ATPS results compared favourably with those predicted on the basis of 34 amino acid residue hydrophobicity scales; assuming exposure of tag residues to solution. Exceptions included LeuAlaGlyValIle and LeuTyrGlyCysIle modifications, which were predicted, assuming full solution exposure, to be more hydrophobic than observed. PMID:15177156

Fexby, Sara; Ihre, Henrik; Van Alstine, James; Bülow, Leif

2004-07-25

158

Baseline Serum Lactate Dehydrogenase Levels for Patients Treated With Intensity-Modulated Radiotherapy for Nasopharyngeal Carcinoma: A Predictor of Poor Prognosis and Subsequent Liver Metastasis  

SciTech Connect

Purpose: To evaluate the prognostic value of baseline serum lactate dehydrogenase (LDH) levels in patients with nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiotherapy (IMRT). Methods and Materials: Cases of NPC (n = 465) that involved treatment with IMRT with or without chemotherapy were retrospectively analyzed. Results: The mean ({+-}SD) and median baseline serum LDH levels for this cohort were 172.77 {+-} 2.28 and 164.00 IU/L, respectively. Levels of LDH were significantly elevated in patients with locoregionally advanced disease (p = 0.016). Elevated LDH levels were identified as a prognostic factor for rates of overall survival (OS), disease-free survival (DFS), and distant metastasis-free survival (DMFS), with p values <0.001 in the univariate analysis and p < 0.001, p = 0.004, and p = 0.003, respectively, in the multivariate analysis. Correspondingly, the prognostic impact of patient LDH levels was found to be statistically significant for rates of OS, DFS, and DMFS (p = 0.028, 0.024, and 0.020, respectively). For patients who experienced subsequent liver failure after treatment, markedly higher pretreatment serum LDH levels were detected compared with patients experiencing distant metastasis events at other sites (p = 0.032). Conclusions: Elevated baseline LDH levels are associated with clinically advanced disease and are a poor prognosticator for OS, DFS, and DMFS for NPC patients. These results suggest that elevated serum levels of LDH should be considered when evaluating treatment options.

Zhou Guanqun; Tang Linglong; Mao Yanping; Chen Lei; Li Wenfei; Sun Ying [Department of Radiation Oncology, Cancer Center, State Key Laboratory of Oncology in South China, Sun Yat-sen University, Guangzhou (China); Liu Lizhi; Li Li [Imaging Diagnosis and Interventional Center, Cancer Center, State Key Laboratory of Oncology in South China, Sun Yat-sen University, Guangzhou (China); Lin Aihua [Department of Medical Statistics and Epidemiology, School of Public Health, Sun Yat-sen University, Guangzhou (China); Ma Jun, E-mail: drjunma@hotmail.com [Department of Radiation Oncology, Cancer Center, State Key Laboratory of Oncology in South China, Sun Yat-sen University, Guangzhou (China)

2012-03-01

159

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

160

[Isoenzymes of lactate and malate dehydrogenases in nuclei of rat embryo fibroblasts infected with oncogenic adenovirus type 12].  

PubMed

The isoenzymatic spectrum of lactatedehydrogenase (LDG; L-lactate: NAD-oxireductase; EC 1.1.1.27) and malatedehydrogenase (MDG; 1-malate: NAD-oxireductase; EC 1.1.1.37) in nuclei of rat embryo fibroblast cells infected with adenovirus type 12 was studied at 3, 5, 8, 18 and 24 days of cultivation. The nuclei were isolated according to the method of Showo et al. modified by Zbarsky and Georgiev. Proteins were fractionated by disc electrophoresis in polyacrylamide gel. In the course of oncovirus-cell interaction three LDG isoenzymes were detected. Changes in the isoenzymatic spectrum of LDG were manifested in increased activity of LDG1 at 5 days after virus penetration into the cell. Partial morphological transformation at 18 days after infection was accompanied by increased activity of LDG1 and LDG2. The morphologically transformed culture was characterised by reduced LDG1 activity. Changes in the spectrum of MDG isoenzymes were detected at 3 days after addition of A-12 virus into the culture. At 24 days postinfection activity of MDG3 was definitely changed in nuclei of REF cells infected with A-12 virus. The revealed disorders in regulation of synthesis of individual isoenzymes are probably due to the epigenomic effect of oncovirus and may be used as indications of tissue culture malignization. PMID:1241176

Ageenko, A I; Vitorgan, Iu E

1975-01-01

161

Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions  

SciTech Connect

The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 {angstrom} resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg{sup 2+} and NADH. This TDH structure from Pseudomonas putida has a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. However, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identification of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate intermediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH.

Malik, Radhika; Viola, Ronald E. (Toledo)

2010-10-28

162

Biocatalytic carbon capture via reversible reaction cycle catalyzed by isocitrate dehydrogenase.  

PubMed

The practice of carbon capture and storage (CCS) requires efficient capture and separation of carbon dioxide from its gaseous mixtures such as flue gas, followed by releasing it as a pure gas which can be subsequently compressed and injected into underground storage sites. This has been mostly achieved via reversible thermochemical reactions which are generally energy-intensive. The current work examines a biocatalytic approach for carbon capture using an NADP(H)-dependent isocitrate dehydrogenase (ICDH) which catalyzes reversibly carboxylation and decarboxylation reactions. Different from chemical carbon capture processes that rely on thermal energy to realize purification of carbon dioxide, the biocatalytic strategy utilizes pH to leverage the reaction equilibrium, thereby realizing energy-efficient carbon capture under ambient conditions. Results showed that over 25 mol of carbon dioxide could be captured and purified from its gas mixture for each gram of ICDH applied for each carboxylation/decarboxylation reaction cycle by varying pH between 6 and 9. This work demonstrates the promising potentials of pH-sensitive biocatalysis as a green-chemistry route for carbon capture. PMID:25152403

Xia, Shunxiang; Frigo-Vaz, Benjamin; Zhao, Xueyan; Kim, Jungbae; Wang, Ping

2014-09-12

163

A transient intermediate in the reaction catalyzed by (S)-mandelate dehydrogenase from Pseudomonas putida.  

PubMed

(S)-Mandelate dehydrogenase from Pseudomonas putida is a member of a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids to alpha-ketoacids. The reductive half-reaction consists of the steps involved in substrate oxidation and FMN reduction. In this study, we investigated the mechanism of this half-reaction in detail. At low temperatures, a transient intermediate was formed in the course of the FMN reduction reaction. This intermediate is characteristic of a charge-transfer complex of oxidized FMN and an electron-rich donor and is formed prior to full reduction of the flavin. The intermediate was not due to binding of anionic substrates or inhibitors. It was only observed with efficient substrates that have high k(cat) values. At higher temperatures, it was formed within the dead time of the stopped-flow instrument. The rate of formation of the intermediate was 3-4-fold faster than its rate of disappearance; the former had a larger isotope effect. This suggests that the charge-transfer donor is an electron-rich carbanion/enolate intermediate that is generated by the base-catalyzed abstraction of the substrate alpha-proton. This is consistent with the observation that the intermediate was not observed with the R277K and R277G mutants, which have been shown to destabilize the carbanion intermediate (Lehoux, I. E., and Mitra, B. (2000) Biochemistry 39, 10055-10065). Thus, the MDH reaction has two rate-limiting steps of similar activation energies: the formation and breakdown of a distinct intermediate, with the latter step being slightly more rate limiting. We also show that MDH is capable of catalyzing the reverse reaction, the reoxidation of reduced MDH by the product ketoacid, benzoylformate. The transient intermediate was observed during the reverse reaction as well, confirming that it is indeed a true intermediate in the MDH reaction pathway. PMID:14596603

Dewanti, Asteriani R; Mitra, Bharati

2003-11-11

164

A thioredoxin fusion protein of VanH, a D-lactate dehydrogenase from Enterococcus faecium: cloning, expression, purification, kinetic analysis, and crystallization.  

PubMed

The gene encoding the vancomycin resistance protein VanH from Enterococcus faecium, a D-lactate dehydrogenase, has been cloned into a thioredoxin expression system (pTRxFus) and expressed as a fusion protein. The use of several other expression systems yielded only inclusion bodies from which no functional protein could be recovered. Experiments to remove the thioredoxin moiety by enterokinase cleavage at the engineered recognition site under a variety of conditions resulted in nonspecific proteolysis and inactivation of the protein. The intact fusion protein was, therefore, used for kinetic studies and crystallization trials. It has been purified to greater than 90% homogeneity by ammonium sulfate precipitation followed by phenyl Sepharose chromatography. Based on k(cat)/KM for pyruvate, it is 20% as active as native VanH. Michaelis constants for NADPH, NADH, and pyruvate, of approximately 3.5 microM, 19.0 microM, and 1.5 mM, respectively, were comparable to those reported for the native VanH (Bugg TDH et al., 1991, Biochemistry 30:10408-10415). Like native VanH, maximum activity of the fusion protein requires the presence of an anion (phosphate or acetate), however, in addition, a strongly reducing environment is needed for optimal efficacy. Competitive inhibition constants for ADP-ribose, NAD+, and oxamate have also been determined. Crystallization by hanging drop vapor diffusion produced two different crystal forms, one hexagonal and the other tetragonal. Flash-frozen crystals of the tetragonal form diffracted to 3.0 A resolution at a synchrotron radiation source. PMID:9605319

Stoll, V S; Manohar, A V; Gillon, W; MacFarlane, E L; Hynes, R C; Pai, E F

1998-05-01

165

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

166

Long term intensive exercise training leads to a higher plasma malate/lactate dehydrogenase (M/L) ratio and increased level of lipid mobilization in horses.  

PubMed

Continuous high intensity training may induce alterations to enzyme activities related to glucose and lipid metabolism in horses. In our study, five Thoroughbred race horses (3 male and 2 female, avg age=5 yrs old) were compared against five riding horses (1 male, 1 female, 3 gelding, avg age=13 yrs old) in terms of energy metabolism, by examining plasma malate (MDH) and lactate (LDH) dehydrogenase activities and M/L ratio. MDH is involved in NADH and ATP generation, whereas LDH can convert NADH back into NAD(+) for ATP generation. An increase in plasma M/L ratio can reflect heightened energy metabolism in the liver and skeletal muscle of horses adapted to continuous intensive exercise. Moreover, plasma lipid metabolism analytes (adiponectin, NEFA, total cholesterol (T-Cho), and triglycerides (TG)) can reflect changes to lipolysis rate, which can also indicate a change in energy metabolism. Overall, race horses demonstrated increased MDH and LDH activity in plasma (4x and 2x greater, respectively), in addition to a plasma M/L ratio twice as high as that of riding horses (2.0 vs 1.0). In addition, race horses also demonstrated significantly higher levels of plasma NEFA (50% greater), TG (2x greater), and T-Cho (20% greater) as compared to riding horses. Therefore, race horse muscles may have adapted to prolonged high intensity endurance exercise by gaining a higher oxidative capacity and an increased capacity for fat utilization as an energy source, resulting in heightened energy metabolism and increased rate of lipid mobilization. PMID:22297553

Li, Gebin; Lee, Peter; Mori, Nobuko; Yamamoto, Ichiro; Arai, Toshiro

2012-06-01

167

Detection of histidine rich protein & lactate dehydrogenase of Plasmodium falciparum in malaria patients by sandwich ELISA using in-house reagents  

PubMed Central

Background & objectives: Despite major control efforts, malaria remains a major public health problem that still causes high mortality rate worldwide especially in Africa and Asia. Accurate and confirmatory diagnosis before treatment initiation is the only way to control the disease. The present study was undertaken to develop reagents using sandwich ELISA for simultaneous detection of PfHRP2 (Plasmodium falciparum histidine rich protein) and PfLDH (P. falciparum lactate dehydrogenase) antigens in the proven malaria cases. Methods: The antibodies were raised against two epitopes of PfHRP2 protein and three unique and unexplored epitopes of PfLDH protein. These antibodies were able to detect PfHRP2 and PfLDH antigens in culture supernatant and parasitized RBC lysate of P. falciparum, respectively up to 50 parasites/?l. The in-house reagents were tested in 200 P. falciparum positive patients residing in Baghpat district of Uttar Pradesh in northern India. Results: Microsphere (PLGA) with CpG ODN were used to generate high titre and high affinity antibodies against selected peptides of PfHRP-2 and pLDH antigen in mice and rabbit. The peptide specific peak titre varied from 12,800 - 102,400 with an affinity ranging 0.73 - 3.0 mM. The indigenously developed reagents are able to detect PfHRP2 and PfLDH antigens as low as 75 parasites/?l of blood with a very high sensitivity (96-100%) and specificity (100%). Interpretation & conclusions: The study highlight the identification of unique epitopes of PfHRP2 and PfLDH, and the generated antibodies against these antigens were used for quantitative estimation of these two antigens using sandwich ELISA. No corresreactivity with P. vivax infected patients was observed with the sera. PMID:24521645

Verma, Priyanka; Biswas, Sukla; Mohan, Teena; Ali, Shakir; Rao, D.N.

2013-01-01

168

A study of salivary lactate dehydrogenase isoenzyme levels in patients with oral leukoplakia and squamous cell carcinoma by gel electrophoresis method  

PubMed Central

Context: The enzyme lactate dehydrogenase (LDH), which is found in almost all the cells of body tissues, can be separated into five fractions and the isoenzyme pattern is believed to vary according to the metabolic requirement of each tissue. LDH concentration in saliva, as an expression of cellular necrosis, could be considered to be a specific indicator for oral lesions that affect the integrity of the oral mucosa. Aim: The present study was designed to evaluate salivary LDH isoenzyme pattern in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) and to correlate between LDH isoenzyme levels and histopathologic grading in selected cases of OL and OSCC. Materials and Methods: Clinically diagnosed 30 cases each of OL and OSCC were selected for the study and 30 healthy individuals of comparable age served as control. Unstimulated whole saliva was aseptically collected and was processed immediately for LDH isoenzymes measurement by agarose gel electrophoresis. Biopsy specimen obtained was processed and stained by hematoxylin and eosin. Sections of OL and OSCC cases were scrutinized histopathologically and appropriately graded for epithelial dysplasia and differentiation of carcinoma respectively. Statistical Analysis Used: Two sample t test for testing the significance of difference between two group means was used. Results and Conclusion: The present salivary analysis for LDH isoenzyme reveals an overall increased salivary LDH isoenzyme level in OL and OSCC cases and a significant correlation between levels of salivary LDH isoenzymes and histopathologic grades of dysplasia in OL and OSCC. Salivary analysis of LDH will definitely provide the clinician and/or the patient himself with an efficient, non invasive and friendly new tool for diagnosis and monitoring of oral precancer and cancer. PMID:25364177

Joshi, Priya Shirish; Golgire, Someshwar

2014-01-01

169

Bioactivity-Guided Identification and Cell Signaling Technology to Delineate the Lactate Dehydrogenase A Inhibition Effects of Spatholobus suberectus on Breast Cancer  

PubMed Central

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

170

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(2)) 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

171

The role of N286 and D320 in the reaction mechanism of human dihydrolipoamide dehydrogenase (E3) center domain  

Microsoft Academic Search

Summary  According to the multiple alignment of various dihydrolipoamide dehydrogenases (E3s) sequences, three human mutant E3s of\\u000a the conserved residues in the center domain, N286D, N286Q, and D320N were created, over-expressed and purified. We characterized\\u000a these mutants to investigate the reaction mechanism of human dihydrolipoamide dehydrogenases. The specific activities of N286D,\\u000a N286Q, and D320N are 30.84%, 24.57% and 48.60% to that

Yi-Chun Wang; Shih-Tsung Wang; Chuan Li; Wen-Hu Liu; Pei-Ru Chen; Ling-Yun Chen; Te-Chung Liu

2007-01-01

172

21 CFR 184.1311 - Ferrous lactate.  

Code of Federal Regulations, 2013 CFR

...crystalline mass. It is prepared by reacting calcium lactate or sodium lactate with ferrous sulfate, direct reaction of lactic acid with iron filings, reaction of ferrous chloride with sodium lactate, or reaction of ferrous sulfate with...

2013-04-01

173

21 CFR 184.1311 - Ferrous lactate.  

Code of Federal Regulations, 2012 CFR

...crystalline mass. It is prepared by reacting calcium lactate or sodium lactate with ferrous sulfate, direct reaction of lactic acid with iron filings, reaction of ferrous chloride with sodium lactate, or reaction of ferrous sulfate with...

2012-04-01

174

21 CFR 184.1311 - Ferrous lactate.  

...crystalline mass. It is prepared by reacting calcium lactate or sodium lactate with ferrous sulfate, direct reaction of lactic acid with iron filings, reaction of ferrous chloride with sodium lactate, or reaction of ferrous sulfate with...

2014-04-01

175

(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

176

Base compositions of genes encoding alpha-actin and lactate dehydrogenase-A from differently adapted vertebrates show no temperature-adaptive variation in G + C content.  

PubMed

There is a long-standing debate in molecular evolution concerning the putative importance of GC content in adapting the thermal stabilities of DNA and RNA. Most studies of this relationship have examined broad-scale compositional patterns, for example, total GC percentages in genomes and occurrence of GC-rich isochores. Few studies have systematically examined the GC contents of individual orthologous genes from differently thermally adapted species. When this has been done, the emphasis has been on comparing large numbers of genes in only a few species. We have approached the GC-adaptation temperature hypothesis in a different manner by examining patterns of base composition of genes encoding lactate dehydrogenase-A (ldh-a) and alpha-actin (alpha-actin) from 51 species of vertebrates whose adaptation temperatures ranged from -1.86 degrees C (Antarctic fishes) to approximately 45 degrees C (desert reptile). No significant positive correlation was found between any index of GC content (GC content of the entire sequence, GC content of the third codon position [GC(3)], and GC content at fourfold degenerate sites [GC(4)]) and any index of adaptation temperature (maximal, mean, or minimal body temperature). For alpha-actin, slopes of regression lines for all comparisons did not differ significantly from zero. For ldh-a, negative correlations between adaptation temperature and total GC content, GC(3), and GC(4) were observed but were shown to be due entirely to phylogenetic influences (as revealed by independent contrast analyses). This comparison of GC content across a wide range of ectothermic ("cold-blooded") and endothermic ("warm-blooded") vertebrates revealed that frogs of the genus Xenopus, which have commonly been used as a representative cold-blooded species, in fact are outliers among ectotherms for the alpha-actin analyses, raising concern about the appropriateness of choosing these amphibians as representative of ectothermic vertebrates in general. Our study indicates that, whereas GC contents of isochores may show variation among different classes of vertebrates, there is no consistent relationship between adaptation temperature and the percentage of thermal stability-enhancing G + C base pairs in protein-coding genes. PMID:12519912

Ream, Rachael A; Johns, Glenn C; Somero, George N

2003-01-01

177

A lactate dehydrogenase ELISA-based assay for the in vitro determination of Plasmodium berghei sensitivity to anti-malarial drugs  

PubMed Central

Background Plasmodium berghei rodent malaria is a well-known model for the investigation of anti-malarial drug efficacy in vivo. However, the availability of drug in vitro assays in P. berghei is reduced when compared with the spectrum of techniques existing for Plasmodium falciparum. New alternatives to the current manual or automated methods described for P. berghei are attractive. The present study reports a new ELISA drug in vitro assay for P. berghei using two monoclonal antibodies against the parasite lactate dehydrogenase (pLDH). Methods This procedure includes a short-in vitro culture, the purification of schizonts and the further generation of synchronized mice infections. Early stages of the parasite are then incubated against different concentrations of anti-malarial drugs using micro-plates. The novelty of this procedure in P. berghei relies on the quantification of the drug activity derived from the amount of pLDH estimated by an ELISA assay using two monoclonal antibodies: 14C1 and 19G7. The IC50s obtained through the ELISA assay were compared with those from the micro-test. Results The initial parameters of the synchronized samples used in the in vitro assays were a parasitaemia of 0.5% and haematocrit of 1%, with an incubation period of 22 hours at 36.5°C. pLDH detection using a 14C1 coating at 10 ?g/ml and 19G7 at 2.5 × 10-3 ?g/ml provided good readouts of optical densities with low background in negative controls and specific detection levels for all parasite stages. IC50s values derived from the ELISA assay for artesunate, chloroquine, amodiaquine and quinine were: 15, 7, 2, and 144 nM, respectively. When artesunate and chloroquine IC50s were evaluated using the micro-test similar values were obtained. Conclusion This ELISA-based in vitro drug assay is easy to implement, fast, and avoids the use radioisotopes or expensive equipment. The utility of this simple assay for screening anti-malarial drug activity against P. berghei in vitro is demonstrated. PMID:23126583

2012-01-01

178

Use of tritiated prostaglandins in metabolism studies. I: Evaluation of the kinetic isotope effect in the prostaglandin dehydrogenase reactions  

Microsoft Academic Search

Although numerous data exist concerning tritium kinetic isotope effect in enzymic reactions, little is related to the metabolism of tritiated prostaglandins. The present study reports an evaluation of the kinetic isotope effect which occurs during the oxidation of 15-hydroxyl group of tritium-labeled prostaglandins E2 and F2 alpha by the 15-hydroxyprostaglandin dehydrogenase and during the oxidation of 9-hydroxyl group of tritium-labeled

C. Moussard; D. Alber; C. Perruche; J. C. Henry

1986-01-01

179

Potentiometric CO titrations of carbon monoxide dehydrogenase and CO-inhibition of the NI-removing reaction with 1,10--phenanthroline  

E-print Network

POTENTIOMETRIC CO TITRATIONS OF CARBON MONOX1DE DEHYDROGENASE AND CO-INHIBITION OF THE Nl-REMOVING REACTION WITH 1, 10-PHENANTHROLINE A Thesis by WILLIAM KENT RUSSELL Submitted to the Office of Graduate Studies of Texas ARM University... in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE December 1996 Major Subject: Chemistry POTENTIOMETRIC CO TITRATIONS OF CARBON MONOXIDE DEHYDROGENASE AND CO-INHIBITION OF THE NI-REMOVlNG REACTION WITH 1, 10-PHENANTHROLINE...

Russell, William Kent

2012-06-07

180

Comparison of results of the CellTiter Blue, the tetrazolium (3-[4,5-dimethylthioazol-2-yl]-2,5-diphenyl tetrazolium bromide), and the lactate dehydrogenase assay applied in brain cells after exposure to advanced glycation endproducts.  

PubMed

Advanced glycation endproducts (AGEs) arise in vivo from the reaction of proteins with sugars or dicarbonyl compounds. They are thought to be involved in the pathogenesis of several diseases such as atherosclerosis, diabetes mellitus, renal failure, and Alzheimer's disease (AD). Several binding molecules for AGEs have been described and it is assumed that many of the effects of AGEs are mediated by receptors like the receptor for AGEs (RAGE). AGEs are known to induce the release of inflammatory cytokines from activated glia in the AD brain and thus AGEs affect the cell viability of neurons and glia. In cell culture experiments controversial effects of AGEs on cell growth and viability were reported by different research groups ranging from stimulation to inhibition of the cell viability. In the present study, the effect of in vitro prepared highly modified AGEs on the viability and the membrane integrity of cultured brain cells was investigated. Three different brain cell lines were treated with glucose human serum albumin AGEs (Glc-AGEs) and methyl glyoxal human serum albumin AGEs (MG-AGEs). To investigate the effect of these model AGEs on cell viability the CellTiter Blue (CTB) and the tetrazolium (3-[4,5-dimethylthioazol-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT) were used. The membrane integrity after exposure to AGEs was assayed using the lactate dehydrogenase (LDH) assay. When using the CTB assay for evaluation all AGEs were found to reduce the viability compared with the native protein in all three cell lines. Additionally, all AGEs were found to affect the membrane integrity compared with the native protein in all cell lines. When using the MTT assay for evaluation only MG-AGEs were found to cause a decrease in the viability in all cell lines used. The results of the MTT assay in Glc-AGEs treated cells varied between the cell lines. To gain a deeper understanding of the cellular responses after exposure of cells to AGEs, the present study compares results obtained when using the CTB, the MTT or the LDH assay in identically AGE treated cells. PMID:17391910

Bigl, Katrin; Schmitt, Annett; Meiners, Ina; Münch, Gerald; Arendt, Thomas

2007-08-01

181

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

182

The role of N286 and D320 in the reaction mechanism of human dihydrolipoamide dehydrogenase (E3) center domain.  

PubMed

According to the multiple alignment of various dihydrolipoamide dehydrogenases (E3s) sequences, three human mutant E3s of the conserved residues in the center domain, N286D, N286Q, and D320N were created, over-expressed and purified. We characterized these mutants to investigate the reaction mechanism of human dihydrolipoamide dehydrogenases. The specific activities of N286D, N286Q, and D320N are 30.84%, 24.57% and 48.60% to that of the wild-type E3 respectively. The FAD content analysis indicated that these mutant E3s about 96.0%, 99.4% and 82.7% of FAD content compared to that of wild-type E3 respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic's data demonstrated that the K(cat) of both forward and reverse reactions of these mutant proteins were decreased. These results suggest that N286 and D320 play a role in the catalytic function of the E3. PMID:17171578

Wang, Yi-Chun; Wang, Shih-Tsung; Li, Chuan; Liu, Wen-Hu; Chen, Pei-Ru; Chen, Ling-Yun; Liu, Te-Chung

2007-03-01

183

Fiber optic biosensors for hydrogen peroxide and L-lactate  

NASA Astrophysics Data System (ADS)

An optical fiber biosensor for the selective determination of hydrogen peroxide has been developed as the base sensor for the construction of multienzyme optodes involving lactate converting enzymes for the analysis of lactic acid. The optode uses the H2O2 dependent oxidation of homovanillic acid by horseradish peroxidase (HRP) as the sensing reaction. The fluorescence of the dimeric product formed is used as the measuring signal related to the concentration of H2O2. HRP was immobilized on a membrane and combined with a bifurcated fiber optic probe. Under optimized conditions the sensor responds linearly to hydrogen peroxide between 1 micrometers ol/l and 0.12 mmol/l and exhibits a half life of 90 days. Using a lactate oxidase-HRP membrane, the sensor is suitable for lactate measurement with a linear range of 3 micrometers ol/l-0.2 mmol/l. To increase the sensitivity for lactate, lactate dehydrogenase was coimmobilized on the sensor membrane. In the presence of NADH the signal for lactate is amplified fourfold through the internal analyte recycling accomplished by the lactate-converting enzymes.

Schubert, Florian; Rinneberg, Herbert H.; Wang, Fang

1995-02-01

184

Reaction norm of fertility traits adjusted for protein and fat production level across lactations in Holstein cattle.  

PubMed

A total of 304,001 artificial insemination outcomes in up to 7 lactations from 142,389 Holstein cows, daughters of 5,349 sires and 101,433 dams, calving between January 1995 and December 2007 in 1,347 herds were studied by a reaction norm model. The (co)variance components for days to first service (DFS), days open, nonreturn rate in the first service (NRFS), and number of services per conception were estimated by 6 models: 3 Legendre polynomial degrees for the genetic effects and adjustment or not for the level of fat plus protein (FP) production recorded at day closest to DFS. For all traits and type of FP adjustment, a second degree polynomial showed the best fit. The use of the adjusted FP model did not increase the level of genetic (co)variance components except for DFS. The heritability for each of the traits was low in general (0.03-0.10) and increased from the first to fourth calving; nevertheless, very important variability was found for the estimated breeding value (EBV) of the sires. The genetic correlations (rg) were close to unity between adjacent calvings, but decreased for most distant parities, ranging from rg=0.36 (for DFS) to rg=0.63 (for NRFS), confirming the existence of heterogeneous genetic (co)variance components and EBV across lactations. The results of the eigen decomposition of rg shows that the first eigenvalue explained between 82 to 92% and the second between 8 to 14% of the genetic variance for all traits; therefore, a deformation of the overall mean trajectory for reproductive performance across the trajectory of the different calving could be expected if selection favored these eigenfunctions. The results of EBV for the 50 best sires showed a substantial reranking and variation in the shape of response across lactations. The more important aspect to highlight, however, is the difference between the EBV of the same sires in different calvings, a characteristic known as plasticity, which is particularly important for DFS and NRFS. This component of fertility adds another dimension to selection for fertility that can be used to change the negative genetic progress of reproductive performance presented in this population of Holstein cows. The use of a reaction norm model should allow producers to obtain more robust cows for maintenance of fertility levels along the whole productive life of the cows. PMID:23664344

Menendez-Buxadera, A; Carabaño, M J; Gonzalez-Recio, O; Cue, R I; Ugarte, E; Alenda, R

2013-07-01

185

Protein kinase A stimulates binding of multiple proteins to a U-rich domain in the 3'-untranslated region of lactate dehydrogenase A mRNA that is required for the regulation of mRNA stability.  

PubMed

We have explored the molecular basis of the cAMP-induced stabilization of lactate dehydrogenase A (LDH-A) mRNA and identified four cytoplasmic proteins of 96, 67, 52, and 50 kDa that specifically bind to a 30-nucleotide uridine-rich sequence in the LDH 3'-untranslated region with a predicted stem-loop structure. Mutational analysis revealed that specific protein binding is dependent upon an intact primary nucleotide sequence in the loop as well as integrity of the adjoining double-stranded stem structure, thus indicating a high degree of primary and secondary structure specificity. The critical stem-loop region is located between nucleotides 1473 and 1502 relative to the mRNA cap site and contains a previously identified cAMP-stabilizing region (CSR) required for LDH-A mRNA stability regulation by the protein kinase A pathway. The 3'-untranslated region binding activity of the proteins is up-regulated after protein kinase A activation, whereas protein dephosphorylation is associated with a loss of binding activity. These results imply a cause and effect relationship between LDH-A mRNA stabilization and CSR-phosphoprotein binding activity. We propose that the U-rich CSR is a recognition signal for CSR-binding proteins and for an mRNA processing pathway that specifically stabilizes LDH mRNA in response to activation of the protein kinase A signal transduction pathway. PMID:9774474

Tian, D; Huang, D; Brown, R C; Jungmann, R A

1998-10-23

186

Bistability in the Isocitrate Dehydrogenase Reaction: An Experimentally Based Theoretical Study  

E-print Network

the occurrence of bistability for parameter values derived from the experiments. Depending on the total to the medium containing IDH and its substrate NADP . We present a theoretical and numerical analysis of a model of biochemical models show that the phenomenon can originate in enzymatic reactions either from positive feedback

Goldbeter, Albert

187

The Reductive Half-reaction of Xanthine Dehydrogenase from Rhodobacter capsulatus: THE ROLE OF GLU232 IN CATALYSIS.  

PubMed

The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu(232) in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu(232) being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme. PMID:25258317

Hall, James; Reschke, Stefan; Cao, Hongnan; Leimkühler, Silke; Hille, Russ

2014-11-14

188

Contribution of K99 and D319 to Substrate Binding and Catalysis in the Saccharopine Dehydrogenase Reaction  

PubMed Central

Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to L-lysine and ?-ketoglutarate. Lysine 99 is within hydrogen-bond distance to the ?-carboxylate of the lysine substrate and D319 is in the vicinity of the carboxamide side chain of NADH. Both are conserved and may be important to the overall reaction. Replacing K99 with M gives decreases of 110-, 80- and 20-fold in the V2/Km values for lysine, ?-ketoglutarate and NADH, respectively. Deuterium isotope effects on V and V/KLys increase, while the solvent deuterium isotope effects decrease compared to the C205S mutant enzyme. Data for K99M suggest a decreased affinity for all reactants and a change in the partition ratio of the imine intermediate to favor hydrolysis. A change in the bound conformation of the imine and/or the distance of the imine carbon to C4 of the nicotinamide ring of NADH is also suggested. Changing D319 to A decreases V2/KNADH by 33-fold. Primary deuterium and solvent deuterium isotope effects decrease compared to C205S suggesting a non-isotope sensitive step has become slower. NADH binds to enzyme first, and sets the site for binding of lysine and ?-ketoglutarate. The slower step is likely the conformational change generated upon binding of NADH. PMID:21819960

Ekanayake, Devi K.; West, Ann H.; Cook, Paul. F.

2011-01-01

189

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. PMID:8142887

Balme, A.; Lederer, F.

1994-01-01

190

13C NMR Characterization of an Exchange Reaction between CO and CO2 Catalyzed by Carbon Monoxide Dehydrogenase  

PubMed Central

Carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidation of CO to CO2 at a nickel?iron?sulfur cluster (the C-cluster). CO oxidation follows a ping-pong mechanism involving two-electron reduction of the C-cluster followed by electron transfer through an internal electron transfer chain to external electron acceptors. We describe 13C NMR studies demonstrating a CODH-catalyzed steady-state exchange reaction between CO and CO2 in the absence of external electron acceptors. This reaction is characterized by a CODH-dependent broadening of the 13CO NMR resonance; however, the chemical shift of the 13CO resonance is unchanged, indicating that the broadening is in the slow exchange limit of the NMR experiment. The 13CO line broadening occurs with a rate constant (1080 s?1 at 20 °C) that is approximately equal to that of CO oxidation. It is concluded that the observed exchange reaction is between 13CO and CODH-bound 13CO2 because 13CO line broadening is pH-independent (unlike steady-state CO oxidation), because it requires a functional C-cluster (but not a functional B-cluster) and because the 13CO2 line width does not broaden. Furthermore, a steady-state isotopic exchange reaction between 12CO and 13CO2 in solution was shown to occur at the same rate as that of CO2 reduction, which is approximately 750-fold slower than the rate of 13CO exchange broadening. The interaction between CODH and the inhibitor cyanide (CN?) was also probed by 13C NMR. A functional C-cluster is not required for 13CN? broadening (unlike for 13CO), and its exchange rate constant is 30-fold faster than that for 13CO. The combined results indicate that the 13CO exchange includes migration of CO to the C-cluster, and CO oxidation to CO2, but not release of CO2 or protons into the solvent. They also provide strong evidence of a CO2 binding site and of an internal proton transfer network in CODH. 13CN? exchange appears to monitor only movement of CN? between solution and its binding to and release from CODH. PMID:18589895

2008-01-01

191

On the reaction mechanism of l-lactate oxidase: Quantitative structure-activity analysis of the reaction with para-substituted l-mandelates  

PubMed Central

The rate constants for reduction of the flavoenzyme, l-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1H- and 2-2H- forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects (1H/2H = 3–7) on the rate constants of flavin reduction were found, indicating that flavin reduction is a direct measure of ?-C-H bond breakage. The rate constants show only a small influence of the electronic characteristics of the substituents, but show a good correlation when combined with some substituent volume parameters. A surprisingly good correlation is found with the molecular mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the flavin N(5) position or a synchronous mechanism in which the ?-C-H is formally abstracted as a H+ while the resulting charge is simultaneously neutralized by another event. PMID:9275167

Yorita, Kazuko; Janko, Karl; Aki, Kenji; Ghisla, Sandro; Palfey, Bruce A.; Massey, Vincent

1997-01-01

192

Utilization of enzyme cascades for complete oxidation of lactate in an enzymatic biofuel cell  

Microsoft Academic Search

Lactate\\/lactic acid has been considered as a biofuel for enzymatic biofuel cells, but only with single enzyme bioanodes containing lactate dehydrogenase. A single enzyme-based bioanode results in the oxidation of lactate to pyruvate, which only allows for 2 of the total of 12 electrons to be harnessed from the lactate leaving the majority of the energy density of the fuel

Daria Sokic-Lazic; Adalgisa Rodrigues de Andrade; Shelley D. Minteer

193

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. PMID:23149136

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

2013-01-01

194

Reconstruction of lactate utilization system in Pseudomonas putida KT2440: a novel biocatalyst for l-2-hydroxy-carboxylate production.  

PubMed

As an important method for building blocks synthesis, whole cell biocatalysis is hindered by some shortcomings such as unpredictability of reactions, utilization of opportunistic pathogen, and side reactions. Due to its biological and extensively studied genetic background, Pseudomonas putida KT2440 is viewed as a promising host for construction of efficient biocatalysts. After analysis and reconstruction of the lactate utilization system in the P. putida strain, a novel biocatalyst that only exhibited NAD-independent d-lactate dehydrogenase activity was prepared and used in l-2-hydroxy-carboxylates production. Since the side reaction catalyzed by the NAD-independent l-lactate dehydrogenase was eliminated in whole cells of recombinant P. putida KT2440, two important l-2-hydroxy-carboxylates (l-lactate and l-2-hydroxybutyrate) were produced in high yield and high optical purity by kinetic resolution of racemic 2-hydroxy carboxylic acids. The results highlight the promise in biocatalysis by the biotechnologically important organism P. putida KT2440 through genomic analysis and recombination. PMID:25373400

Wang, Yujiao; Lv, Min; Zhang, Yingxin; Xiao, Xieyue; Jiang, Tianyi; Zhang, Wen; Hu, Chunhui; Gao, Chao; Ma, Cuiqing; Xu, Ping

2014-01-01

195

Reconstruction of lactate utilization system in Pseudomonas putida KT2440: a novel biocatalyst for l-2-hydroxy-carboxylate production  

PubMed Central

As an important method for building blocks synthesis, whole cell biocatalysis is hindered by some shortcomings such as unpredictability of reactions, utilization of opportunistic pathogen, and side reactions. Due to its biological and extensively studied genetic background, Pseudomonas putida KT2440 is viewed as a promising host for construction of efficient biocatalysts. After analysis and reconstruction of the lactate utilization system in the P. putida strain, a novel biocatalyst that only exhibited NAD-independent d-lactate dehydrogenase activity was prepared and used in l-2-hydroxy-carboxylates production. Since the side reaction catalyzed by the NAD-independent l-lactate dehydrogenase was eliminated in whole cells of recombinant P. putida KT2440, two important l-2-hydroxy-carboxylates (l-lactate and l-2-hydroxybutyrate) were produced in high yield and high optical purity by kinetic resolution of racemic 2-hydroxy carboxylic acids. The results highlight the promise in biocatalysis by the biotechnologically important organism P. putida KT2440 through genomic analysis and recombination. PMID:25373400

Wang, Yujiao; Lv, Min; Zhang, Yingxin; Xiao, Xieyue; Jiang, Tianyi; Zhang, Wen; Hu, Chunhui; Gao, Chao; Ma, Cuiqing; Xu, Ping

2014-01-01

196

Amperometric determination of lactate with novel trienzyme/poly(carbamoyl) sulfonate hydrogel-based sensor.  

PubMed

A novel trienzyme sensor for the amperometric determination of lactate was constructed by immobilizing salicylate hydroxylase (SHL, E.C. 1.14.13.1), l-lactate dehydrogenase (LDH, E.C. 1.1.1.27), and pyruvate oxidase (PyOD, E.C. 1.2.3.3) on a Clark-type oxygen electrode. The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. LDH catalyzes the specific dehydrogenation of lactate consuming NAD(+). SHL catalyzes the irreversible decarboxylation and the hydroxylation of salicylate in the presence of oxygen and NADH produced by LDH. PyOD decarboxylates pyruvate using oxygen and phosphate. SHL and PyOD force the equilibrium of dehydrogenation of lactate by LDH to the product side by consuming NADH and pyruvate, respectively. Dissolved oxygen acts as an essential material for both PyOD and SHL during their respective enzymatic reactions. Therefore, an amplified signal, caused by the consumptions of dissolved oxygen by the two enzymes, was observed in the measurement of lactate. Regeneration of cofactor was found in the trienzyme system. A Teflon membrane was used to fabricate the sensor in order to avoid interferences. The sensor has a fast response (2s) and short recovery times (2 min). The total test time for a measurement by using this lactate sensor (4 min) was faster than using a commercial lactate testing kit (up to 10 min). The sensor has a linear range between 10 and 400 microM lactate, with a detection limit of 4.3 microM. A good agreement (R2 = 0.9984) with a commercial lactate testing kit was obtained in beverage sample measurements. PMID:15142609

Kwan, Roger C H; Hon, Phoebe Y T; Mak, Karen K W; Renneberg, Reinhard

2004-07-15

197

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

198

Lactate metabolism in the fetal rabbit lung  

SciTech Connect

Lactate is frequently overlooked as a potential substrate for the fetal lung, even though it is present in the fetal circulation in concentrations as high as 8 mM. These high concentrations, coupled with the relatively low levels of glucose in the fetal blood, may indicate that lactate can substitute for glucose in pulmonary energy generation and phospholipid synthesis. A series of experiments was therefore undertaken in order to investigate the role of lactate in perinatal pulmonary development. Explants from 30 day gestation fetal rabbit lungs were incubated in Krebs-Ringer bicarbonate buffer supplemented with 3 mM (U-/sup 14/C)-glucose and varying levels of lactate. In the absence of medium lactate, fetal rabbit lung explants were capable of producing lactate at a rate of approximately 200 etamoles/mg protein/hour. The addition of lactate to the bathing medium immediately reduced net lactate production and above 4 mM, fetal rabbit lung explants became net utilizers of lactate. Media lactate concentrations of 2.5 mM, 5 mM and 10 mM also decreased glucose incorporation into total tissue disaturated phosphatidylcholine by approximately 20%, 35%, and 45%, respectively. Glucose incorporation into surfactant phosphatidylcholine was also reduced by approximately 50%, when lactate was present in the incubation medium at a concentration of 5 mM. Additional experiments also revealed that fetal lung lactate dehydrogenase activity was almost twice that found in the adult rabbit lung. These data indicate that lactate may be an important carbon source for the developing lung and could be a significant component in the manufacture of surfactant phosphatidylcholine during late gestation.

Engle, M.J.; Brown, D.J.; Dooley, M.

1986-05-01

199

The crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A complexed with the enzyme reaction product throws light on its enzymatic function.  

PubMed Central

Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. A description is given of the crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A (DHODA) complexed with the product of the enzyme reaction orotate. The structure of the complex to 2.0 A resolution has been compared with the structure of the native enzyme. The active site of DHODA is known to contain a water filled cavity buried beneath a highly conserved and flexible loop. In the complex the orotate displaces the water molecules from the active site and stacks above the DHODA flavin isoalloxazine ring, causing only small movements of the surrounding protein residues. The orotate is completely buried beneath the protein surface, and the orotate binding causes a significant reduction in the mobility of the active site loop. The orotate is bound by four conserved asparagine side chains (Asn 67, Asn 127, Asn 132, and Asn 193), the side chains of Lys 43 and Ser 194, and the main chain NH groups of Met 69, Gly 70, and Leu 71. Of these the Lys 43 side chain makes hydrogen bonds to both the flavin isoalloxazine ring and the carboxylate group of the orotate. Potential interactions with bound dihydroorotate are considered using the orotate complex as a basis for molecular modeling. The role of Cys 130 as the active site base is discussed, and the sequence conservation of the active site residues across the different families of DHODs is reviewed, along with implications for differences in substrate binding and in the catalytic mechanisms between these families. PMID:9655329

Rowland, P.; Bjornberg, O.; Nielsen, F. S.; Jensen, K. F.; Larsen, S.

1998-01-01

200

D-lactate metabolism in the alga, Chlamydomonas Reinhardtii  

SciTech Connect

(/sup 14/C)D-lactate rapidly accumulates in Chlamydomonas cells under anaerobic conditions from the sugar-phosphate pools which are labeled during photosynthesis with /sup 14/CO/sub 2/. A soluble D-lactate dehydrogenase (30 ..mu..mol NADH oxidized/h/mg Chl), which functions only in the direction of pyruvate reduction, has been partially purified and characterized. The D-lactate is reoxidized in Chlamydomonas by a mitochondrial membrane-bound dehydrogenase. This enzyme is known in the plant literature as glycolate dehydrogenase, an enzyme of the oxidative photosynthetic carbon (C/sub 2/) cycle. This dehydrogenase may be linked to the mitochondrial electron transport chain, although the direct electron acceptor is unknown. Therefore, D-lactate accumulation may be, in part, due to the shut down of electron transport during anaerobiosis. In vivo chase experiments have shown that the D-lactate turns over rapidly when algal cells, which have been grown with air levels of CO/sub 2/ (0.04%), are returned to aerobic conditions in the light. Such turnover is not observed in cells which had been grown with 1 to 5% CO/sub 2/. Cells grown with high CO/sub 2/ have lower levels of glycolate dehydrogenase activity. They are currently using mutants of Chlamydomonas deficient in mitochondrial respiration to study the role of D-lactate oxidation in these algae.

Husic, D.W.; Tolbert, N.E.

1986-05-01

201

Pyruvate into lactate and back: From the Warburg effect to symbiotic energy fuel exchange in cancer cells  

Microsoft Academic Search

Tumor cells fuel their metabolism with glucose and glutamine to meet the bioenergetic and biosynthetic demands of proliferation. Hypoxia and oncogenic mutations drive glycolysis, with the pyruvate to lactate conversion being promoted by increased expression of lactate dehydrogenase A and inactivation of pyruvate dehydrogenase. The NAD+ pool is consecutively regenerated and supports the high glycolytic flux required to produce anabolic

Olivier Feron

2009-01-01

202

Lactic acid-producing yeast cells having nonfunctional L- or D-lactate:ferricytochrome C oxidoreductase cells  

DOEpatents

Yeast cells having an exogenous lactate dehydrogenase gene ae modified by reducing L- or D-lactate:ferricytochrome c oxidoreductase activity in the cell. This leads to reduced consumption of lactate by the cell and can increase overall lactate yields in a fermentation process. Cells having the reduced L- or D-lactate:ferricytochrome c oxidoreductase activity can be screened for by resistance to organic acids such as lactic or glycolic acid.

Miller, Matthew (Boston, MA); Suominen, Pirkko (Maple Grove, MN); Aristidou, Aristos (Highland Ranch, CO); Hause, Benjamin Matthew (Currie, MN); Van Hoek, Pim (Camarillo, CA); Dundon, Catherine Asleson (Minneapolis, MN)

2012-03-20

203

Utilization of Lactate Isomers by Propionibacterium freudenreichii subsp. shermanii: Regulatory Role for Intracellular Pyruvate.  

PubMed

Five strains of Propionibacterium freudenreichii subsp. shermanii utilized the l-(+) isomer of lactate at a faster rate than they did the d-(-) isomer when grown with a mixture of lactate isomers under a variety of conditions. ATCC 9614, grown anaerobically in defined medium containing 160 mM dl-lactate, utilized only 4 and 15% of the d-(-)-lactate by the time 50 and 90%, respectively, of the l-(+)-lactate was used. The intracellular pyruvate concentration was high (>100 mM) in the initial stages of lactate utilization, when either dl-lactate or the l-(+) isomer was the starting substrate. The concentration of this intermediate dropped during dl-lactate fermentation such that when only d-(-)-lactate remained, the concentration was <20 mM. When only the d-(-) isomer was initially present, a similar relatively low concentration of intracellular pyruvate was present, even at the start of lactate utilization. The NAD-independent lactate dehydrogenase activities in extracts showed different kinetic properties with regard to pyruvate inhibition, depending upon the lactate isomer present. Pyruvate gave a competitive inhibitor pattern with l-(+)-lactate and a mixed-type inhibitor pattern with d-(-)-lactate. It is suggested that these properties of the lactate dehydrogenases and the intracellular pyruvate concentrations explain the preferential use of the l-(+) isomer. PMID:16347134

Crow, V L

1986-08-01

204

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

205

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 PMID:8407843

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

1993-01-01

206

Structural and Kinetic Evidence That Catalytic Reaction of Human UDP-glucose 6-Dehydrogenase Involves Covalent Thiohemiacetal and Thioester Enzyme Intermediates*  

PubMed Central

Biosynthesis of UDP-glucuronic acid by UDP-glucose 6-dehydrogenase (UGDH) occurs through the four-electron oxidation of the UDP-glucose C6 primary alcohol in two NAD+-dependent steps. The catalytic reaction of UGDH is thought to involve a Cys nucleophile that promotes formation of a thiohemiacetal enzyme intermediate in the course of the first oxidation step. The thiohemiacetal undergoes further oxidation into a thioester, and hydrolysis of the thioester completes the catalytic cycle. Herein we present crystallographic and kinetic evidence for the human form of UGDH that clarifies participation of covalent catalysis in the enzymatic mechanism. Substitution of the putative catalytic base for water attack on the thioester (Glu161) by an incompetent analog (Gln161) gave a UGDH variant (E161Q) in which the hydrolysis step had become completely rate-limiting so that a thioester enzyme intermediate accumulated at steady state. By crystallizing E161Q in the presence of 5 mm UDP-glucose and 2 mm NAD+, we succeeded in trapping a thiohemiacetal enzyme intermediate and determined its structure at 2.3 Å resolution. Cys276 was covalently modified in the structure, establishing its role as catalytic nucleophile of the reaction. The thiohemiacetal reactive C6 was in a position suitable to become further oxidized by hydride transfer to NAD+. The proposed catalytic mechanism of human UGDH involves Lys220 as general base for UDP-glucose alcohol oxidation and for oxyanion stabilization during formation and breakdown of the thiohemiacetal and thioester enzyme intermediates. Water coordinated to Asp280 deprotonates Cys276 to function as an aldehyde trap and also provides oxyanion stabilization. Glu161 is the Brønsted base catalytically promoting the thioester hydrolysis. PMID:22123821

Egger, Sigrid; Chaikuad, Apirat; Klimacek, Mario; Kavanagh, Kathryn L.; Oppermann, Udo; Nidetzky, Bernd

2012-01-01

207

Mechanistic and Computational Studies of the Reductive Half-Reaction of Tyrosine to Phenylalanine Active Site Variants of d-Arginine Dehydrogenase.  

PubMed

The flavin-mediated enzymatic oxidation of a CN bond in amino acids can occur through hydride transfer, carbanion, or polar nucleophilic mechanisms. Previous results with d-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) using multiple deuterium kinetic isotope effects (KIEs) and computational studies established preferred binding of the substrate protonated on the ?-amino group, with cleavages of the NH and CH bonds occurring in asynchronous fashion, consistent with the three possible mechanisms. The hydroxyl groups of Y53 and Y249 are ?4 Å from the imino and carboxylate groups of the reaction product iminoarginine, suggesting participation in binding and catalysis. In this study, we have investigated the reductive half-reactions of the Y53F and Y249F variants of PaDADH using substrate and solvent deuterium KIEs, solvent viscosity and pH effects, and quantum mechanical/molecular mechanical computational approaches to gain insights into the catalytic roles of the tyrosines and evaluate whether their mutations affect the transition state for substrate oxidation. Both Y53F and Y249F enzymes oxidized d-arginine with steady-state kinetic parameters similar to those of the wild-type enzyme. Rate constants for flavin reduction (kred) with d-leucine, a slow substrate amenable to rapid kinetics, were 3-fold smaller than the wild-type value with similar pKa values for an unprotonated group of ?10.0. Similar pKa values were observed for (app)Kd in the variant and wild-type enzymes. However, cleavage of the substrate NH and CH bonds in the enzyme variants occurred in synchronous fashion, as suggested by multiple deuterium KIEs on kred. These data can be reconciled with a hydride transfer mechanism, but not with carbanion and polar nucleophilic mechanisms. PMID:25243743

Gannavaram, Swathi; Sirin, Sarah; Sherman, Woody; Gadda, Giovanni

2014-10-21

208

Phosphorylant capacity study and lactate mitochondrial oxidation in frozen bovine sperm.  

PubMed

Frozen-stored bovine sperm-pellets of proven fertility were used, and the response to respiratory chain effectors was studied, thus demonstrating the energy conservation capacity. It was further observed that the assayed suspensions used lactate oxidatively, which proves the LDH-X mitochondrial activity (the presence of oxidative substrates is fundamental in capacitation and acrosome reaction processes). The suspensions were treated with 10mM phosphate buffer hypotonic medium to eliminate plasmalema and cytoplasmic content. Lactate respiration was sensitive to respiratory chain effectors, such as oligomycin and antimycin. To evaluate the LDH-X contribution to mitochondrial respiration, lipoate dehydrogenase was inhibited through 5-methoxyindole-2-carboxylic acid (MICA) in the presence of pyruvate-malate and citrate-malate, obtaining with the addition of lactate, oxygen uptakes of 18% and 51% with respect to respiration with the mentioned substrates. In the MICA dose-effect curve, a major sensitivity to inhibitor in active state mitochondrial respiration is obtained when pyruvate-malate is used. Lactate competence with pyruvate by mitochondrial LDH-X was observed. The results obtained would allow the thorough study of the necessity of oxidative energy in the capacitation and fertilization processes, and of the LDH-X role in frozen-stored bovine sperm. PMID:2402176

Beconi, M T; Beorlegui, N B; Sarmiento, N K; Mora, N G

1990-01-01

209

Comprehensive review on lactate metabolism in human health.  

PubMed

Metabolic pathways involved in lactate metabolism are important to understand the physiological response to exercise and the pathogenesis of prevalent diseases such as diabetes and cancer. Monocarboxylate transporters are being investigated as potential targets for diagnosis and therapy of these and other disorders. Glucose and alanine produce pyruvate which is reduced to lactate by lactate dehydrogenase in the cytoplasm without oxygen consumption. Lactate removal takes place via its oxidation to pyruvate by lactate dehydrogenase. Pyruvate may be either oxidized to carbon dioxide producing energy or transformed into glucose. Pyruvate oxidation requires oxygen supply and the cooperation of pyruvate dehydrogenase, the tricarboxylic acid cycle, and the mitochondrial respiratory chain. Enzymes of the gluconeogenesis pathway sequentially convert pyruvate into glucose. Congenital or acquired deficiency on gluconeogenesis or pyruvate oxidation, including tissue hypoxia, may induce lactate accumulation. Both obese individuals and patients with diabetes show elevated plasma lactate concentration compared to healthy subjects, but there is no conclusive evidence of hyperlactatemia causing insulin resistance. Available evidence suggests an association between defective mitochondrial oxidative capacity in the pancreatic ?-cells and diminished insulin secretion that may trigger the development of diabetes in patients already affected with insulin resistance. Several mutations in the mitochondrial DNA are associated with diabetes mellitus, although the pathogenesis remains unsettled. Mitochondrial DNA mutations have been detected in a number of human cancers. d-lactate is a lactate enantiomer normally formed during glycolysis. Excess d-lactate is generated in diabetes, particularly during diabetic ketoacidosis. d-lactic acidosis is typically associated with small bowel resection. PMID:24929216

Adeva-Andany, M; López-Ojén, M; Funcasta-Calderón, R; Ameneiros-Rodríguez, E; Donapetry-García, C; Vila-Altesor, M; Rodríguez-Seijas, J

2014-07-01

210

Genetics Home Reference: Dihydropyrimidine dehydrogenase deficiency  

MedlinePLUS

... symptoms, are vulnerable to severe, potentially life-threatening toxic reactions to certain drugs called fluoropyrimidines that are ... with dihydropyrimidine dehydrogenase deficiency and build up to toxic levels in the body (fluoropyrimidine toxicity). Severe inflammation ...

211

Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions  

SciTech Connect

Shewanella oneidensis MR-1 is a facultative anaerobe growing by coupling organic matter oxidation to reduction of wide range of electron acceptors. Here we quantitatively assessed lactate and pyruvate metabolism of these bacteria under three distinct conditions: electron acceptor limited growth on lactate with O2 and fumarate, and pyruvate fermentation, which does not sustain growth but allows cells to survive for prolonged period. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of all ATP needed for growth depending on the electron acceptor nature and availability. While being indispensible for growth, respiration of fumarate does not contribute much to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions S. oneidensis MR-1 carried out incomplete substrate oxidation, and TCA cycle did not contribute significantly to substrate oxidation. Pyruvate dehydrogenase reaction was not involved in lactate metabolism under O2 limitation, however was important for anaerobic growth probably supplying reducing equivalents for biosynthesis. Unexpectedly, obtained results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination between substrate-level phosphorylation and a respiratory process, where pyruvate serves as electron donor and electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by recently described new type of oxidative NAD(P)H independent D-lactate dehydrogenase (Dld-II). Based on involved enzymes localization we hypothesize that pyruvate reduction coupled to formate oxidation may be accompanied by proton motive force generation.

Pinchuk, Grigoriy E.; Geydebrekht, Oleg V.; Hill, Eric A.; Reed, Jennifer L.; Konopka, Allan; Beliaev, Alex S.; Fredrickson, Jim K.

2011-12-30

212

Efficient Production of Pyruvate from DL-Lactate by the Lactate-Utilizing Strain Pseudomonas stutzeri SDM  

PubMed Central

Background The platform chemical lactate is currently produced mainly through the fermentation of sugars presented in biomass. Besides the synthesis of biodegradable polylactate, lactate is also viewed as a feedstock for the green chemistry of the future. Pyruvate, another important platform chemical, can be produced from lactate through biocatalysis. Methodology/Principal Findings It was established that whole cells of Pseudomonas stutzeri SDM catalyze lactate oxidation with lactate-induced NAD-independent lactate dehydrogenases (iLDHs) through the inherent electron transfer chain. Unlike the lactate oxidation processes observed in previous reports, the mechanism underlying lactate oxidation described in the present study excluded the costliness of the cofactor regeneration step and production of the byproduct hydrogen peroxide. Conclusions/Significance Biocatalysis conditions were optimized by using the cheap dl-lactate as the substrate and whole cells of the lactate-utilizing P. stutzeri SDM as catalyst. Under optimal conditions, the biocatalytic process produced pyruvate at a high concentration (48.4 g l?1) and a high yield (98%). The bioconversion system provides a promising alternative for the green production of pyruvate. PMID:22792404

Gao, Chao; Qiu, Jianhua; Ma, Cuiqing; Xu, Ping

2012-01-01

213

[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

214

Reduction of Coenzyme Q by Succinic Acid Dehydrogenase  

Microsoft Academic Search

Crane, Hatefi, Lester and Widmer1,2 have shown that coenzyme Q is reduced by mitochondria or derivative particles like electron-transporting particles, or succinic dehydrogenase complex, and that this reaction is sensitive to antimycin A. We have now succeeded in reducing coenzyme Q with a soluble preparation of succinic acid dehydrogenase, and this reaction was not sensitive to antimycin A.

Józef Heller; Ludmila Szarkowska; Czeslawa Petryszyn

1961-01-01

215

A comparative proteomic analysis of Bacillus coagulans in response to lactate stress during the production of L-lactic acid.  

PubMed

The growth rate and maximum biomass of Bacillus coagulans 2-6 were inhibited by lactate; inhibition by sodium lactate was stronger than by calcium lactate. The differences of protein expressions by B. coagulans 2-6 under the lactate stress were determined using two-dimensional electrophoresis coupled with mass spectrometric identification. Under the non-stress condition, calcium lactate stress and sodium lactate stress, the number of detected protein spots was 1,571 ± 117, 1,281 ± 231 and 904 ± 127, respectively. Four proteins with high expression under lactate stress were identified: lactate dehydrogenase, cysteine synthase A, aldo/keto reductase and ribosomal protein L7/L12. These proteins are thus potential targets for the reconstruction of B. coagulans to promote its resistance to lactate stress. PMID:25214213

Wang, Xiuwen; Qin, Jiayang; Wang, Landong; Xu, Ping

2014-12-01

216

Regulation of foot muscle glutamate dehydrogenase during  

E-print Network

19/12/2012 1 Regulation of foot muscle glutamate dehydrogenase during land snail estivation Ryan is an important player in amino acid metabolism, with links to the Krebs and urea cycle GDH Glutamate + NAD(P)+ KG compared to Control Km NH4 Km KG Vmax Forward Reaction Reverse Reaction Glutamate + NAD(P)+ KG + NH4

Storey, Kenneth B.

217

Regulation of heart muscle pyruvate dehydrogenase kinase  

PubMed Central

1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [32P]phosphate from [?-32P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100?m) and cyclic 3?:5?-nucleotides (at 10?m) had no significant effect on kinase activity. 3. The Km for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76?m. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The Km for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9–25.4?m. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The Km for pyruvate in the pyruvate dehydrogenase reaction was 35.5?m. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25–500?m. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate (endogenous or added at 2 or 10?m) the kinase activity was enhanced by low concentrations of pyruvate (25–100?m) and inhibited by a high concentration (500?m). Activation of the kinase reaction was not seen when sodium pyrophosphate was substituted for thiamin pyrophosphate. 7. Under the conditions of the kinase assay, pig heart pyruvate dehydrogenase forms 14CO2 from [1-14C]pyruvate in the presence of thiamin pyrophosphate. Previous work suggests that the products may include acetoin. Acetoin activated the kinase reaction in the presence of thiamin pyrophosphate but not with sodium pyrophosphate. It is suggested that acetoin formation may contribute to activation of the kinase reaction by low pyruvate concentrations in the presence of thiamin pyrophosphate. 8. Pyruvate effected the conversion of pyruvate dehydrogenase phosphate into pyruvate dehydrogenase in rat heart mitochondria incubated with 5mm-2-oxoglutarate and 0.5mm-l-malate as respiratory substrates. It is suggested that this effect of pyruvate is due to inhibition of the pyruvate dehydrogenase kinase reaction in the mitochondrion. 9. Pyruvate dehydrogenase kinase activity was inhibited by high concentrations of Mg2+ (15mm) and by Ca2+ (10nm–10?m) at low Mg2+ (0.15mm) but not at high Mg2+ (15mm). PMID:4462746

Cooper, Ronald H.; Randle, Philip J.; Denton, Richard M.

1974-01-01

218

Glycoprotein Expression in Human Milk During Lactation  

PubMed Central

While milk proteins have been studied for decades, strikingly little effort has been applied to determining how the post-translational modifications (PTMs) of these proteins may change during the course of lactation. PTMs, particularly glycosylation, can greatly influence protein structure, function, and stability and can particularly influence the gut where their degradation products are potentially bioactive. In this work, previously undiscovered temporal variations in both expression and glycosylation of the glycoproteome of human milk are observed. Lactoferrin, one of the most abundant glycoproteins in human milk, is shown to be dynamically glycosylated during the first ten days of lactation. Variations in expression or glycosylation levels are also demonstrated for several other abundant whey proteins, including tenascin, bile salt-stimulated lipase, xanthine dehydrogenase, and mannose receptor. PMID:20415418

Froehlich, John W.; Dodds, Eric D.; Barboza, Mariana; McJimpsey, Erica L.; Seipert, Richard R.; Francis, Jimi; An, Hyun Joo; Freeman, Samara; German, J. Bruce; Lebrilla, Carlito B.

2010-01-01

219

Isolation of a lactic dehydrogenase-A-deficient CHO-K1 mutant by nylon cloth replica plating  

Microsoft Academic Search

A mutant Chinese hamster ovary cell deficient in lactate dehydrogenase A activity has been isolated using a nonselective technique. The method uses histochemical staining to examine colonies directly for enzyme activity and nylon cloth replica plating to recover particular clones. The mutant cell has an apparent Km (pyruvate to lactate) that is nearly tenfold higher than the parental cell, while

T. D. Stamato; Carol Jones

1977-01-01

220

LACTATE-DEGRADING SYSTEM IN BUTYRIBACTERIUM RETTGERI SUBJECT TO GLUCOSE REPRESSION  

PubMed Central

Wittenberger, Charles L. (National Institute of Dental Research, U.S. Public Health Service, Bethesda, Md.), and Ann S. Haaf. Lactate-degrading system in Butyribacterium rettgeri subject to glucose repression. J. Bacteriol. 88:896–903. 1964.—The ability of Butyribacterium rettgeri to utilize lactate as the main energy source for growth requires the formation of a lactate-degrading system. The precise nature of this system is unknown, but preliminary evidence suggests that cellular acquisition of lactate-decomposing activity involves the formation of a nonpyridine nucleotide-linked lactic dehydrogenase. This enzyme, which can couple lactate oxidation to the reduction of ferricyanide [K3Fe(CN)6-lactic de-hydrogenase (LDH)], is absent from glucose-grown cells; this observation appears to account for the inability of such cells to decompose lactate even though they may form lactate from glucose. The formation of K3Fe(CN)6-LDH in growing cultures requires the addition of lipoic acid to the medium, and is repressed by glucose, pyruvate, or fructose. When any of the latter substrates are included in the growth medium with lactate, nicotinamide adenine dinucleotide-linked LDH activity is present in cells at markedly higher levels than it is in cells grown on lactate alone. PMID:14219052

Wittenberger, Charles L.; Haaf, Ann S.

1964-01-01

221

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

222

21 CFR 862.1440 - Lactate dehydrogenase test system.  

Code of Federal Regulations, 2011 CFR

...diseases such as acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial infarction, and tumors of the lung or kidneys. (b) Classification. Class II (special controls). The device is exempt...

2011-04-01

223

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

224

RNA silencing of lactate dehydrogenase gene in Rhizopus oryzae  

PubMed Central

Rhizopus oryzae is a filamentous fungus, belonging to the order Mucorales. It can ferment a wide range of carbohydrates hydrolyzed from lignocellulosic materials and even cellobiose to produce ethanol. However, R. oryzae also produces lactic acid as a major metabolite, which reduces the yield of ethanol. In this study, we show that significant reduction of lactic acid production could be achieved by short (25nt) synthetic siRNAs targeting the ldhA gene. The average yield of lactic acid production by R. oryzae during the batch fermentation process, where glucose had been used as a sole carbon source, diminished from 0.07gm/gm in wild type to 0.01gm/gm in silenced samples. In contrast, the average yield of ethanol production increased from 0.39gm/gm in wild type to 0.45gm/gm in silenced samples. These results show 85.7% (gm/gm) reduction in lactic acid production as compared with the wild type R. oryzae, while an increase of 15.4% (gm/gm) in ethanol yield. PMID:21769297

Gheinani, Ali Hashemi; Jahromi, Neda Haghayegh; Feuk-Lagerstedt, Elisabeth; Taherzadeh, Mohammad J

2011-01-01

225

Direct and Nitroxyl (HNO)-Mediated Reactions of Acyloxy Nitroso Compounds with the Thiol- Containing Proteins Glyceraldehyde 3- Phosphate Dehydrogenase and Alkyl Hydroperoxide Reductase Subunit C  

PubMed Central

Nitroxyl (HNO) reacts with thiols and this reactivity requires the use of donors with 1-nitrosocyclohexyl acetate, pivalate and trifluoroacetate forming a new group. These acyloxy nitroso compounds inhibit glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by forming a reduction reversible active site disulfide and a reduction irreversible sulfinic acid or sulfinamide modification at Cys 244. Addition of these acyloxy nitroso compounds to AhpC C165S yields a sulfinic acid and sulfinamide modification. A potential mechanism for these transformations includes nucleophilic addition of the protein thiol to a nitroso compound to yield an N-hydroxysulfenamide, which reacts with thiol to give disulfide or rearranges to sulfinamides. Known HNO donors produce the un-substituted protein sulfinamide as the major product while the acetate and pivalate give substituted sulfinamides that hydrolyze to sulfinic acids. These results suggest that nitroso compounds form a general class of thiol-modifying compounds allowing their further exploration. PMID:23895568

Mitroka, Susan; Shoman, Mai E.; DuMond, Jenna F.; Bellavia, Landon; Aly, Omar M.; Abdel-Aziz, Mohamed; Kim-Shapiro, Daniel B.; King, S. Bruce

2013-01-01

226

The reactions of D-glyceraldehyde 3-phosphate with thiols and the holoenzyme of D-glyceraldehyde 3-phosphate dehydrogenase and of inorganic phosphate with the acyl-holoenzyme.  

PubMed Central

D-Glyceraldehyde 3-phosphate forms adducts with thiols. These adducts, which are presumed to be hemithioacetals, equilibrate rapidly with the unhydrated form of the aldehyde, which is the subtrate for D-glyceraldehyde 3-phosphate dehydrogenase. The adduct provides a substrate buffer system whereby a constant low free aldehyde concentration can be maintained during the oxidation of aldehyde by the enzyme and NAD+. With this system, the kinetics of the association of the aldehyde with the enzyme were examined. The rate profile for this reaction is a single exponential process, showing that all four active sites of the enzyme have equivalent and independent reactivity towards the aldehyde, with an apparent second-order rate constant of 5 X 10(7)M-1-S-1 at pH8.0 and 21 degrees C. The second-order rate constant becomes 8 X 10(7)M-1-S-1 when account is taken of the forward and reverse catalytic rate constants of the dehydrogenase. The pH-dependence of the observed rate constant is consistent with a requirement for the unprotonated form of a group of pK 6.1, which is the pK observed for second ionization of glyceraldehyde 3-phosphate. The rate of phosphorolysis of the acyl-enzyme intermediate during the steady-state oxidative phosphorylation of the aldehyde was studied, and is proportional to the total Pi concentration up to at least 1 mM-Pi at pH 7.5. The pH-dependence of the rate of NADH generation under these conditions can be explained by the rate law d[NADA]/dt = k[acy] holoenzyme][PO4(3-)-A1, where thioester bond, although kinetically indistinguishable rate equations for the reaction are possible. The rates of the phosphorolysis reaction and of the aldehyde-association reaction decrease with increasing ionic strength, suggesting that the active site of the enzyme has cationic groups which are involved in the reaction of the enzyme with anionic substrates. Images Fig. 1. Fig. 2. Fig. 6. Fig. 7. PMID:12740

Armstrong, J M; Trentham, D R

1976-01-01

227

The d-2-Hydroxyacid Dehydrogenase Incorrectly Annotated PanE Is the Sole Reduction System for Branched-Chain 2-Keto Acids in Lactococcus lactis? †  

PubMed Central

Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branched-chain 2-keto acids to 2-hydroxyacids, are of interest in a variety of fields, including cheese flavor formation via amino acid catabolism. In this study, we used both targeted and random mutagenesis to identify the genes responsible for the reduction of 2-keto acids derived from amino acids in Lactococcus lactis. The gene panE, whose inactivation suppressed hydroxyisocaproate dehydrogenase activity, was cloned and overexpressed in Escherichia coli, and the recombinant His-tagged fusion protein was purified and characterized. The gene annotated panE was the sole gene responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine, while ldh, encoding l-lactate dehydrogenase, was responsible for the reduction of the 2-keto acids derived from phenylalanine and methionine. The kinetic parameters of the His-tagged PanE showed the highest catalytic efficiencies with 2-ketoisocaproate, 2-ketomethylvalerate, 2-ketoisovalerate, and benzoylformate (Vmax/Km ratios of 6,640, 4,180, 3,300, and 2,050 U/mg/mM, respectively), with NADH as the exclusive coenzyme. For the reverse reaction, the enzyme accepted d-2-hydroxyacids but not l-2-hydroxyacids. Although PanE showed the highest degrees of identity to putative NADP-dependent 2-ketopantoate reductases (KPRs), it did not exhibit KPR activity. Sequence homology analysis revealed that, together with the d-mandelate dehydrogenase of Enterococcus faecium and probably other putative KPRs, PanE belongs to a new family of d-2-hydroxyacid dehydrogenases which is unrelated to the well-described d-2-hydroxyisocaproate dehydrogenase family. Its probable physiological role is to regenerate the NAD+ necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds. PMID:19047348

Chambellon, Emilie; Rijnen, Liesbeth; Lorquet, Frederique; Gitton, Christophe; van Hylckama Vlieg, Johan E. T.; Wouters, Jeroen A.; Yvon, Mireille

2009-01-01

228

Identification of octopine dehydrogenase from Mytilus galloprovincialis.  

PubMed

A cDNA encoding the putative octopine dehydrogenase (OcDH) from the mussel Mytilus galloprovincialis was cloned and sequenced. The complete coding region was expressed in the bacteria Escherichia coli and the recombinant protein was purified. The M. galloprovincialis OcDH appears to have the highest affinity for the amino acid substrate L-arginine (88.22%), compared to L-alanine (9.04%) and glycine (2.74%). This enzyme showed no activity when taurine or ?-alanine was used as substrate. These data strongly support that this recombinant enzyme is octopine dehydrogenase and not another opine dehydrogenase such as alanopine or strombine dehydrogenases. The superimposition of the theoretical three-dimensional model of the M. galloprovincialis OcDH and the crystal structure of its homologous counterpart from the great scallop Pecten maximus showed interesting changes in the amino acid binding site which could explain the differences found in the substrate affinity between the two molluscs. A phylogenetic analysis was performed comparing M. galloprovincialis OcDH and annotated sequences representing the five opine dehydrogenase (OpDH) protein family members. The phylogenetic tree which was obtained clustered the OpDH enzymes according to the evolutionary relationships of the species and not to the biochemical reaction catalysed. Octopine dehydrogenase has been identified in the Mytilidae family for the first time, having previously only been established in one other marine invertebrate (P. maximus). PMID:21791249

Vázquez-Dorado, Sandra; Sanjuan, Andrés; Comesaña, Angel S; de Carlos, Alejandro

2011-10-01

229

6-Phosphogluconate Dehydrogenase Mechanism  

PubMed Central

The reductive carboxylation of ribulose-5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (6PGDH) from Candida utilis was investigated using kinetic isotope effects. The intrinsic isotope effect for proton abstraction from Ru5P was found at 4.9 from deuterium isotope effects on V and V/K and from tritium isotope effects on V/K. The presence of 6-phosphogluconate (6PG) in the assay mixture changes the magnitude of the observed isotope effects. In the absence of 6PG D(V/K) and D(V) are 1.68 and 2.46, respectively, whereas the presence of 6PG increases D(V/K) to 2.84 and decreases D(V) to 1.38. A similar increase of T(V/K) is observed as 6PG builds up in the reaction mixture. These data indicate that in the absence of 6PG, a slow step, which precedes the chemical process, is rate-limiting for the reaction, whereas in the presence of 6PG, the rate-limiting step follows the isotope-sensitive step. Kinetic analysis of reductive carboxylation shows that 6PG at low concentrations decreases the Km of Ru5P, whereas at higher concentrations, the usual competitive pattern is observed. These data indicate that full activity of 6PGDH is achieved when one subunit carries out the catalysis and the other subunit carries an unreacted 6PG. Thus, 6PG is like an allosteric activator of 6PGDH. PMID:20452987

Hanau, Stefania; Montin, Katy; Cervellati, Carlo; Magnani, Morena; Dallocchio, Franco

2010-01-01

230

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

231

Metabolism of D-lactate and structurally related organic acids in Chlamydomonas reinhardtii  

SciTech Connect

During the initial minutes of anaerobiosis, /sup 14/C-labeled D-lactate, derived from the photosynthetic sugar phosphate pool, accumulated in the unicellular green alga, Chlamydomonas reinhardtii. The production of the D-isomer of lactate by algae is in contrast to plant and mammalian cells in which L-lactate is formed. After initial lactate formation, Chlamydomonas exhibits a mixed-acid type fermentation, thereby avoiding lactate accumulation and enabling the cells to tolerate extended periods of anaerobiosis. A pyruvate reductase which catalyzes the formation of D-lactate in Chlamydomonas was partially purified and characterized. Lactate produced anaerobically was metabolized only when Chlamydomonas cells were returned to aerobic conditions, and reoxidation of the D-lactate was apparently catalyzed by a mitochondrial membrane-bound dehydrogenase, rather than by the soluble pyruvate reductase. Mutants of Chlamydomonas, deficient in mitochondrial respiration, were used to demonstrate that lactate metabolism was linked to the mitochondrial electron transport chain. In addition, the oxidation of glycolate, a structural analog of lactate, was also linked to mitochondrial electron transport in vivo.

Husic, D.W.

1986-01-01

232

Alternative schemes of butyrate production in Butyrivibrio fibrisolvens and their relationship to acetate utilization, lactate production, and phylogeny  

Microsoft Academic Search

Butyrivibrio fibrisolvens strains D1 and A38 produced little lactate, but strain 49 converted as much as 75% of its glucose to lactate. Strain 49 had\\u000a tenfold more lactate dehydrogenase activity than strains D1 or A38, this activity was stimulated by fructose 1,6-bisphosphate,\\u000a and had a pH optimum of 6.25. A role for fructose 1,6-bisphosphate or pH regulation of lactate production

Francisco Diez-Gonzalez; Daniel R. Bond; Elizabeth Jennings; James B. Russell

1999-01-01

233

Lactation in Islam.  

PubMed

Preservation and promotion of breastfeeding in Islamic countries could be increased by stressing the religious importance of this practice as prescribed in Islamic religious teachings. The child's right to be breastfed is affirmed by the Quaran, the source of Islamic law and morality. Quranic verse 2:233 recommends a 2 year period of lactation. According to Islam a nursing mother is entitled to receive compensation from the father for nursing the child. The father, though, has the option to engage a paid or unpaid wet-nurse for the child, in which case the mother looses her right to be paid for nursing even is she volunteered to breastfeed. The mother's right to nurse a child without compensation is prior to a father's right to engage a wet-nurse. In another Islamic source the moral importance of breastfeeding is stressed. The mother receives the reward of a good deed for every single drop she gives her child. Islamic precepts on lactation influenced Arabian medicine. Avicenna's view that children should be breastfed for 2 years was approvingly quoted by European physicians in the 17th century. Major Arabian medical texts contain chapters on lactation, on tests for quality of breast milk, and on diets and drugs for improving lactation. Research at Al-Azhar University is directed toward finding a contraceptive that will not inhabit lactation and will not affect the quality of breast milk. PMID:12266219

Hefnawi, F I

1982-01-01

234

Combinatorial engineering of ldh-a and bcl-2 for reducing lactate production and improving cell growth in dihydrofolate reductase-deficient Chinese hamster ovary cells  

Microsoft Academic Search

In Chinese hamster ovary (CHO) cells, rapid glucose metabolism normally leads to inefficient use of glucose, most of which\\u000a is converted to lactate during cell cultures. Since lactate accumulation during the culture often exerts a negative effect\\u000a on cell growth and valuable product formation, several genetic engineering approaches have been developed to suppress lactate\\u000a dehydrogenase-A (LDH-A), the enzyme converting pyruvate

Min Kyoung Jeon; Da Young Yu; Gyun Min Lee

235

[Temperature-switched high-efficiency D-lactate production from glycerol].  

PubMed

Glycerol from oil hydrolysis industry is being considered as one of the abundent raw materials for fermentation industry. In present study, the aerobic and anaerobic metabolism and growth properties on glycerol by Esherichia coli CICIM B0013-070, a D-lactate over-producing strain constructed previously, at different temperatures were investigated, followed by a novel fermentation process, named temperature-switched process, was established for D-lactate production from glycerol. Under the optimal condition, lactate yield was increased from 64.0% to 82.6%. Subsequently, the yield of D-lactate from glycerol was reached up to 88.9% while a thermo-inducible promoter was used to regulate D-lactate dehydrogenase transcription. PMID:23631124

Tian, Kangming; Zhou, Li; Chen, Xianzhong; Shen, Wei; Shi, Guiyang; Singh, Suren; Lu, Fuping; Wang, Zhengxiang

2013-01-01

236

Inhibition of lactate-induced swelling by dichloroacetate in human astrocytoma cells.  

PubMed

High levels of tissue lactate exacerbate tissue damage that results from cerebral ischemia and reperfusion injury that follows. Post-ischemic treatment with dichloroacetate (DCA) facilitates a decrease in lactate in the central nervous system (CNS) of animals during reperfusion following experimental ischemia, thus it may help to ameliorate ischemic cell damage. It has been suggested that the lactate lowering effect is mediated through a stimulatory effect of DCA on pyruvate dehydrogenase (PDHC) activity. We have studied such a hypothesis in a human astrocytoma derived cell line, UC-11MG. Under conditions resembling those of the ischemic tissue (i.e. high lactate and low pH) these cells accumulate lactate, driven by the inwardly directed proton gradient, and swell as a consequence of the osmotic effect of intracellular lactate. We have demonstrated that DCA increases PDHC activity and also reduces lactate-induced swelling. However, we also found that these two effects could be uncoupled and that the ability of DCA to prevent swelling is still present in the absence of any stimulation of PDHC. We also demonstrated that DCA competitively inhibits the uptake of lactate (Ki = 1.9 mM) and increases the efflux of lactate in a trans-acting manner that suggests the presence of a lactate-DCA exchange. We present a mechanism by which reduction in the rate of lactate uptake could account for the observed inhibition of swelling. This effect of DCA on lactate transport indicates another possible mechanism of action for DCA in facilitating the decrease in lactate observed in vivo during reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1814583

Tomsig, J L; Gruenstein, E; Dimlich, R V

1991-12-24

237

Ethanol intake during lactation  

Microsoft Academic Search

Lactating rats, with litters adjusted to 8 pups on day 1, were divided into 4 groups: control animals (C), which received water and Nuvilab chow ad libitum, and ethanol animals (E), which received 20% (E20), 10% (E10), or 5% (E5) ethanol diluted in the drinking water and Nuvilab chow ad libitum. On day 12 of life, the pups were weighed

L. M Oyama; R. C Couto; G. E. C Couto; A. R Dâmaso; C. M Oller do Nascimento

2000-01-01

238

Brain alcohol dehydrogenase.  

PubMed

Significant alcohol dehydrogenase activity has been demonstrated in the soluble fraction of rat brain and is very similar to the liver enzyme in kinetic properties and responses to inhibitors. A cerebral mechanism that oxidizes ethanol may play a significant role in local adjustments during exposure to ethanol and in the pathogenesis of the neural disorders associated with chronic alcohol ingestion or withdrawal. PMID:4300045

Raskin, N H; Sokoloff, L

1968-10-01

239

Recycling of NAD + using coimmobilized alcohol dehydrogenase and E. coli  

Microsoft Academic Search

The use of immobilized enzymes has opened the possibility of large scale utilization of NAD+-linked dehydrogenases, but the applications of this technique were limited by the necessity of providing the large amounts\\u000a of NAD+ required by its stoichiometric consumption in the reaction. After immobilization of alcohol dehydrogenase and intactE. coli by glutaraldehyde in the presence of serum albumin, the respiratory

C. Burstein; H. Ounissi; M. D. Legoy; G. Gellf; D. Thomas

1981-01-01

240

Properties of Alanine Dehydrogenase and Aspartase from Propionibacterium freudenreichii subsp. shermanii.  

PubMed

During lactate fermentation by Propionibacterium freudenreichii subsp. shermanii ATCC 9614, the only amino acid metabolized was aspartate. After lactate exhaustion, alanine was one of the two amino acids to be metabolized. For every 3 mol of alanine metabolized, 2 mol of propionate, 1 mol each of acetate and CO(2), and 3 mol of ammonia were formed. The specific activity of alanine dehydrogenase was 0.08 U/mg of protein during lactate fermentation, and it increased to 0.9 U/mg of protein after lactate exhaustion. Alanine dehydrogenase and aspartase, key enzymes in the metabolism of alanine and aspartate, respectively, were partially purified, and some of their properties were studied. Alanine dehydrogenase had a pH optimum of 9.2 to 9.6 and high K(m) values for both NAD (1 to 4 mM) and alanine (7 to 20 mM). Activity was inhibited by low concentrations of pyruvate and NADH. The pH optimum of aspartase decreased from approximately 7.5 to approximately 6.4 when the MgCl(2) and aspartate concentrations were decreased. Plots of aspartate concentration versus activity showed either hyperbolic or sigmoidal kinetics (interaction coefficient, up to a value of 3.1), depending on pH and MgCl(2) concentration. MgCl(2) was either an activator or an inhibitor, depending on pH and its concentration. Aspartase activity was inhibited by low concentrations of fumarate. The properties of alanine dehydrogenase and aspartase are consistent with the finding that aspartate is metabolized during lactate fermentation, while alanine is only fermented after lactate exhaustion and then at a slow rate. PMID:16347414

Crow, V L

1987-08-01

241

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. PMID:23665046

Foster, Christy A.; Mick, Gail J.; Wang, Xudong; McCormick, Kenneth

2014-01-01

242

Sorbitol production from lactose by engineered Lactobacillus casei deficient in sorbitol transport system and mannitol-1-phosphate dehydrogenase  

Microsoft Academic Search

Sorbitol is a sugar alcohol largely used in the food industry as a low-calorie sweetener. We have previously described a sorbitol-producing\\u000a Lactobacillus casei (strain BL232) in which the gutF gene, encoding a sorbitol-6-phosphate dehydrogenase, was expressed from the lactose operon. Here, a complete deletion of\\u000a the ldh1 gene, encoding the main l-lactate dehydrogenase, was performed in strain BL232. In a

Reinout De Boeck; Luz Adriana Sarmiento-Rubiano; Inmaculada Nadal; Vicente Monedero; Gaspar Pérez-Martínez; María J. Yebra

2010-01-01

243

Succinate Dehydrogenase 1  

PubMed Central

A procedure was developed for the partial purification of succinate dehydrogenase from mung bean (Vigna radiata L.) hypocotyls and soybean (Glycine max [L] Merr. v. Ransom) cotyledons. The procedure utilized a Triton X-100 extraction followed by ammonium sulfate precipitation. The final fraction was enriched in two polypeptides with approximate molecular weights of 67,000 and 30,000 daltons, exhibited a pH optima of 7.0 to 7.5, contained a b-type cytochrome, and exhibited the characteristic ferredoxin-type and high potential iron-sulfur protein-type electron paramagnetic resonance signals reported for the iron-sulfur centers of mammalian succinate dehydrogenase. Inhibition constants of 1.15 and 24.6 micromolar for oxaloacetate and malonate, respectively, were obtained. PMID:16662722

Burke, John J.; Siedow, James N.; Moreland, Donald E.

1982-01-01

244

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

245

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

246

Lactating Mother and Psychotropic Drugs  

PubMed Central

Usage of psychotropics during pregnancy and lactation has always been a topic of debate and controversy. The debate stems from the potential adverse effects on the growing fetus or infants due to the transfer of psychotropic drugs through placenta or breast milk of mothers receiving them; and the problem of discontinuing psychotropics in lactating mother considering chances of relapse. However, most of the psychotropics are found to be relatively safe when used cautiously during the lactation phase. This article describes available data on the use of psychotropics in lactating mothers, in particular, in relation to the safety profile of infants. PMID:21327172

Tripathi, B. M.; Majumder, Pradipta

2010-01-01

247

Light modulation of glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase by photosynthetic electron flow in pea chloroplasts  

SciTech Connect

Light activation of NADP-linked glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) and light inactivation of glucose-6-P dehydrogenase (EC 1.1.1.49) appear to be modulated within pea leaf chloroplasts by mediators which are reduced by photosynthetic electron flow from the photosystem I reaction center. Dichlorophenyl-1,1-dimethylurea inhibition of this modulation can be completely reversed by ascorbate plus 2,6-dichlorophenolindophenol in broken chloroplasts, but not in intact chloroplasts. Intact chloroplasts are impermeable to 2,6-dichlorophenolindophenol at pH 7.5. Studies on the effect of light in reconstituted chloroplasts with photosystem I-enriched particles in the place of whole thylakoids revealed that photosystem I participants in the light modulation of NADP-linked glyceraldehyde-3-P dehydrogenase and of glucose-6-P dehydrogenase.

Akamba, L.M.; Anderson, L.E.

1981-02-01

248

Lactate Regulates Rat Male Germ Cell Function through Reactive Oxygen Species  

PubMed Central

Besides giving structural support, Sertoli cells regulate the fate of germ cells by supplying a variety of factors. These factors include hormones, several pro- and anti-apoptotic agents and also energetic substrates. Lactate is one of the compounds produced by Sertoli cells, which is utilized as an energetic substrate by germ cells, particularly spermatocytes and spermatids. Beyond its function as an energy source, some studies have proposed a role of lactate in the regulation of gene expression not strictly related to the energetic state of the cells. The general hypothesis that motivated this investigation was that lactate affects male germ cell function, far beyond its well-known role as energetic substrate. To evaluate this hypothesis we investigated: 1) if lactate was able to regulate germ cell gene expression and if reactive oxygen species (ROS) participated in this regulation, 2) if different signal transduction pathways were modified by the production of ROS in response to lactate and 3) possible mechanisms that may be involved in lactate stimulation of ROS production. In order to achieve these goals, cultures of germ cells obtained from male 30-day old rats were exposed to 10 or 20 mM lactate. Increases in lactate dehydrogenase (LDH) C and monocarboxylate transporter (MCT)2 expression, in Akt and p38-MAPK phosphorylation levels and in ROS production were observed. These effects were impaired in the presence of a ROS scavenger. Lactate stimulated ROS production was also inhibited by a LDH inhibitor or a NAD(P)H oxidase (NOX) inhibitor. NOX4 expression was identified in male germ cells. The results obtained herein are consistent with a scenario where lactate, taken up by germ cells, becomes oxidized to pyruvate with the resultant increase in NADH, which is a substrate for NOX4. ROS, products of NOX4 activity, may act as second messengers regulating signal transduction pathways and gene expression. PMID:24498241

Galardo, Maria Noel; Regueira, Mariana; Riera, Maria Fernanda; Pellizzari, Eliana Herminia; Cigorraga, Selva Beatriz; Meroni, Silvina Beatriz

2014-01-01

249

Methylenetetrahydrofolate dehydrogenase from Clostridium formicoaceticum and methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase (combined) from Clostridium thermoaceticum  

SciTech Connect

Methylenetetrahydrofolate dehydrogenase is widely distributed and has been found in every cell type investigated. The NAD-specific enzyme has been purified to homogeneity from Clostridium formicoaceticum and the NADP-specific enzyme has been obtained from Clostridium thermoaceticum. Other sources of the NADP-specific enzyme are Streptococcus species, Escherichia coli, Clostridium cylindrosporum, Salmonella typhimurium, yeast, liver from various animals, calf thymus, and plants. The NAD-specific enzyme has been demonstrated in Acetobacterium woodii, some methane bacteria, and in Ehrlich ascites tumor cells. Of considerable interest are the observations that in porcine and ovine livers, as well as in yeast, methylenetetrahydrofolate dehydrogenase purified to homogeneity also contains methylenetetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase activities. Now it appears that the purified methylenetetrahydrofolate dehydrogenase from C. thermoaceticum also has cyclohydrolase but not synthetase activity. Methylenetetrahydrofolate dehydrogenase has been discussed previously in this series, as has methenyltetrahydrofolate cyclohydrolase. In C. formicoaceticum and C. thermoaceticum these tetrahydrofolate-dependent enzymes participate in a sequence of metabolic reactions by which carbon dioxide is reduced to the methyl group of 5-methyltetrahydrofolate which in turn is utilized for the synthesis of acetate. This pathway provides the mechanism for disposing of reducing equivalents generated in glycolysis.

Ljungdahl, L.G.; O'Brien, W.E.; Moore, M.R.; Liu, M.T.

1980-01-01

250

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. PMID:16535705

Mansfield, S. D.; De Jong, E.; Saddler, J. N.

1997-01-01

251

Investigations of the lactate minimum test.  

PubMed

We evaluated: the agreement between lactate minimum and maximal lactate steady state (MLSS) cycling powers (study 1); whether rates of change of blood lactate concentration during the lactate minimum test reflect that of constant power exercise (study 2); whether the lactate minimum power is influenced by the muscle groups used to elevate blood lactate concentration (study 3). Study 1: 32 subjects performed a lactate minimum test comprising a lactate elevation phase, recovery phase, and incremental phase (five 4 min stages); MLSS was subsequently determined. Study 2: 8 subjects performed a lactate minimum test and five 22 min constant power tests at the incremental phase exercise intensities. Study 3: 10 subjects performed two identical lactate minimum tests, except during the second test the lactate elevation phase comprised arm-cranking. Lactate minimum and MLSS powers demonstrated good agreement (mean bias+/-95% limits of agreement: 2+/-22 W). Rates of change of blood lactate concentration during each incremental phase stage and corresponding constant power test did not correlate. Lactate minimum power was lowered when arm-cranking was used during the lactate elevation phase (157+/-29 vs. 168+/-21 W; p<0.05). The lactate elevation phase modifies blood lactate concentration responses during the incremental phase, thus good agreement between lactate minimum and MLSS powers seems fortuitous. PMID:19199204

Johnson, M A; Sharpe, G R; Brown, P I

2009-06-01

252

Hamster sperm capacitation: role of pyruvate dehydrogenase A and dihydrolipoamide dehydrogenase.  

PubMed

Recently, we demonstrated that pyruvate dehydrogenase A2 (PDHA2) is tyrosine phosphorylated in capacitated hamster spermatozoa. In this report, using bromopyruvate (BP), an inhibitor of PDHA, we demonstrated that hamster sperm hyperactivation was blocked regardless of whether PDHA was inhibited prior to or after the onset of hyperactivation, but the acrosome reaction was blocked only if PDHA was inhibited prior to the onset of the acrosome reaction. Further, inhibition of PDHA activity did not inhibit capacitation-associated protein tyrosine phosphorylation observed in hamster spermatozoa. It is demonstrated that the essentiality of PDHA for sperm capacitation is probably dependent on its ability to generate effectors of capacitation such as reactive oxygen species (ROS) and cAMP, which are significantly decreased in the presence of BP. MICA (5-methoxyindole-2-carboxylic acid, a specific inhibitor of dihydrolipoamide dehydrogenase [DLD]), another component of the pyruvate dehydrogenase complex (PDHc), also significantly inhibited ROS generation and cAMP levels thus implying that these enzymes of the PDHc are required for ROS and cAMP generation. Furthermore, dibutryl cyclic adenosine monophosphate could significantly reverse the inhibition of hyperactivation observed in the presence of BP and inhibition of acrosome reaction observed in the presence of BP or MICA. The calcium ionophore, A23187, could also significantly reverse the inhibitory effect of BP and MICA on sperm acrosome reaction. These results establish that PDHA is required for hamster sperm hyperactivation and acrosome reaction, and DLD is required for hamster acrosome reaction. This study also provides evidence that ROS, cAMP, and calcium are involved downstream to PDHA. PMID:18401010

Kumar, Vivek; Kota, Venkatesh; Shivaji, S

2008-08-01

253

Genomic reconstruction of Shewanella oneidensis MR-1 metabolism reveals previously uncharacterized machinery for lactate utilization  

SciTech Connect

The ability to utilize lactate as a sole source of carbon and energy is one of the key metabolic signatures of Shewanellae, a diverse group of dissimilatory metal reducing bacteria commonly found in aquatic and sedimentary environments. Nonetheless, homology searches failed to recognize orthologs of previously described bacterial D- or L-lactate oxidizing enzymes (Escherichia coli genes dld and lldD) in any of the 13 analyzed genomes of Shewanella spp. Using comparative genomic techniques, we identified a conserved chromosomal gene cluster in Shewanella oneidensis MR-1 (locus tag: SO1522-SO1518) containing lactate permease and candidate genes for both D- and L-lactate dehydrogenase enzymes. The predicted D-LDH gene (dldD, SO1521) is a distant homolog of FAD-dependent lactate dehydrogenase from yeast, whereas the predicted L-LDH is encoded by three genes with previously unknown functions (lldEGF, SO1520-19-18). Through a combination of genetic and biochemical techniques, we experimentally confirmed the predicted physiological role of these novel genes in S. oneidensis MR-1 and carried out successful functional validation studies in Escherichia coli and Bacillus subtilis. We conclusively showed that dldD and lldEFG encode fully functional D-and L-LDH enzymes, which catalyze the oxidation of the respective lactate stereoisomers to pyruvate. Notably, the S. oneidensis MR-1 LldEFG enzyme is the first described example of a multi-subunit lactate oxidase. Comparative analysis of >400 bacterial species revealed the presence of LldEFG and Dld in a broad range of diverse species accentuating the potential importance of these previously unknown proteins in microbial metabolism.

Pinchuk, Grigoriy E.; Rodionov, Dmitry A.; Yang, Chen; Li, Xiaoqing; Osterman, Andrei L.; Dervyn, Etienne; Geydebrekht, Oleg V.; Reed, Samantha B.; Romine, Margaret F.; Collart, Frank R.; Scott, J.; Fredrickson, Jim K.; Beliaev, Alex S.

2009-02-24

254

Single-step bioluminescence analyses of enzymes, using Cibacrone Blue chromatography for removal of interfering dehydrogenases.  

PubMed

To provide for bioluminescence measurements of the enzymatic activities of dehydrogenases, disturbing contaminants were removed from a bacterial luciferase extract by chromatography, using Blue Sepharose CL-6B, a cross-linked agarose to which Cibacrone Blue F3G-A is covalently attached. This compound has a strong affinity to the dinucleotide fold, which is a region in enzymes binding NAD(H) or NADP(H). In contrast to the absorbed dehydrogenases, both luciferase and oxidoreductase were easily eluted and appeared close to the main bulk of UV-absorbing but analytically less important material. A rapid recording of the elution of luciferase was accomplished with a new electrochemical bioluminescence assay. Due to this and the early elution of the desired material, it could be chromatographed, recognized and collected in less than two hours. Thereby the light-yielding capacity of the sensitive material was well preserved. For bioluminescence assay solutions composed of pooled oxidoreductase-luciferase fractions, FMN and a long chain aldehyde were prepared and supplemented with NAD+ and either lactate, malate or 3-hydroxybutyrate. The analyses were carried out in a single step performance by adding the enzyme sample to the luciferase solution. Minute amounts of lactate dehydrogenase, malate dehydrogenase and 3-hydroxybutyrate dehydrogenase yielded a linear light response permitting assay in the lower part of the femtomole region. In case a dehydrogenase does not occur as a contaminant of a commercial luciferase preparation, purification with Cibacrone Blue can be omitted as demonstrated for glucose-6-phosphate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6633514

Brolin, S E

1983-01-01

255

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

256

Synthesis of Triptorelin Lactate Catalyzed by Lipase in Organic Media  

PubMed Central

Triptorelin lactate was successfully synthesized by porcine pancreatic lipase (PPL) in organic solvents. The effects of acyl donor, substrate ratio, organic solvent, temperature, and water activity were investigated. Under the optimum conditions, a yield of 30% for its ester could be achieved in the reaction for about 48 h. PMID:22949842

Zhuang, Hong; Wang, Zhi; Wang, Jiaxin; Zhang, Hong; Xun, Erna; Chen, Ge; Yue, Hong; Tang, Ning; Wang, Lei

2012-01-01

257

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

258

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

259

Protein engineering of formate dehydrogenase  

Microsoft Academic Search

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is one of the best enzymes for the purpose of NADH regeneration in dehydrogenase-based synthesis of optically active compounds. Low operational stability and high production cost of native FDHs limit their application in commercial production of chiral compounds. The review summarizes the results on engineering of bacterial and yeast FDHs aimed at improving their

Vladimir I. Tishkov; Vladimir O. Popov

2006-01-01

260

Malate dehydrogenase in bovine spermatozoa  

E-print Network

can be used as a terminal electron acceptor in a me- ' dium containing substrate, cofactor, methylene blue, dia- phorase and hydrazine for localization of dehydrogenase activity after starch gel electrophoresis. The use of PMS was suggested... can be used as a terminal electron acceptor in a me- ' dium containing substrate, cofactor, methylene blue, dia- phorase and hydrazine for localization of dehydrogenase activity after starch gel electrophoresis. The use of PMS was suggested...

Lin, Hozong Robert

2012-06-07

261

Purification and Characterization of an Arene cis-Dihydrodiol Dehydrogenase Endowed with Broad Substrate Specificity toward Polycyclic Aromatic Hydrocarbon Dihydrodiols  

Microsoft Academic Search

Initial reactions involved in the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) include a ring-dihydroxylation catalyzed by a dioxygenase and a subsequent oxidation of the dihydrodiol products by a dehydrogenase. In this study, the dihydrodiol dehydrogenase from the PAH-degrading Sphingomonas strain CHY-1 has been characterized. The bphB gene encoding PAH dihydrodiol dehydrogenase (PDDH) was cloned and overexpressed as a His-tagged

Yves Jouanneau; Christine Meyer

2009-01-01

262

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. PMID:16666209

Kimmerer, Thomas W.; Stringer, Mary A.

1988-01-01

263

Characterization of (R)-2-Hydroxyisocaproate Dehydrogenase and a Family III Coenzyme A Transferase Involved in Reduction of l-Leucine to Isocaproate by Clostridium difficile  

PubMed Central

The strictly anaerobic pathogenic bacterium Clostridium difficile occurs in the human gut and is able to thrive from fermentation of leucine. Thereby the amino acid is both oxidized to isovalerate plus CO2 and reduced to isocaproate. In the reductive branch of this pathway, the dehydration of (R)-2-hydroxyisocaproyl-coenzyme A (CoA) to (E)-2-isocaprenoyl-CoA is probably catalyzed via radical intermediates. The dehydratase requires activation by an ATP-dependent one-electron transfer (J. Kim, D. Darley, and W. Buckel, FEBS J. 272:550-561, 2005). Prior to the dehydration, a dehydrogenase and a CoA transferase are supposed to be involved in the formation of (R)-2-hydroxyisocaproyl-CoA. Deduced amino acid sequences of ldhA and hadA from the genome of C. difficile showed high identities to d-lactate dehydrogenase and family III CoA transferase, respectively. Both putative genes encoding the dehydrogenase and CoA transferase were cloned and overexpressed in Escherichia coli; the recombinant Strep tag II fusion proteins were purified to homogeneity and characterized. The substrate specificity of the monomeric LdhA (36.5 kDa) indicated that 2-oxoisocaproate (Km = 68 ?M, k cat = 31 s?1) and NADH were the native substrates. For the reverse reaction, the enzyme accepted (R)- but not (S)-2-hydroxyisocaproate and therefore was named (R)-2-hydroxyisocaproate dehydrogenase. HadA showed CoA transferase activity with (R)-2-hydroxyisocaproyl-CoA as a donor and isocaproate or (E)-2-isocaprenoate as an acceptor. By site-directed mutagenesis, the conserved D171 was identified as an essential catalytic residue probably involved in the formation of a mixed anhydride with the acyl group of the thioester substrate. However, neither hydroxylamine nor sodium borohydride, both of which are inactivators of the CoA transferase, modified this residue. The dehydrogenase and the CoA transferase fit well into the proposed pathway of leucine reduction to isocaproate. PMID:16957230

Kim, Jihoe; Darley, Daniel; Selmer, Thorsten; Buckel, Wolfgang

2006-01-01

264

Characterization of (R)-2-hydroxyisocaproate dehydrogenase and a family III coenzyme A transferase involved in reduction of L-leucine to isocaproate by Clostridium difficile.  

PubMed

The strictly anaerobic pathogenic bacterium Clostridium difficile occurs in the human gut and is able to thrive from fermentation of leucine. Thereby the amino acid is both oxidized to isovalerate plus CO(2) and reduced to isocaproate. In the reductive branch of this pathway, the dehydration of (R)-2-hydroxyisocaproyl-coenzyme A (CoA) to (E)-2-isocaprenoyl-CoA is probably catalyzed via radical intermediates. The dehydratase requires activation by an ATP-dependent one-electron transfer (J. Kim, D. Darley, and W. Buckel, FEBS J. 272:550-561, 2005). Prior to the dehydration, a dehydrogenase and a CoA transferase are supposed to be involved in the formation of (R)-2-hydroxyisocaproyl-CoA. Deduced amino acid sequences of ldhA and hadA from the genome of C. difficile showed high identities to d-lactate dehydrogenase and family III CoA transferase, respectively. Both putative genes encoding the dehydrogenase and CoA transferase were cloned and overexpressed in Escherichia coli; the recombinant Strep tag II fusion proteins were purified to homogeneity and characterized. The substrate specificity of the monomeric LdhA (36.5 kDa) indicated that 2-oxoisocaproate (K(m) = 68 muM, k(cat) = 31 s(-1)) and NADH were the native substrates. For the reverse reaction, the enzyme accepted (R)- but not (S)-2-hydroxyisocaproate and therefore was named (R)-2-hydroxyisocaproate dehydrogenase. HadA showed CoA transferase activity with (R)-2-hydroxyisocaproyl-CoA as a donor and isocaproate or (E)-2-isocaprenoate as an acceptor. By site-directed mutagenesis, the conserved D171 was identified as an essential catalytic residue probably involved in the formation of a mixed anhydride with the acyl group of the thioester substrate. However, neither hydroxylamine nor sodium borohydride, both of which are inactivators of the CoA transferase, modified this residue. The dehydrogenase and the CoA transferase fit well into the proposed pathway of leucine reduction to isocaproate. PMID:16957230

Kim, Jihoe; Darley, Daniel; Selmer, Thorsten; Buckel, Wolfgang

2006-09-01

265

Prolactin inhibition at the end of lactation programs for a central hypothyroidism in adult rat.  

PubMed

Malnutrition during lactation is associated with hypoprolactinemia and failure in milk production. Adult rats whose mothers were malnourished presented higher body weight and serum tri-iodothyronine (T(3)). Maternal hypoprolactinemia at the end of lactation caused higher body weight in adult life, suggesting an association between maternal prolactin (PRL) level and programming of the offspring's adult body weight. Here, we studied the consequences of the maternal PRL inhibition at the end of lactation by bromocriptine (BRO) injection, a dopaminergic agonist, upon serum TSH and thyroid hormones, thyroid iodide uptake, liver mitochondrial alpha-glycerophosphate dehydrogenase (mGPD), liver and pituitary de-iodinase activities (D1 and/or D2), and in vitro post-TRH TSH release in the adult offspring. Wistar lactating rats were divided into BRO - injected with 1 mg/twice a day, daily for the last 3 days of lactation, and C - control, saline-injected with the same frequency. At 180 days of age, the offspring were injected with (125)I i.p. and after 2 h, they were killed. Adult animals whose mothers were treated with BRO at the end of lactation presented lower serum TSH (-51%), T(3) (-23%), and thyroxine (-21%), lower thyroid (125)I uptake (-41%), liver mGPD (-55%), and pituitary D2 (-51%) activities, without changes in the in vitro post-TRH TSH release. We show that maternal PRL suppression at the end of lactation programs a hypometabolic state in adulthood, in part due to a thyroid hypofunction, caused by a central hypothyroidism, probably due to decreased TRH secretion. We suggest that PRL during lactation can regulate the hypothalamus-pituitary-thyroid axis and programs its function. PMID:18490438

Bonomo, Isabela Teixeira; Lisboa, Patrícia Cristina; Passos, Magna Cottini Fonseca; Alves, Simone Bezerra; Reis, Adelina Martha; de Moura, Egberto Gaspar

2008-08-01

266

Cadmium chloride inhibits lactate gluconeogenesis in isolated human renal proximal tubules: a cellular metabolomic approach with 13C-NMR.  

PubMed

As part of a study on cadmium nephrotoxicity, we studied the effect of cadmium chloride (CdCl2) in isolated human renal proximal tubules metabolizing the physiological substrate lactate. Dose-effect experiments showed that 10-500 ?M CdCl2 reduced lactate removal, glucose production and the cellular levels of ATP, coenzyme A, acetyl-coenzyme A and of reduced glutathione in a dose-dependent manner. After incubation with 5 mM L: -[1-(13)C]-, or L: -[2-(13)C]-, or L: -[3-(13)C] lactate or 5 mM L: -lactate plus 25 mM NaH(13)CO3 as substrates, substrate utilization and product formation were measured by both enzymatic and carbon 13 NMR methods. Combination of enzymatic and NMR measurements with a mathematical model of lactate metabolism previously validated showed that 100 ?M CdCl2 caused an inhibition of flux through lactate dehydrogenase and alanine aminotransferase and through the entire gluconeogenic pathway; fluxes were diminished by 19% (lactate dehydrogenase), 28% (alanine aminotransferase), 28% (pyruvate carboxylase), 42% (phosphoenolpyruvate carboxykinase), and 52% (glucose-6-phosphatase). Such effects occurred without altering the oxidation of the lactate carbons or fluxes through enzymes of the tricarboxylic acid cycle despite a large fall of the cellular ATP level, a marker of the energy status and of the viability of the renal cells. These results that were observed at clinically relevant tissue concentrations of cadmium provide a biochemical basis for a better understanding of the cellular mechanism of cadmium-induced renal proximal tubulopathy in humans chronically exposed to cadmium. PMID:21153630

Faiz, Hassan; Conjard-Duplany, Agnès; Boghossian, Michelle; Martin, Guy; Baverel, Gabriel; Ferrier, Bernard

2011-09-01

267

Nutritional aspects of human lactation*  

PubMed Central

This paper reviews the literature on the incidence and duration of breast-feeding in various countries, the volume and composition of breast milk, the health and nutrition of breast-fed babies as judged by growth and morbidity, maternal nutritional requirements during lactation, and the effect of prolonged lactation on maternal health. It appears that lactation can be as well sustained by impoverished as by affluent mothers, and that even in communities where malnutrition is common the average growth of infants is satisfactory up to the age of about 3 months on a diet of breast milk alone. Breast milk appears to have specific anti-infective properties, but prolonged breast-feeding will not prevent infections among older infants reared in a poor environment. The authors believe that breast-feeding is the best form of nutrition for the young infant and deplore its decline in modern industrial societies. The recommendations of various FAO/WHO Expert Groups on nutritional intakes during lactation are summarized. The need for an increased daily energy intake of 4.2 MJ (1 000 kcal) is questioned, and an increase of 2.5 MJ (600 kcal) is suggested. Data on the effect of prolonged lactation on the health of the mother are scanty; body weight appears to be maintained even among poorly nourished mothers. The authors stress the need for well-planned and technically adequate studies of the material and psychological factors involved in breast feeding. PMID:816479

Thomson, A. M.; Black, A. E.

1975-01-01

268

Midtrimester abortion by ethacridine lactate.  

PubMed

This article discusses a clinical trial with the abortifacient agent ethacridine lactate as it was used for midtrimester abortion in Calcutta during the period January-July 1980. Results are then compared with intraamniotic hypertonic saline. 130 subjects were divided into 2 groups--Group 1 (60 women) were terminated with ethacridine lactate and group 2 (70 women) were terminated with saline. In cases where the patient complained of pain, analgesia was administered. In both groups, the largest concentration of women fell in the age groups 16-20 and 21-25. Similarly, single women were the largest representation in both groups although the saline group included more widows. Ethacridine lactate can be administered earlier in the 2nd trimester than saline. With it, expulsion occurred within 36 hours in 56.6% of the cases as compared with 22.9% in group 2. Both groups required the same amount of assistance with oxytocin. In group 1, there were only 3 cases (5%) of minor complications whereas in group 2, 19 cases (27.1%) developed complications. This alone strongly recommends ethacridine lactate as the preferred abortifacient. The success rate was 98%. Thus, ethacridine lactate appears to be a safe and effective agent for pregnancy termination during the 2nd trimester. PMID:7142727

Goswami, B K; Raha, A; Gupta, A; Mukherjee, K

1982-07-01

269

Hypoxia stimulates lactate disposal in rainbow trout.  

PubMed

Current understanding of lactate metabolism in fish is based almost entirely on the interpretation of concentration measurements that cannot be used to infer changes in flux. The goals of this investigation were: (1) to quantify baseline lactate fluxes in rainbow trout (Oncorhynchus mykiss) under normoxic conditions; (2) to establish how changes in rates of lactate appearance (R(a)) and disposal (R(d)) account for the increase in blood lactate elicited by hypoxia; and (3) to identify the tissues responsible for lactate production. R(a) and R(d) lactate of rainbow trout were measured in vivo by continuous infusion of [U-(14)C]lactate in trout exposed to 25% O(2) saturation or maintained in normoxia for 90 min. In normoxic fish, R(a) lactate decreased from 18.2 to 13.1 ?mol kg(-1) min(-1) and R(d) lactate from 19.0 to 12.8. R(a) and R(d) were always matched, thereby maintaining a steady baseline blood lactate concentration of ?0.8 mmol l(-1). By contrast, the hypoxic fish increased blood lactate to 8.9 mmol l(-1) and R(a) lactate from 18.4 to 36.5 ?mol kg(-1) min(-1). This stimulation of anaerobic glycolysis was unexpectedly accompanied by a 52% increase in R(d) lactate from 19.9 to 30.3 ?mol kg(-1) min(-1). White muscle was the main producer of lactate, which accumulated to 19.2 ?mol g(-1) in this tissue. This first study of non-steady-state lactate kinetics in fish shows that the increase in lactate disposal elicited by hypoxia plays a strategic role in reducing the lactate load on the circulation. Without this crucial response, blood lactate accumulation would double. PMID:21037059

Omlin, Teye; Weber, Jean-Michel

2010-11-15

270

Functional Replacement of the Escherichia coliD-(-)Lactate Dehydrogenase Gene (ldhA) with the L-(+)Lactate Dehydrogenase Gene (ldhL) from Pediococcus acidilactici  

Microsoft Academic Search

The microbial production of L-()-lactic acid is rapidly expanding to allow increased production of poly- lactic acid (PLA), a renewable, biodegradable plastic. The physical properties of PLA can be tailored for specific applications by controlling the ratio of L-() and D-() isomers. For most uses of PLA, the L-() isomer is more abundant. As an approach to reduce costs associated

Shengde Zhou; K. T. Shanmugam; L. O. Ingram

2003-01-01

271

Exogenous lactate supply affects lactate kinetics of rainbow trout, not swimming performance.  

PubMed

Intense swimming causes circulatory lactate accumulation in rainbow trout because lactate disposal (Rd) is not stimulated as strongly as lactate appearance (Ra). This mismatch suggests that maximal Rd is limited by tissue capacity to metabolize lactate. This study uses exogenous lactate to investigate what constrains maximal Rd and minimal Ra. Our goals were to determine how exogenous lactate affects: 1) Ra and Rd of lactate under baseline conditions or during graded swimming, and 2) exercise performance (critical swimming speed, Ucrit) and energetics (cost of transport, COT). Results show that exogenous lactate allows swimming trout to boost maximal Rd lactate by 40% and reach impressive rates of 56 ?mol·kg(-1)·min(-1). This shows that the metabolic capacity of tissues for lactate disposal is not responsible for setting the highest Rd normally observed after intense swimming. Baseline endogenous Ra (resting in normoxic water) is not significantly reduced by exogenous lactate supply. Therefore, trout have an obligatory need to produce lactate, either as a fuel for oxidative tissues and/or from organs relying on glycolysis. Exogenous lactate does not affect Ucrit or COT, probably because it acts as a substitute for glucose and lipids rather than extra fuel. We conclude that the observed 40% increase in Rd lactate is made possible by accelerating lactate entry into oxidative tissues via monocarboxylate transporters (MCTs). This observation together with the weak expression of MCTs and the phenomenon of white muscle lactate retention show that lactate metabolism of rainbow trout is significantly constrained by transmembrane transport. PMID:25121611

Omlin, Teye; Langevin, Karolanne; Weber, Jean-Michel

2014-10-15

272

Malate Dehydrogenases of Pisum sativum  

PubMed Central

Mitochondria and leaf microbodies isolated from leaves of pea (Pisum sativum) by sucrose density gradient centrifugation were each shown to have a unique form (isoenzyme) of malate dehydrogenase (EC 1.1.1.37) based on chromatographic and kinetic properties. Root organelle preparations were shown to contain only a mitochondrial malate dehydrogenase with physical and kinetic properties similar to the leaf form. The absence of a detectable root microbody malate dehydrogenase similar to the leaf enzyme, which is intermediate in electrophoretic and chromatographic properties between the mitochondrial and soluble isoenzymes, was confirmed by diethylaminoethyl cellulose column chromatography and starch-gel electrophoresis of total homogenates from leaf and root tissue. These findings tend to support the role of the leaf microbody isoenzyme in a pathway unique to photosynthetic tissue. Images PMID:16658469

Zschoche, William C.; Ting, Irwin P.

1973-01-01

273

Lactate dehydrogenase regulation of the metmyoglobin reducing system to improve color stability of bovine muscles through lactate enhancement  

E-print Network

-B activity of bovine M.- Longissimus lumborum (LD), Semimembranosus (SM), and Psoas major (PM) steaks at 14 d (end of storage and display) at 1?C ............................................................ 73 4.3 LSMeans for TRA of bovine... M.- Longissimus lumborum (LD), Semimembranosus (SM), and Psoas major (PM) steaks at 14 d (end of storage and display) at 1?C ............................................................ 74 4.4 Pictures for bovine M...

Kim, Yuan Hwan

2009-05-15

274

Alcohol Dehydrogenases: Identification and Names for Gene Families  

Microsoft Academic Search

Plant gene products that have been described as `alcohol dehydrogenases' are surveyed and related to their CPGN nomenclature. Most are Zn-dependent medium chain dehydrogenases, including `classical' alcohol dehydrogenase (Adh1), glutathione-dependent formaldehyde dehydrogenase (Fdh1), cinnamyl alcohol dehydrogenase (Cad2), and benzyl alcohol dehydrogenase (Bad1). Plant gene products belonging to the short-chain dehydrogenase class should not be called alcohol dehydrogenases unless such activity

1999-01-01

275

Is lactation nature's contraceptive? Data from Samoa.  

PubMed

Data from a Samoan menstruation study suggest that lactation, even intensive on-demand lactation, does not inhibit menstruation or conception. This paper explores the applied and theoretical implications of continuing to accept lactation as a universally effective fertility control mechanism. Such thinking can have disastrous implications for family planning programs, and it keeps us from challenging long-held assumptions about lactation's role in population growth in early populations. PMID:1514124

Fitzgerald, M H

1992-01-01

276

Neuroendocrine mechanisms of lactational infertility in women  

Microsoft Academic Search

The current knowledge on the mechanisms of lactational infertility, discussed during a symposium of investigators in this subject, is reviewed. Three periods of lactation are examined: the first weeks postpartum, the period of extended lactational amenorrhea and the recovery of ovarian function. In the first postpartum weeks the inhibition of ovarian function is accounted by diminished pituitary response to GnRH,

S DIAZI; M SERON-FERRE; HB CROXATTO; J VELDHUIS

1995-01-01

277

Superabsorbed alcohol dehydrogenase—a new catalyst for asymmetric reductions  

Microsoft Academic Search

A new immobilisate of alcohol dehydrogenase (ADH) is described in which all components for the reaction, i.e. enzyme, the\\u000a coenzyme NADP+, the buffer and other cofactors (trace elements), are immobilized together. It is an all-inclusive catalyst. The support\\u000a is a cheap, commercially-available, superabsorbent polymer. The immobilisation is easy to achieve. The superabsorbed ADH is,\\u000a even when dried, a stable and

Günter E. Jeromin

2009-01-01

278

Formation of d(-)-1,2-propanediol and d(-)-lactate from glucose by Clostridium sphenoides under phosphate limitation  

Microsoft Academic Search

Clostridium sphenoides was grown on glucose in a phosphate-limited medium. Below 80 µM phosphate two new products were formed in addition to ethanol, acetate, H2 and CO2: d(-)-1,2-propanediol and d(-)-lactate. These compounds were apparently synthesized via the methylglyoxal by-pass. The activity of the enzymes involvedmethylglyoxal synthase, methylglyoxal reductase, 1,2-propanediol dehydrogenase and glyoxalase-could be demonstrated in cell extracts of C. sphenoides.

Khue Tran-Din; Gerhard Gottschalk

1985-01-01

279

The Occurrence of Glycolate Dehydrogenase and Glycolate Oxidase in Green Plants  

PubMed Central

Homogenates of various lower land plants, aquatic angiosperms, and green algae were assayed for glycolate oxidase, a peroxisomal enzyme present in green leaves of higher plants, and for glycolate dehydrogenase, a functionally analogous enzyme characteristic of certain green algae. Green tissues of all lower land plants examined (including mosses, liverworts, ferns, and fern allies), as well as three freshwater aquatic angiosperms, contained an enzyme resembling glycolate oxidase, in that it oxidized l- but not d-lactate in addition to glycolate, and was insensitive to 2 mm cyanide. Many of the green algae (including Chlorella vulgaris, previously claimed to have glycolate oxidase) contained an enzyme resembling glycolate dehydrogenase, in that it oxidized d- but not l-lactate, and was inhibited by 2 mm cyanide. Other green algae had activity characteristic of glycolate oxidase and, accordingly, showed a substantial glycolate-dependent O2 uptake. It is pointed out that this distribution pattern of glycolate oxidase and glycolate dehydrogenase among the green plants may have phylogenetic significance. Activities of catalase, a marker enzyme for peroxisomes, were also determined and were generally lower in the algae than in the land plants or aquatic angiosperms. Among the algae, however, there were no consistent correlations between levels of catalase and the type of enzyme which oxidized glycolate. PMID:16658555

Frederick, Sue Ellen; Gruber, Peter J.; Tolbert, N. E.

1973-01-01

280

Amino acid sequence of ovine 6-phosphogluconate dehydrogenase.  

PubMed

The amino acid sequence of the NADP+-dependent enzyme ovine 6-phosphogluconate dehydrogenase has been determined by conventional direct protein sequence analysis of peptides resulting from digestion of the protein with trypsin and chemical cleavages with cyanogen bromide, hydroxylamine, and iodosobenzoic acid. The polypeptide contains 466 amino acids and its NH2 terminus is acetylated. The Candida utilis enzyme is inactivated by reaction of pyridoxal phosphate with two lysine residues (Minchiotti, L., Ronchi, S., and Rippa, M. (1981) Biochim. Biophys. Acta 657, 232-242). These residues are conserved in the ovine enzyme. In contrast to NAD+ dehydrogenases which have weakly related sequences and spatially related folds in their nucleotide-binding sites, no significant sequence homologies were detected between 6-phosphogluconate dehydrogenase and any of three other NADP+-requiring enzymes, glutamate dehydrogenase, p-hydroxybenzoate hydroxylase, and dihydrofolate reductase. This is in accord with structural data that show no spatial relationship between NADP+-binding sites in these enzymes. PMID:6685125

Carne, A; Walker, J E

1983-11-10

281

Expression of NAD + -dependent formate dehydrogenase in Enterobacter aerogenes and its involvement in anaerobic metabolism and H 2 production  

Microsoft Academic Search

An expression system for NAD+-dependent formate dehydrogenase gene (fdh1), from Candida boidinii, was constructed and cloned into Enterobacter aerogenes IAM1183. With the fdh1 expression, the total H2 yield was attributed to a decrease in activity of the lactate pathway and an increase of the formate pathway flux due to\\u000a the NADH regeneration. Analysis of the redox state balance and ethanol-to-acetate

Yuan Lu; Hongxin Zhao; Chong Zhang; Qiheng Lai; Xi Wu; Xin-Hui Xing

2009-01-01

282

Attention and blood lactate levels in equestrians performing show jumping.  

PubMed

In equestrian show jumping, attention is particularly important to ensure maximum accuracy. Due to the anaerobic nature of the jumping and its requirement for precision coordination between human and horse, there may be a relation between the onset of lactic threshold and decrease in attention. In 12 healthy and injury-free equestrians (6 men, 6 women), the effects (blood lactate and glucose) of a show jumping course (250 m long with eight vertical obstacles with a height of 1.15 m height) on capacity and selectivity of attention was assessed. A typical reaction time paradigm and test of divided attention were administered. At the end of the course a significant increase of blood lactate was observed, whereas blood glucose did not significantly change. A deterioration of attention (intensity and selectivity) and a worsening of performance with increasing of blood lactate were observed. The present results led to the conclusion that the increase in blood lactate that occurs in riders executing a show jumping course is associated with worsening of both attentive capabilities and performance. PMID:25068743

Perciavalle, Valentina; Di Corrado, Donatella; Scuto, Claudia; Perciavalle, Vincenzo; Coco, Marinella

2014-06-01

283

Highly efficient asymmetric reduction of arylpropionic aldehydes by horse liver alcohol dehydrogenase through dynamic kinetic resolution.  

PubMed

The enantioselective synthesis of (2S)-2-phenylpropanol and (2S)-2-(4-iso-butylphenyl)propanol ((S)-Ibuprofenol) has been achieved by means of Horse Liver Alcohol Dehydrogenase (HLADH) in buffered aqueous solution or buffered organic solvent mixtures; under the reaction conditions, a dynamic kinetic resolution (DKR) process was realized with good reaction yields and enantiomeric ratios. PMID:17912408

Giacomini, Daria; Galletti, Paola; Quintavalla, Arianna; Gucciardo, Gabriele; Paradisi, Francesca

2007-10-21

284

Medications in Pregnancy and Lactation  

Microsoft Academic Search

One of the least-developed areas of clinical pharmacology and drug research is the use of medication during pregnancy and lactation. This article is the first in a two-part series designed to familiarize physicians with many aspects of the drugs they commonly prescribe for pregnant and breast-feeding women. Almost every pregnant woman is exposed to some type of medication during pregnancy.

Catalin S. Buhimschi; Carl P. Weiner

2009-01-01

285

Inhibition of inosine monophosphate dehydrogenase by sesquiterpene lactones.  

PubMed

Inosine monophosphate (IMP) dehydrogenase had previously been determined to be a likely target enzyme for the sesquiterpene lactones, a class of potential anti-neoplastic drugs. IMP dehydrogenase was purified approx. 770-fold from the P-388 lymphocytic leukemia tumor cell line. The Km values for the substrates, IMP and NAD, were determined to be 12 microM and 25 microM, respectively. Xanthine monophosphate (XMP) was shown to be a competitive inhibitor with a Ki of 67 microM. Mycophenolic acid gave mixed-type inhibition with a Ki of 8 nM for the noncompetitive component and a Ki of 2 nM for the competitive component. Dissociation constants (Kd) and rate constants for inhibition of IMP dehydrogenase by nine different sesquiterpene lactones were determined. The highest Kd was seen with 2,3-dihydrohelenalin while the lowest Kd was observed with bis-helenalinyl malonate. Binding of the drugs by IMP dehydrogenase increased as the size of the drug increased. Also, changes in structure at position 6 had a relatively large effect on the Kd. There was no correlation with hydrophobicity, as determined by octanol/water partition. The first-order rate constants for the reaction of the sesquiterpene lactones with IMP dehydrogenase (k1) and the second-order rate constants for the reaction of the sesquiterpene lactones with glutathione (k2) were also determined. The rate constants for most of the sesquiterpene lactones with the alpha-methylene-gamma-lactone moiety were similar and were approximately twice as great as the rate constants for those sesquiterpene lactones with only the alpha, beta-unsaturated cyclopentenone ring. Microlenin had approximately 5-times the reactivity of the other sesquiterpene lactones towards IMP dehydrogenase, but had approximately the same reactivity towards glutathione, suggesting that it was bound to the enzyme in a way which facilitated its reaction with one or more essential sulfhydryls. The same procedure was used for a series of N-substituted maleimide compounds with the N-substituent ranging in size from a methyl group to a benzyl group. The binding of the maleimide compounds was generally tighter than for the sesquiterpene lactones and there was an increase in binding with size. PMID:2889474

Page, J D; Chaney, S G; Hall, I H; Lee, K H; Holbrook, D J

1987-11-01

286

Maximal lactate steady state in kayaking.  

PubMed

A fixed blood lactate value of 4?mM was commonly used to calculate workload at maximal lactate steady state (MLSS) in kayaking. Our purpose was to measure the actual blood lactate value at MLSS and workload at MLSS in kayaking and assess the validity of using a fixed blood lactate value to calculate the workload at MLSS. 8 junior kayakers (15.1±1.2 years; 179.9±7.3?cm; 72.3±4.9?kg) participated in an incremental workload test and 4-6 sub-maximal constant workload tests (duration of 30?min) on a kayaking ergometer. Blood lactate was measured to calculate the blood lactate value and workload at MLSS. The blood lactate value at MLSS in kayaking was 5.4±0.7?mM. The measured workload at MLSS (112±22 watts) was significantly greater than the calculated workload using a lactate value of 4?mM (104±18 watts, p=0.016). The measured MLSS workload was not significantly different from the calculated workload using a fixed lactate value of 5.4?mM (115±19 watts, p=0.16) or 5.0?mM (113±19 watts, p=0.78) in the incremental tests. A fixed blood lactate value of 5?mM instead of 4?mM might be a better estimate in kayaking given the incremental workload test used in this study. PMID:24886924

Li, Y; Niessen, M; Chen, X; Hartmann, U

2014-10-01

287

Variations in the activity of several enzymes in the mammary glands of non-pregnant, pregnant and lactating rabbits  

PubMed Central

1. The enzymes phosphofructokinase (EC 2.7.1.11), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), ATP–citrate lyase (EC 4.1.3.8), acetyl-CoA carboxylase (EC 6.4.1.2) and acetyl-CoA synthetase (EC 6.2.1.1) were assayed in rabbit mammary glands at various stages of the pregnancy–lactation cycle. 2. The activities of all enzymes were low during pregnancy and, with the exception of phosphofructokinase, in non-pregnant animals. Two- to ten-fold increases in enzyme activities occurred over the first 20 days of lactation. Although milk yield was considerably decreased, the enzyme activities remained elevated in late lactation (45 days after parturition). 3. These findings are discussed in relation to mammary-gland metabolism and compared with similar observations previously made on ruminants and other small mammals. PMID:4244890

Hartmann, P. E.; Jones, E. A.

1970-01-01

288

The importance of alcohol dehydrogenase in regulation of ethanol metabolism in rat liver cells.  

PubMed Central

We used titration with the inhibitors tetramethylene sulphoxide and isobutyramide to assess quantitatively the importance of alcohol dehydrogenase in regulation of ethanol oxidation in rat hepatocytes. In hepatocytes isolated from starved rats the apparent Flux Control Coefficient (calculated assuming a single-substrate irreversible reaction with non-competitive inhibition) of alcohol dehydrogenase is 0.3-0.5. Adjustment of this coefficient to allow for alcohol dehydrogenase being a two-substrate reversible enzyme increases the value by 1.3-1.4-fold. The final value of the Flux Control Coefficient of 0.5-0.7 indicates that alcohol dehydrogenase is a major rate-determining enzyme, but that other factors also have a regulatory role. In hepatocytes from fed rats the Flux Control Coefficient for alcohol dehydrogenase decreases with increasing acetaldehyde concentration. This suggests that, as acetaldehyde concentrations rise, control of the pathway shifts from alcohol dehydrogenase to other enzymes, particularly aldehyde dehydrogenase. There is not a single rate-determining step for the ethanol metabolism pathway and control is shared among several steps. PMID:1898355

Page, R A; Kitson, K E; Hardman, M J

1991-01-01

289

NAD(+)-dependent D-2-hydroxyisocaproate dehydrogenase of Lactobacillus delbrueckii subsp. bulgaricus. Gene cloning and enzyme characterization.  

PubMed

A genomic library from Lactobacillus delbrueckii subsp. bulgaricus was used to complement an Escherichia coli mutant strain deficient for both lactate dehydrogenase and pyruvate formate lyase, and thus unable to grow anaerobically. One recombinant clone was found to display a broad specificity NAD(+)-dependent D-2-hydroxyacid dehydrogenase activity. The corresponding gene (named hdhD) was subcloned and sequenced. The deduced amino acid sequence of the encoded enzyme indicates a 333-residue protein closely related to D-2-hydroxyisocaproate (i.e. 2-hydroxy-4-methyl-pentanoate) dehydrogenase (D-HO-HxoDH) of Lactobacillus casei and other NAD(+)-dependent D-lactate dehydrogenases (D-LDH) from several other bacterial species. The hdhD gene was overexpressed under the control of the lambda phage PL promoter and the enzyme was purified with a two-step method. The L. delbrueckii subsp. bulgaricus enzyme, like that of L. casei, was shown to be active on a wide variety of 2-oxoacid substrates except those having a branched beta-carbon. PMID:7925358

Bernard, N; Johnsen, K; Ferain, T; Garmyn, D; Hols, P; Holbrook, J J; Delcour, J

1994-09-01

290

Conformational changes and catalysis by alcohol dehydrogenase.  

PubMed

As shown by X-ray crystallography, horse liver alcohol dehydrogenase undergoes a global conformational change upon binding of NAD(+) or NADH, involving a rotation of the catalytic domain relative to the coenzyme binding domain and the closing up of the active site to produce a catalytically efficient enzyme. The conformational change requires a complete coenzyme and is affected by various chemical or mutational substitutions that can increase the catalytic turnover by altering the kinetics of the isomerization and rate of dissociation of coenzymes. The binding of NAD(+) is kinetically limited by a unimolecular isomerization (corresponding to the conformational change) that is controlled by deprotonation of the catalytic zinc-water to produce a negatively-charged zinc-hydroxide, which can attract the positively-charged nicotinamide ring. The deprotonation is facilitated by His-51 acting through a hydrogen-bonded network to relay the proton to solvent. Binding of NADH also involves a conformational change, but the rate is very fast. After the enzyme binds NAD(+) and closes up, the substrate displaces the hydroxide bound to the catalytic zinc; this exchange may involve a double displacement reaction where the carboxylate group of a glutamate residue first displaces the hydroxide (inverting the tetrahedral coordination of the zinc), and then the exogenous ligand displaces the glutamate. The resulting enzyme-NAD(+)-alcoholate complex is poised for hydrogen transfer, and small conformational fluctuations may bring the reactants together so that the hydride ion is transferred by quantum mechanical tunneling. In the process, the nicotinamide ring may become puckered, as seen in structures of complexes of the enzyme with NADH. The conformational changes of alcohol dehydrogenase demonstrate the importance of protein dynamics in catalysis. PMID:19583966

Plapp, Bryce V

2010-01-01

291

Conformational Changes and Catalysis by Alcohol Dehydrogenase§  

PubMed Central

As shown by X-ray crystallography, horse liver alcohol dehydrogenase undergoes a global conformational change upon binding of NAD+ or NADH, involving a rotation of the catalytic domain relative to the coenzyme binding domain and the closing up of the active site to produce a catalytically efficient enzyme. The conformational change requires a complete coenzyme and is affected by various chemical or mutational substitutions that can increase the catalytic turnover by altering the kinetics of the isomerization and rate of dissociation of coenzymes. The binding of NAD+ is kinetically limited by a unimolecular isomerization (corresponding to the conformational change) that is controlled by deprotonation of the catalytic zinc-water to produce a negatively-charged zinc-hydroxide, which can attract the positively-charged nicotinamide ring. The deprotonation is facilitated by His-51 acting through a hydrogen-bonded network to relay the proton to solvent. Binding of NADH also involves a conformational change, but the rate is very fast. After the enzyme binds NAD+ and closes up, the substrate displaces the hydroxide bound to the catalytic zinc; this exchange may involve a double displacement reaction where the carboxylate group of a glutamate residue first displaces the hydroxide (inverting the tetrahedral coordination of the zinc), and then the exogenous ligand displaces the glutamate. The resulting enzyme-NAD+-alcoholate complex is poised for hydrogen transfer, and small conformational fluctuations may bring the reactants together so that the hydride ion is transferred by quantum mechanical tunneling. In the process, the nicotinamide ring may become puckered, as seen in structures of complexes of the enzyme with NADH. The conformational changes of alcohol dehydrogenase demonstrate the importance of protein dynamics in catalysis. PMID:19583966

Plapp, Bryce V.

2009-01-01

292

Evaluation of Antigen Detection Tests, Microscopy, and Polymerase Chain Reaction for Diagnosis of Malaria in Peripheral Blood in Asymptomatic Pregnant Women in Nanoro, Burkina Faso  

PubMed Central

Rapid diagnostics tests (RDTs) detect malaria specific antigen(s) in the circulation, even when parasites are sequestered in the placenta and not visible by microscopy. However, research on their diagnostic accuracy during pregnancy is limited. Pregnant women (n = 418) were screened for malaria during routine antenatal care by using two RDTs that detect histidine-rich protein 2 (HRP2) or Plasmodium lactate dehydrogenase, and enzyme-linked immunosorbent assays with antibodies that detect dihydrofolate reductase–thymidylate synthase or heme-detoxification protein, and compared with real-time polymerase chain reaction (RT-PCR) and microscopy for evaluation of their diagnostic accuracy. Prevalence of malaria infection was high (53% by PCR). The RT-PCR and the HRP2 RDT detected most cases of malaria during pregnancy, whereas microscopy, the Plasmodium lactate dehydrogenase RDT, and enzyme-linked immunosorbent assays for dihydrofolate reductase–thymidylate synthase and heme-detoxification protein antibodies did not detect several low-density infections. Therefore, the HRP2 RDT could be a useful tool in high-transmission areas for diagnosis of malaria in asymptomatic pregnant women. PMID:22859362

Kattenberg, Johanna H.; Tahita, Christian M.; Versteeg, Inge A. J.; Tinto, Halidou; Traore/Coulibaly, Maminata; D'Alessandro, Umberto; Schallig, Henk D. F. H.; Mens, Petra F.

2012-01-01

293

Evaluation of antigen detection tests, microscopy, and polymerase chain reaction for diagnosis of malaria in peripheral blood in asymptomatic pregnant women in Nanoro, Burkina Faso.  

PubMed

Rapid diagnostics tests (RDTs) detect malaria specific antigen(s) in the circulation, even when parasites are sequestered in the placenta and not visible by microscopy. However, research on their diagnostic accuracy during pregnancy is limited. Pregnant women (n = 418) were screened for malaria during routine antenatal care by using two RDTs that detect histidine-rich protein 2 (HRP2) or Plasmodium lactate dehydrogenase, and enzyme-linked immunosorbent assays with antibodies that detect dihydrofolate reductase-thymidylate synthase or heme-detoxification protein, and compared with real-time polymerase chain reaction (RT-PCR) and microscopy for evaluation of their diagnostic accuracy. Prevalence of malaria infection was high (53% by PCR). The RT-PCR and the HRP2 RDT detected most cases of malaria during pregnancy, whereas microscopy, the Plasmodium lactate dehydrogenase RDT, and enzyme-linked immunosorbent assays for dihydrofolate reductase-thymidylate synthase and heme-detoxification protein antibodies did not detect several low-density infections. Therefore, the HRP2 RDT could be a useful tool in high-transmission areas for diagnosis of malaria in asymptomatic pregnant women. PMID:22859362

Kattenberg, Johanna H; Tahita, Christian M; Versteeg, Inge A J; Tinto, Halidou; Traoré Coulibaly, Maminata; D'Alessandro, Umberto; Schallig, Henk D F H; Mens, Petra F

2012-08-01

294

Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions  

SciTech Connect

Shewanella oneidensis MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of wide range of electron acceptors. Here, we quantitatively assessed lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor limited growth on lactate with O2; lactate with fumarate; and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensible for growth, respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions S. oneidensis MR-1 carried out incomplete substrate oxidation, whereby the TCA cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under O2 limitation but was required for anaerobic growth likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as electron donor and electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by recently described new type of oxidative NAD(P)H independent D-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by proton motive force generation.

Pinchuk, Grigoriy E.; Geydebrekht, Oleg V.; Hill, Eric A.; Reed, Jennifer L.; Konopka, Allan; Beliaev, Alex S.; Fredrickson, Jim K.

2011-12-01

295

The Crystal Structure of a Ternary Complex of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa Provides New Insight Into the Reaction Mechansim and Shows A Novel Binding Mode of the 2'-Phosphate of NADP+ and A Novel Cation Binding Site  

SciTech Connect

In the human pathogen Pseudomonas aeruginosa, the NAD(P)+-dependent betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors-abundant at infection sites-and producing glycine betaine and NADPH, potentially protective against the high-osmolarity and oxidative stresses prevalent in the infected tissues. Disruption of the PaBADH gene negatively affects the growth of bacteria, suggesting that this enzyme could be a target for antibiotic design. PaBADH is one of the few ALDHs that efficiently use NADP+ and one of the even fewer that require K+ ions for stability. Crystals of PaBADH were obtained under aerobic conditions in the presence of 2-mercaptoethanol, glycerol, NADP+ and K+ ions. The three-dimensional structure was determined at 2.1-A resolution. The catalytic cysteine (C286, corresponding to C302 of ALDH2) is oxidized to sulfenic acid or forms a mixed disulfide with 2-mercaptoethanol. The glutamyl residue involved in the deacylation step (E252, corresponding to E268 of ALDH2) is in two conformations, suggesting a proton relay system formed by two well-conserved residues (E464 and K162, corresponding to E476 and K178, respectively, of ALDH2) that connects E252 with the bulk water. In some active sites, a bound glycerol molecule mimics the thiohemiacetal intermediate; its hydroxyl oxygen is hydrogen bonded to the nitrogen of the amide groups of the side chain of the conserved N153 (N169 of ALDH2) and those of the main chain of C286, which form the 'oxyanion hole.' The nicotinamide moiety of the nucleotide is not observed in the crystal, and the adenine moiety binds in the usual way. A salt bridge between E179 (E195 of ALDH2) and R40 (E53 of ALDH2) moves the carboxylate group of the former away from the 2?-phosphate of the NADP+, thus avoiding steric clashes and/or electrostatic repulsion between the two groups. Finally, the crystal shows two K+ binding sites per subunit. One is in an intrasubunit cavity that we found to be present in all known ALDH structures. The othersingle bondnot described before for any ALDH but most likely present in most of themsingle bondis located in between the dimeric unit, helping structure a region involved in coenzyme binding and catalysis. This may explain the effects of K+ ions on the activity and stability of PaBADH.

Gonzalez-Segura, L.; Rudino-Pinera, E; Munoz-Clares, R; Horjales, E

2009-01-01

296

Ligand-induced conformational changes and a reaction intermediate in branched-chain 2-oxo acid dehydrogenase (E1) from Thermus thermophilus HB8, as revealed by X-ray crystallography.  

PubMed

The alpha(2)beta(2) tetrameric E1 component of the branched-chain 2-oxo acid (BCOA) dehydrogenase multienzyme complex is a thiamin diphosphate (ThDP)-dependent enzyme. E1 catalyzes the decarboxylation of a BCOA concomitant with the formation of the alpha-carbanion/enamine intermediate, 2-(1-hydroxyalkyl)-ThDP, followed by transfer of the 1-hydroxyalkyl group to the distal sulfur atom on the lipoamide of the E2 component. In order to elucidate the catalytic mechanism of E1, the alpha- and beta-subunits of E1 from Thermus thermophilus HB8 have been co-expressed in Escherichia coli, purified and crystallized as a stable complex, and the following crystal structures have been analyzed: the apoenzyme (E1(apo)), the holoenzyme (E1(holo)), E1(holo) in complex with the substrate analogue 4-methylpentanoate (MPA) as an ES complex model, and E1(holo) in complex with 4-methyl-2-oxopentanoate (MOPA) as the alpha-carbanion/enamine intermediate (E1(ceim)). Binding of cofactors to E1(apo) induces a disorder-order transition in two loops adjacent to the active site. Furthermore, upon binding of MPA to E1(holo), the loop comprised of Gly121beta-Gln131beta moves close to the active site and interacts with MPA. The carboxylate group of MPA is recognized mainly by Tyr86beta and N4' of ThDP. The hydrophobic moiety of MPA is recognized by Phe66alpha, Tyr95alpha, Met128alpha and His131alpha. As an intermediate, MOPA is decarboxylated and covalently linked to ThDP, and the conformation of the protein loop is almost the same as in the substrate-free (holoenzyme) form. These results suggest that E1 undergoes an open-closed conformational change upon formation of the ES complex with a BCOA, and the mobile region participates in the recognition of the carboxylate group of the BCOA. ES complex models of E1(holo).MOPA and of E1(ceim).lipoamide built from the above structures suggest that His273alpha and His129beta' are potential proton donors to the carbonyl group of a BCOA and to the proximal sulfur atom on the lipoamide, respectively. PMID:15033367

Nakai, Tadashi; Nakagawa, Noriko; Maoka, Nobuko; Masui, Ryoji; Kuramitsu, Seiki; Kamiya, Nobuo

2004-04-01

297

Lactate formation in Caldicellulosiruptor saccharolyticus is regulated by the energy carriers pyrophosphate and ATP.  

PubMed

Caldicellulosiruptor saccharolyticus displays superior H(2) yields on a wide range of carbon sources provided that lactate formation is avoided. Nevertheless, a low lactate flux is initiated as the growth rate declined in the transition to the stationary phase, which coincides with a drastic decrease in the glucose consumption and acetate production fluxes. In addition, the decrease in growth rate was accompanied by a sudden increase and then decrease in NADH levels. The V'(MAX) of the lactate dehydrogenase (LDH) doubled when the cells entered the stationary phase. Kinetic analysis revealed that at the metabolic level LDH activity is regulated through (i) competitive inhibition by pyrophosphate (PPi, k(i)=1.7 mM) and NAD (k(i)=0.43 mM) and (ii) allosteric activation by FBP (300%), ATP (160%) and ADP (140%). From these data a MWC-based model was derived. Simulations with this model could explain the observed lactate shift by displaying how the sensitivity of LDH activity to NADH/NAD ratio varied with different PP(i) concentrations. Moreover, the activation of LDH by ATP indicates that C. saccharolyticus uses LDH as a means to adjusts its flux of ATP and NADH production. To our knowledge, this is the first time PPi is observed as an effector of LDH. PMID:20060925

Willquist, Karin; van Niel, Ed W J

2010-05-01

298

Dichloroacetate increases glucose use and decreases lactate in developing rat brain  

SciTech Connect

Dichloroacetate (DCA) activates pyruvate dehydrogenase (PDH) by inhibiting PDH kinase. Neutralized DCA (100 mg/kg) or saline was intravenously administered to 20 to 25-day-old rats (50-75g). Fifteen minutes later a mixture of {sup 6-14}C glucose and {sup 3}H fluorodeoxyglucose (FDG) was administered intravenously and the animals were sacrificed by microwave irradiation (2450 MHz, 8.0 kW, 0.6-0.8 sec) after 2 or 5 min. Brain regional rates of glucose use and metabolite levels were determined. DCA-treated rats had increased rates of glucose use in all regions studied (cortex, thalamus, striatum, and brain stem), with an average increase of 41%. Lactate levels were lower in all regions, by an average of 35%. There were no significant changes in levels of ATP, creatine phosphate, or glycogen in any brain region. Blood levels of lactate did not differ significantly between the DCA- and the saline-treated groups. Blood glucose levels were higher in the DCA group. In rats sacrificed by freeze-blowing, DCA treatment caused lower brain levels of both lactate and pyruvate. These results cannot be explained by any systemic effect of DCA. Rather, it appears that in the immature rat, DCA treatment results in activation of brain PDH, increased metabolism of brain pyruvate and lactate, and a resulting increase in brain glycolytic rate.

Miller, A.L.; Hatch, J.P.; Prihoda, T.J. (Univ. of Texas Health Science Center, San Antonio (USA))

1990-12-01

299

Increased production of succinic acid in Escherichia coli by overexpression of malate dehydrogenase  

Microsoft Academic Search

Escherichia coli NZN111 is a double mutant with inactivated lactate dehydrogenase and pyruvate formate-lyase. It cannot utilize glucose anaerobically\\u000a because of its unusually high intracellular NADH\\/NAD+ ratio. We have now constructed a recombinant strain, E. coli NZN111\\/pTrc99a-mdh, which, during anaerobic fermentation, produced 4.3 g succinic acid l?1 from 13.5 g glucose l?1. The NADH\\/NAD+ ratio decreased from 0.64 to 0.26. Furthermore, dual-phase fermentation (aerobic growth

Li-ya LiangRong-ming; Rong-ming Liu; Jiang-feng Ma; Ke-quan Chen; Min Jiang; Ping Wei

300

Lactate metabolism in fetal type II pneumocytes  

Microsoft Academic Search

Fetal rat lung type II pneumocytes in organotypic culture produce significant quantities of both pyruvate and lactate, even\\u000a when maintained under aerobic conditions (5% CO2\\/air). A hypoxic atmosphere of 95% nitrogen\\/5% CO2 increased lactate production by 35%, while cells in a hyperoxic environment (95% oxygen\\/5% CO2) manufactured lactate at a rate similar to controls. Cells incubated under aerobic conditions following

Michael J. Engle; D. Jeannette Brown; Anne F. Dehring

1986-01-01

301

The effect of exercise on lactate metabolism  

PubMed Central

1. An I.V. injection of 5 ?c [U-14C]sodium L(+)-lactate was given to four subjects at rest and again 10 min after beginning a 40-50 min period of heavy exercise at an estimated 62-72% of their maximum aerobic power (V?O2 max.). Both blood lactate concentration and V?O2 remained relatively constant after the first few minutes of exercise. 2. In all subjects both at rest and during exercise blood lactate and total radioactivity were measured at frequent intervals after injection of [14C]lactate. Timed expired gas collections were made and the quantity of 14CO2 present in each collection measured. In two subjects the specific activity of lactate and of glucose isolated from blood was also measured. 3. It was found that during 30 min of exercise 35-68% of the administered [14C]lactate was recovered as 14CO2 in the expired gas, whereas at rest only 3-7% was recovered in the same period. 4. After injection of [14C]lactate the blood 14C concentration and the specific activity of the blood lactate declined very rapidly. This decline was more rapid during exercise than at rest. 5. In the two subjects in whom it was measured the specific activity of blood glucose was lower during exercise than at rest. 6. These results show that both at rest and during heavy exercise, lactate is removed from the blood and metabolized, and that during exercise this metabolism is much more rapid. 7. In the light of these findings the sustained blood lactate concentration observed in these experiments is regarded as representing a dynamic equilibrium between the production and metabolism of lactate during exercise. The results give no support to the hypothesis that lactate is produced only during the first few minutes of submaximal work. PMID:4715350

Hubbard, Judith L.

1973-01-01

302

Bacterial 2,3-butanediol dehydrogenases  

Microsoft Academic Search

Enterobacter aerogenes, Aeromonas hydrophila, Serratia marcescens and Staphylococcus aureus possessing L(+)-butanediol dehydrogenase produced mainly meso-butanediol and small amounts of optically active butanediol; Acetobacter suboxydans, Bacillus polymyxa and Erwinia carotovora containing D(-)-butanediol dehydrogenase produced more optically active butanediol than meso-butanediol. Resting and growing cells of these organisms oxidized only one enantiomer of racemic butanediol. The D(-)-butanediol dehydrogenase from Bacillus polymyxa was

Hanni Höhn-Bentz; F. Radler

1978-01-01

303

The stress signalling pathway nuclear factor E2-related factor 2 is activated in the liver of sows during lactation  

PubMed Central

Background It has recently been shown that the lactation-induced inflammatory state in the liver of dairy cows is accompanied by activation of the nuclear factor E2-related factor 2 (Nrf2) pathway, which regulates the expression of antioxidant and cytoprotective genes and thereby protects tissues from inflammatory mediators and reactive oxygen species (ROS). The present study aimed to study whether the Nrf2 pathway is activated also in the liver of lactating sows. Findings Transcript levels of known Nrf2 target genes, UGT1A1 (encoding glucuronosyltransferase 1 family, polypeptide A1), HO-1 (encoding heme oxygenase 1), NQO1 (encoding NAD(P)H dehydrogenase, quinone 1), GPX1 (encoding glutathione peroxidase), PRDX6 (encoding peroxiredoxin 6), TXNRD1 (encoding thioredoxin reductase 1), and SOD (encoding superoxide dismutase), in the liver are significantly elevated (between 1.7 and 3.1 fold) in lactating sows compared to non-lactating sows. The inflammatory state in the liver was evidenced by the finding that transcript levels of genes encoding acute phase proteins, namely haptoglobin (HP), fibrinogen ? (FGG), complement factor B (CFB), C-reactive protein (CRP) and lipopolysaccharide-binding protein (LBP), were significantly higher (2 to 8.7 fold) in lactating compared to non-lactating sows. Conclusions The results of the present study indicate that the Nrf2 pathway in the liver of sows is activated during lactation. The activation of Nrf2 pathway during lactation in sows might be interpreted as a physiologic means to counteract the inflammatory process and to protect the liver against damage induced by inflammatory signals and ROS. PMID:23039904

2012-01-01

304

Affinity Chromatography of Lactate Dehydrogenase: An Experiment for the Undergraduate Biochemistry Laboratory.  

ERIC Educational Resources Information Center

Discusses a laboratory technique of enzyme purification by affinity chromatography as part of an undergraduate biochemical methodology course. Provides preparation details of the rat muscle homogenate and reagents. Proposes column requirements and assaying information. (MVL)

Anderson, Alexander J.

1988-01-01

305

Effect of a Marathon Run on Serum Lipoproteins, Creatine Kinase, and Lactate Dehydrogenase in Recreational Runners  

ERIC Educational Resources Information Center

The objective of this study was to determine the effect of a marathon run on serum lipid and lipoprotein concentrations and serum muscle enzyme activities and follow their recovery after the run. These blood concentrations were measured before, immediately after, and serially after a marathon run in 15 male recreational runners. The triglyceride…

Kobayashi, Yoshio; Takeuchi, Toshiko; Hosoi, Teruo; Yoshizaki, Hidekiyo; Loeppky, Jack A.

2005-01-01

306

Serum lactate dehydrogenase level as a prognostic factor in Hodgkin's disease  

Microsoft Academic Search

The efficacy of currently available treatments for Hodgkin's disease (HD) has led to a substantial modification in the prognosis of this disease; nevertheless there is still a group of patients that cannot be cured with conventional treatments and who will be candidates for alternative therapy. In the present work we analysed the prognostic influence of the most relevant clinico-biological characteristics

R García; JM Hernández; M González; J Galende; MC del Cañizo; L Vázquez; JF San Miguel

1993-01-01

307

The expression of lactate dehydrogenase in Zea mays seedlings under hypoxic and anoxic conditions  

E-print Network

and style of Plant Physiology. regenerates the NAD' needed to maintain glycolysis and the minimal production . of ATP. Some tissues such as the anoxic leaves of Schoenoplectus lacustris, Scirpus maritimus and Typha latifolia (Barclay and Crawford, 1982... and style of Plant Physiology. regenerates the NAD' needed to maintain glycolysis and the minimal production . of ATP. Some tissues such as the anoxic leaves of Schoenoplectus lacustris, Scirpus maritimus and Typha latifolia (Barclay and Crawford, 1982...

MacAlpine, David Michael

2012-06-07

308

Pressure-induced thermostabilization of glutamate dehydrogenase from the hyperthermophile  

E-print Network

Pressure-induced thermostabilization of glutamate dehydrogenase from the hyperthermophile glutamate dehydrogenases ~GDHs!: the native enzyme from the hyperthermophile Pyrococcus furiosus ~Pf spin resonance; glutamate dehydrogenase; glycerol; pressure; Pyrococcus furiosus; stability

Vetriani, Costantino

309

Improved Production of 2,3-Butanediol in Bacillus amyloliquefaciens by Over-Expression of Glyceraldehyde-3-Phosphate Dehydrogenase and 2,3-butanediol Dehydrogenase  

PubMed Central

Background Previously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production. Methodology/Principal Findings In the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD+ to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD+. In this study, to improve 2,3-BD production, we first over-produced NAD+-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h. Conclusions/Significance Co-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms. PMID:24098433

Yang, Taowei; Rao, Zhiming; Zhang, Xian; Xu, Meijuan; Xu, Zhenghong; Yang, Shang-Tian

2013-01-01

310

Betaine aldehyde dehydrogenase in sorghum.  

PubMed Central

The ability to synthesize and accumulate glycine betaine is wide-spread among angiosperms and is thought to contribute to salt and drought tolerance. In plants glycine betaine is synthesized by the two-step oxidation of choline via the intermediate betaine aldehyde, catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). Two sorghum (Sorghum bicolor) cDNA clones, BADH1 and BADH15, putatively encoding betaine aldehyde dehydrogenase were isolated and characterized. BADH1 is a truncated cDNA of 1391 bp. BADH15 is a full-length cDNA clone, 1812 bp in length, predicted to encode a protein of 53.6 kD. The predicted amino acid sequences of BADH1 and BADH15 share significant homology with other plant BADHs. The effects of water deficit on BADH mRNA expression, leaf water relations, and glycine betaine accumulation were investigated in leaves of preflowering sorghum plants. BADH1 and BADH15 mRNA were both induced by water deficit and their expression coincided with the observed glycine betaine accumulation. During the course of 17 d, the leaf water potential in stressed sorghum plants reached -2.3 MPa. In response to water deficit, glycine betaine levels increased 26-fold and proline levels increased 108-fold. In severely stressed plants, proline accounted for > 60% of the total free amino acid pool. Accumulation of these compatible solutes significantly contributed to osmotic potential and allowed a maximal osmotic adjustment of 0.405 MPa. PMID:8934627

Wood, A J; Saneoka, H; Rhodes, D; Joly, R J; Goldsbrough, P B

1996-01-01

311

Reconstruction of an Acetogenic 2,3-Butanediol Pathway Involving a Novel NADPH-Dependent Primary-Secondary Alcohol Dehydrogenase  

PubMed Central

Acetogenic bacteria use CO and/or CO2 plus H2 as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogen Clostridium autoethanogenum is known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize the C. autoethanogenum enzymes for lactate and 2,3-butanediol biosynthesis. The putative C. autoethanogenum lactate dehydrogenase was active when expressed in Escherichia coli. The 2,3-butanediol pathway was reconstituted in E. coli by cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resulting E. coli strain produced 1.1 ± 0.2 mM 2R,3R-butanediol (23 ?M h?1 optical density unit?1), which is comparable to the level produced by C. autoethanogenum during growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, that C. autoethanogenum can act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway from C. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential of C. autoethanogenum as a platform for sustainable chemical production. PMID:24657865

Köpke, Michael; Gerth, Monica L.; Maddock, Danielle J.; Mueller, Alexander P.; Liew, FungMin

2014-01-01

312

Calcium deficiency, pregnancy, and lactation in rats  

Microsoft Academic Search

Summary The calcium homeostatic mechanism was challenged in adult female rats by feeding them a calcium-deficient diet containing oxalate, and by subjecting them to pregnancy and lactation. The regimen caused a substantial weight loss, especially in those animals which reared their young well. Severe hypocalcaemia was observed in the lactating rats. Serum-P was slightly elevated. The content of hydroxyproline in

P. Rasmussen

1977-01-01

313

21 CFR 184.1207 - Calcium lactate.  

Code of Federal Regulations, 2010 CFR

...as GRAS § 184.1207 Calcium lactate. (a) Calcium lactate (C6 H10 CaO6 .xH2 O, where...commercially by the neutralization of lactic acid with calcium carbonate or calcium hydroxide. (b) The ingredient...

2010-04-01

314

21 CFR 184.1207 - Calcium lactate.  

...as GRAS § 184.1207 Calcium lactate. (a) Calcium lactate (C6 H10 CaO6. xH2 O, where...commercially by the neutralization of lactic acid with calcium carbonate or calcium hydroxide. (b) The ingredient...

2014-04-01

315

21 CFR 184.1207 - Calcium lactate.  

Code of Federal Regulations, 2012 CFR

...as GRAS § 184.1207 Calcium lactate. (a) Calcium lactate (C6 H10 CaO6 .xH2 O, where...commercially by the neutralization of lactic acid with calcium carbonate or calcium hydroxide. (b) The ingredient...

2012-04-01

316

21 CFR 184.1207 - Calcium lactate.  

Code of Federal Regulations, 2013 CFR

...as GRAS § 184.1207 Calcium lactate. (a) Calcium lactate (C6 H10 CaO6 .xH2 O, where...commercially by the neutralization of lactic acid with calcium carbonate or calcium hydroxide. (b) The ingredient...

2013-04-01

317

21 CFR 184.1207 - Calcium lactate.  

Code of Federal Regulations, 2011 CFR

...as GRAS § 184.1207 Calcium lactate. (a) Calcium lactate (C6 H10 CaO6 .xH2 O, where...commercially by the neutralization of lactic acid with calcium carbonate or calcium hydroxide. (b) The ingredient...

2011-04-01

318

Adaptations of Maternal Adipose Tissue to Lactation  

Microsoft Academic Search

The ability to store substantial amounts of energy as lipid in adipose tissue has allowed development of a variety of strategies in wild animals to meet the considerable metabolic challenge of lactation. The ability to use adipose tissue energy has also been critical for development of the exceptional rates of milk production achieved in the dairy cow. Lactation thus results

Richard G. Vernon; Caroline M. Pond

1997-01-01

319

[Lactate oxidation by Hansenula anomala cells].  

PubMed

Lactate oxidation by freeze-dried cells of Hansenula anomala was studied in the presence of exogenous electron acceptors, potassium ferricyanide (PF) and phenazine metosulfate (PMS). The product of oxidation is pyruvate. The apparent Michaelis constants for lactate, PF and PMS are 1.2, 0.6 and 0.05, respectively. The pH optimum of lactate oxidation is 8.0. The freeze-dried cells of the yeast are inactivated by 5 mM bromopyruvate by 50% within 1.5 min. Treatment of the intact cells with ultrasound activates them. The respiratory function of the cells is characterized with a KM(app.) of 0.05 mM in the presence of lactate. PF has no effect on respiration. A scheme is proposed for lactate oxidation by the cells with damaged membranes whose amount after lyophilization is 10--20%. PMID:39226

Kulis, Iu Iu; Kadziauskene, K V; Vidzhiuna?te, R A

1979-01-01

320

Reduction of reactive oxygen species ameliorates metabolism-secretion coupling in islets of diabetic GK rats by suppressing lactate overproduction.  

PubMed

We previously demonstrated that impaired glucose-induced insulin secretion (IS) and ATP elevation in islets of Goto-Kakizaki (GK) rats, a nonobese model of diabetes, were significantly restored by 30-60-min suppression of endogenous reactive oxygen species (ROS) overproduction. In this study, we investigated the effect of a longer (12 h) suppression of ROS on metabolism-secretion coupling in ?-cells by exposure to tempol, a superoxide (O2(-)) dismutase mimic, plus ebselen, a glutathione peroxidase mimic (TE treatment). In GK islets, both H2O2 and O2(-) were sufficiently reduced and glucose-induced IS and ATP elevation were improved by TE treatment. Glucose oxidation, an indicator of Krebs cycle velocity, also was improved by TE treatment at high glucose, whereas glucokinase activity, which determines glycolytic velocity, was not affected. Lactate production was markedly increased in GK islets, and TE treatment reduced lactate production and protein expression of lactate dehydrogenase and hypoxia-inducible factor 1? (HIF1?). These results indicate that the Warburg-like effect, which is characteristic of aerobic metabolism in cancer cells by which lactate is overproduced with reduced linking to mitochondria metabolism, plays an important role in impaired metabolism-secretion coupling in diabetic ?-cells and suggest that ROS reduction can improve mitochondrial metabolism by suppressing lactate overproduction through the inhibition of HIF1? stabilization. PMID:23349483

Sasaki, Mayumi; Fujimoto, Shimpei; Sato, Yuichi; Nishi, Yuichi; Mukai, Eri; Yamano, Gen; Sato, Hiroki; Tahara, Yumiko; Ogura, Kasane; Nagashima, Kazuaki; Inagaki, Nobuya

2013-06-01

321

Lactation curves of commercial ewes rearing lambs.  

PubMed

Three-hour milk production measurements determined by machine milking at 3-d intervals throughout a 63-d lactation period were used to describe lactation curves for crossbred ewes lambing at 1 and 2 yr of age and rearing single and twin lambs. Age of ewe, type of rearing, and day of lactation affected (P < 0.05) milk production. Over the 63-d lactation, average daily milk production was 2.56 and 2.63 kg, respectively, for 1- and 2-yr-old ewes rearing single lambs and 2.73 and 3.47 kg, respectively, for 1- and 2-yr-old ewes rearing twins. Milk production of 2-yr-old ewes rearing twin lambs peaked at 21 d of lactation, and that of 1- and 2-yr-old ewes rearing singles peaked between 27 and 30 d of lactation. The largest differences in the lactation curves among age and rearing ewe classes were found in early lactation. These differences were reduced by midlactation, and by late lactation, milk production for all ewes was similar. Diurnal variation in milk production by ewes was evaluated in an 8 x 8 Latin square design. Diurnal variation in milk yield measurements of eight mature ewes, each bearing and rearing twin lambs, was similar between d 21 and 24 of lactation. Time of milk production measurements within a day did not affect yield determinations. Extrapolation from 3-h production estimates to daily milk production is valid in determining a ewe's milk contribution in support of lamb growth. PMID:11831522

Cardellino, R A; Benson, M E

2002-01-01

322

Pyruvate dehydrogenase deficiency: molecular basis for intrafamilial heterogeneity.  

PubMed

Two half-brothers and their mother had symptomatic pyruvate dehydrogenase complex deficiency. The infants had severe congenital lactic acidosis, seizures, and apneic spells and died at the ages 3 and 4 months. The mother was less symptomatic with mental retardation, truncal ataxia, and dysarthria. The residual pyruvate dehydrogenase activities in cultured skin fibroblasts from the 2 infants and their mother were 7, 15, and 10% of control values. Immunoblot analysis showed negligible amounts of E1 alpha and E1 beta subunits of the complex. Northern blot analysis for the E1 alpha subunit showed normal results. In the 2 sons, complementary DNA sequence analysis revealed a cytosine to thymine mutation in exon 4, resulting in a change of arginine 127 to tryptophan in the E1 alpha subunit. Restriction enzyme analysis of the polymerase chain reaction product representing exon 4 of the E1 alpha gene revealed that the mother was a heterozygotes. Complementary DNA restriction analysis and methylation analysis of the X chromosome DXS255 loci revealed skewed activation of the mutant allele, consistent with the deficient pyruvate dehydrogenase activity in the mother's fibroblasts. The milder maternal phenotype is consistent with variable X-inactivation patterns in different organs of female heterozygotes. PMID:8024267

Fujii, T; Van Coster, R N; Old, S E; Medori, R; Winter, S; Gubits, R M; Matthews, P M; Brown, R M; Brown, G K; Dahl, H H

1994-07-01

323

Treatment of mastitis during lactation  

PubMed Central

Treatment of mastitis should be based on bacteriological diagnosis and take national and international guidelines on prudent use of antimicrobials into account. In acute mastitis, where bacteriological diagnosis is not available, treatment should be initiated based on herd data and personal experience. Rapid bacteriological diagnosis would facilitate the proper selection of the antimicrobial. Treating subclinical mastitis with antimicrobials during lactation is seldom economical, because of high treatment costs and generally poor efficacy. All mastitis treatment should be evidence-based, i.e., the efficacy of each product and treatment length should be demonstrated by scientific studies. Use of on-farm written protocols for mastitis treatment promotes a judicious use of antimicrobials and reduces the use of antimicrobials. PMID:22081939

2009-01-01

324

Substrate specificity of the pyruvate dehydrogenase complex from Escherichia coli.  

PubMed

The investigation of the substrate specificity of the pyruvate dehydrogenase complex from Escherichia coli allows a description of the binding region of pyruvate. Substrate analogs with electronegative substitutions in the methyl group show a strong competitive inhibition of the overall reaction of the pyruvate dehydrogenase complex. The most efficient inhibitor is fluoropyruvate which has a more than 100-fold higher affinity for the enzyme than pyruvate (Ki = 1.4 x 10(-6) M) does. The affinity of alpha-keto acids decreases with increasing chain length. Branched chain alpha-keto acids are even less effective inhibitors (Ki = approximately 0.02 M). alpha-Ketobutyrate is the only alpha-keto acid which is able to substitute for pyruvate as a substrate in the overall reaction of the enzyme complex. The Km value (3 mM) is 10-fold greater than that for pyruvate. The steady state kinetics of the overall reaction of alpha-ketobutyrate exhibits the same cooperativity (nh = 1.9) as seen with pyruvate. Small modifications of the carbonyl or the carboxyl group of pyruvate prevent binding completely. Binding of pyruvate to the pyruvate dehydrogenase complex may thus require interaction with two independent electrophilic centers. The acceptance of the methyl group seems not so much due to lipophilic interactions as to a steric effect. The experiments were carried out with an enzyme which was purified by a modified procedure which is faster and more convenient than previous methods. The procedure is applicable up to 0.5 liter of crude extract. PMID:7005225

Bisswanger, H

1981-01-25

325

Alkalizing Reactions Streamline Cellular Metabolism in Acidogenic Microorganisms  

PubMed Central

An understanding of the integrated relationships among the principal cellular functions that govern the bioenergetic reactions of an organism is necessary to determine how cells remain viable and optimise their fitness in the environment. Urease is a complex enzyme that catalyzes the hydrolysis of urea to ammonia and carbonic acid. While the induction of urease activity by several microorganisms has been predominantly considered a stress-response that is initiated to generate a nitrogen source in response to a low environmental pH, here we demonstrate a new role of urease in the optimisation of cellular bioenergetics. We show that urea hydrolysis increases the catabolic efficiency of Streptococcus thermophilus, a lactic acid bacterium that is widely used in the industrial manufacture of dairy products. By modulating the intracellular pH and thereby increasing the activity of ?-galactosidase, glycolytic enzymes and lactate dehydrogenase, urease increases the overall change in enthalpy generated by the bioenergetic reactions. A cooperative altruistic behaviour of urease-positive microorganisms on the urease-negative microorganisms within the same environment was also observed. The physiological role of a single enzymatic activity demonstrates a novel and unexpected view of the non-transcriptional regulatory mechanisms that govern the bioenergetics of a bacterial cell, highlighting a new role for cytosol-alkalizing biochemical pathways in acidogenic microorganisms. PMID:21152088

Arioli, Stefania; Ragg, Enzio; Scaglioni, Leonardo; Fessas, Dimitrios; Signorelli, Marco; Karp, Matti; Daffonchio, Daniele; De Noni, Ivano; Mulas, Laura; Oggioni, Marco; Guglielmetti, Simone; Mora, Diego

2010-01-01

326

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. PMID:24079682

Andersen, Lars W.; Mackenhauer, Julie; Roberts, Jonathan C.; Berg, Katherine M.; Cocchi, Michael N.; Donnino, Michael W.

2014-01-01

327

Lactational State Modifies Alcohol Pharmacokinetics in Women  

PubMed Central

Background Given the physiological adaptations of the digestive system during lactation, the present study tested the hypothesis that lactation alters alcohol pharmacokinetics. Methods Lactating women who were exclusively breastfeeding a 2- to 5-month-old infant and 2 control groups of nonlactating women were studied. The first control group consisted of women who were exclusively formula-feeding similarly aged infants, whereas the other consisted of women who had never given birth. A within-subjects design study was conducted such that women drank a 0.4 g/kg dose of alcohol following a 12-hour overnight fast during one test session (fasted condition) or 60 minutes after consuming a standard breakfast during the other (fed condition). Blood alcohol concentration (BAC) levels and mood states were obtained at fixed intervals before and after alcohol consumption. Results Under both conditions, the resultant BAC levels at each time point were significantly lower and the area under the blood alcohol time curve were significantly smaller in lactating women when compared with the 2 groups of nonlactating women. That such changes were due to lactation per se and not due to recent parturient events was suggested by the finding that alcohol pharmacokinetics of nonlactating mothers, who were tested at a similar time postpartum, were no different from women who had never given birth. Despite lower BAC levels in lactating mothers, there were no significant differences among the 3 groups of women in the stimulant effects of alcohol. However, lactating women did differ in the sedative effects of alcohol when compared with nulliparous but not formula-feeding mothers. That is, both groups of parous women felt sedated for shorter periods of time when compared with nulliparous women. Conclusions The systemic availability of alcohol was diminished during lactation. However, the reduced availability of alcohol in lactating women did not result in corresponding changes in the subjective effects of alcohol. PMID:17433009

Pepino, Marta Yanina; Steinmeyer, Allison L.; Mennella, Julie A.

2008-01-01

328

Mathematical modelling of the dehydrogenase catalyzed hexanol oxidation with coenzyme regeneration by NADH oxidase  

Microsoft Academic Search

The hexanol oxidation catalyzed by alcohol dehydrogenase from baker's yeast (YADH) has been investigated with two different forms of the biocatalyst: the isolated YADH as well as the YADH in the permeabilized whole cells. It was found that in this reaction, equilibrium is shifted to the reduction side. Hence, to increase the conversion it was necessary to regenerate NAD+. For

Ana Vrsalovi? Prese?ki; ?ur?a Vasi?-Ra?ki

2009-01-01

329

Pyruvate dehydrogenase complex: Metabolic link to ischemic brain injury and target of oxidative stress  

Microsoft Academic Search

The mammalian pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme complex (greater than 7 million Daltons) that catalyzes the oxida- tive 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 metab- olism. PDHC enzyme activity and immunoreactivity are lost in selectively

Erica Martin; Robert E. Rosenthal; Gary Fiskum

2005-01-01

330

Expression pattern and biochemical characteristics of a major epidermal retinol dehydrogenase  

Microsoft Academic Search

The biological functions of vitamin A in the epidermis are mediated by all-trans retinoic acid, which is biosynthesized from retinol in two oxidative reactions. The first step involves enzymatic conversion of retinol to retinaldehyde. The physiological significance and relative contributions of the various retinol dehydrogenases to the oxidation of retinol in epidermal cells remain unclear. We report the characterization of

Nedialka G Markova; A Pinkas-Sarafova; N Karaman-Jurukovska; V Jurukovski; M Simon

2003-01-01

331

Androsterone 3?-substituted derivatives as inhibitors of type 3 17?-hydroxysteroid dehydrogenase  

Microsoft Academic Search

Androsterone derivatives substituted at position 3 were synthesized starting from dihydrotestosterone in a short sequence of reactions. They proved to be potent inhibitors (IC50=57–147nM) of type 3 17?-hydroxysteroid dehydrogenase, a key enzyme of steroidogenesis, which catalyzes the transformation of androstenedione to steroid active androgen testosterone.

Béatrice Tchédam Ngatcha; Van Luu-The; Donald Poirier

2000-01-01

332

Minireview: Cellular Redox State Regulates Hydroxysteroid Dehydrogenase Activity and Intracellular Hormone Potency  

Microsoft Academic Search

Hydroxysteroid dehydrogenases (HSDs) interconvert potent and relatively inactive forms of individual steroid hormones using nicotinamide cofactors NADPH\\/NADP and NADH\\/ NAD (nicotinamide adenine dinucleotide (phosphate), re- duced\\/oxidized forms). Although reactions with purified en- zymes in vitro may be driven in either direction depending on the assay conditions, HSD enzymes appear to function in one direction or the other in intact cells.

Anil K. Agarwal; Richard J. Auchus

2005-01-01

333

In vitro effects of bicarbonate and bicarbonate-lactate buffered peritoneal dialysis solutions on mesothelial and neutrophil function.  

PubMed

The inclusion of bicarbonate in the formulation of peritoneal dialysis solutions may avoid the in vitro impairment of certain cell functions seen with acidic lactate-based fluids. The supranormal physiological levels of HCO3- and PCO2 inherent in such formulations may, however, not be biocompatible. This study compared the in vitro biocompatibility of a pH 5.2 lactate-based formulation with formulations containing either 40 mM lactate at pH 7.4, 38 mM HCO3- at pH 6.8 (PCO2 at approximately 240 mm Hg) or 7.4 (PCO2 at approximately 60 mm Hg), and 25 mM HCO3- plus 15 mM lactate at pH 6.8 (PCO2 at approximately 160 mm Hg) or 7.4 (PCO2 at approximately 40 mm Hg). Significant release of lactate dehydrogenase or decreases in ATP content by human peritoneal mesothelial cells (HPMC) and human peripheral polymorphonuclear leukocytes (PMN) after a 30-min exposure to each test solution was only seen with the pH 5.2 lactate-based fluid. The ATP content of HPMC exposed to this fluid returned to control levels after 30 min of recovery in M199 control medium but showed a trend toward decreasing ATP content at 240 min. Similarly, interleukin (IL)-1 beta-induced IL-6 synthesis by HPMC was also only significantly reduced by the pH 5.2 lactate solution. PMN chemiluminescence was unaffected by 30-min exposure to all test solutions except for the pH 5.2 lactate formulation. Staphylococcus epidermidis phagocytosis was reduced to between 46 to 57% of control with all test solutions except the pH 5.2 lactate solution, which further suppressed the chemiluminescence response to 17% of control. These data suggest that short exposure to supranormal physiological levels of HCO3- and PCO2 does not impair HPMC or PMN viability and function. Furthermore, neutral pH lactate-containing solutions show equivalent biocompatibility to bicarbonate-based ones. PMID:8785390

Topley, N; Kaur, D; Petersen, M M; Jörres, A; Williams, J D; Faict, D; Holmes, C J

1996-02-01

334

Safety of dermatologic medications in pregnancy and lactation: Part II. Lactation.  

PubMed

Dermatologists are frequently faced with questions from women who are breastfeeding about the safety of commonly prescribed topical and systemic medications during lactation. Safety data in lactation, particularly regarding medications that are unique to dermatology, are limited and can be difficult to locate. We have consolidated the available safety data in a single reference guide for clinicians. We review literature pertaining to the safety of common dermatologic therapies in lactation and offer recommendations based on the available evidence. PMID:24528912

Butler, Daniel C; Heller, Misha M; Murase, Jenny E

2014-03-01

335

Original article Insulin and/or dexamethasone regulation of lactate  

E-print Network

Original article Insulin and/or dexamethasone regulation of lactate production and its relationship, the lactate production was two times greater in sheep than in cow AT. Insulin increased lactate production in the two species, whereas DEX decreased it. DEX, in the presence of insulin, increased lactate production

Paris-Sud XI, Université de

336

Effect of sodium gluconate on the solubility of calcium lactate  

Microsoft Academic Search

Calcium and lactate are present in excess of their solubility in Cheddar cheese. Consequently, calcium lactate crystals (CLC) are a common defect in Cheddar cheese. A novel approach for preventing CLC is the addition of sodium gluconate. Sodium gluconate has the potential to increase the solubility of calcium and lactate by forming soluble complexes with calcium and lactate ions, and

C. Phadungath; L. E. Metzger

2011-01-01

337

Tungsten and molybdenum regulation of formate dehydrogenase expression in Desulfovibrio vulgaris Hildenborough.  

PubMed

Formate is an important energy substrate for sulfate-reducing bacteria in natural environments, and both molybdenum- and tungsten-containing formate dehydrogenases have been reported in these organisms. In this work, we studied the effect of both metals on the levels of the three formate dehydrogenases encoded in the genome of Desulfovibrio vulgaris Hildenborough, with lactate, formate, or hydrogen as electron donors. Using Western blot analysis, quantitative real-time PCR, activity-stained gels, and protein purification, we show that a metal-dependent regulatory mechanism is present, resulting in the dimeric FdhAB protein being the main enzyme present in cells grown in the presence of tungsten and the trimeric FdhABC? protein being the main enzyme in cells grown in the presence of molybdenum. The putatively membrane-associated formate dehydrogenase is detected only at low levels after growth with tungsten. Purification of the three enzymes and metal analysis shows that FdhABC? specifically incorporates Mo, whereas FdhAB can incorporate both metals. The FdhAB enzyme has a much higher catalytic efficiency than the other two. Since sulfate reducers are likely to experience high sulfide concentrations that may result in low Mo bioavailability, the ability to use W is likely to constitute a selective advantage. PMID:21498650

da Silva, Sofia M; Pimentel, Catarina; Valente, Filipa M A; Rodrigues-Pousada, Claudina; Pereira, Inês A C

2011-06-01

338

Tungsten and Molybdenum Regulation of Formate Dehydrogenase Expression in Desulfovibrio vulgaris Hildenborough ?  

PubMed Central

Formate is an important energy substrate for sulfate-reducing bacteria in natural environments, and both molybdenum- and tungsten-containing formate dehydrogenases have been reported in these organisms. In this work, we studied the effect of both metals on the levels of the three formate dehydrogenases encoded in the genome of Desulfovibrio vulgaris Hildenborough, with lactate, formate, or hydrogen as electron donors. Using Western blot analysis, quantitative real-time PCR, activity-stained gels, and protein purification, we show that a metal-dependent regulatory mechanism is present, resulting in the dimeric FdhAB protein being the main enzyme present in cells grown in the presence of tungsten and the trimeric FdhABC3 protein being the main enzyme in cells grown in the presence of molybdenum. The putatively membrane-associated formate dehydrogenase is detected only at low levels after growth with tungsten. Purification of the three enzymes and metal analysis shows that FdhABC3 specifically incorporates Mo, whereas FdhAB can incorporate both metals. The FdhAB enzyme has a much higher catalytic efficiency than the other two. Since sulfate reducers are likely to experience high sulfide concentrations that may result in low Mo bioavailability, the ability to use W is likely to constitute a selective advantage. PMID:21498650

da Silva, Sofia M.; Pimentel, Catarina; Valente, Filipa M. A.; Rodrigues-Pousada, Claudina; Pereira, Ines A. C.

2011-01-01

339

Pyruvate Dehydrogenase Complex Activity Controls Metabolic and Malignant Phenotype in Cancer Cells*  

PubMed Central

High lactate generation and low glucose oxidation, despite normal oxygen conditions, are commonly seen in cancer cells and tumors. Historically known as the Warburg effect, this altered metabolic phenotype has long been correlated with malignant progression and poor clinical outcome. However, the mechanistic relationship between altered glucose metabolism and malignancy remains poorly understood. Here we show that inhibition of pyruvate dehydrogenase complex (PDC) activity contributes to the Warburg metabolic and malignant phenotype in human head and neck squamous cell carcinoma. PDC inhibition occurs via enhanced expression of pyruvate dehydrogenase kinase-1 (PDK-1), which results in inhibitory phosphorylation of the pyruvate dehydrogenase ? (PDH?) subunit. We also demonstrate that PDC inhibition in cancer cells is associated with normoxic stabilization of the malignancy-promoting transcription factor hypoxia-inducible factor-1? (HIF-1?) by glycolytic metabolites. Knockdown of PDK-1 via short hairpin RNA lowers PDH? phosphorylation, restores PDC activity, reverts the Warburg metabolic phenotype, decreases normoxic HIF-1? expression, lowers hypoxic cell survival, decreases invasiveness, and inhibits tumor growth. PDK-1 is an HIF-1-regulated gene, and these data suggest that the buildup of glycolytic metabolites, resulting from high PDK-1 expression, may in turn promote HIF-1 activation, thus sustaining a feed-forward loop for malignant progression. In addition to providing anabolic support for cancer cells, altered fuel metabolism thus supports a malignant phenotype. Correction of metabolic abnormalities offers unique opportunities for cancer treatment and may potentially synergize with other cancer therapies. PMID:18541534

McFate, Thomas; Mohyeldin, Ahmed; Lu, Huasheng; Thakar, Jay; Henriques, Jeremy; Halim, Nader D.; Wu, Hong; Schell, Michael J.; Tsang, Tsz Mon; Teahan, Orla; Zhou, Shaoyu; Califano, Joseph A.; Jeoung, Nam Ho; Harris, Robert A.; Verma, Ajay

2008-01-01

340

Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase.  

PubMed

Formaldehyde, a major industrial chemical, is classified as a carcinogen because of its high reactivity with DNA. It is inactivated by oxidative metabolism to formate in humans by glutathione-dependent formaldehyde dehydrogenase. This NAD(+)-dependent enzyme belongs to the family of zinc-dependent alcohol dehydrogenases with 40 kDa subunits and is also called ADH3 or chi-ADH. The first step in the reaction involves the nonenzymatic formation of the S-(hydroxymethyl)glutathione adduct from formaldehyde and glutathione. When formaldehyde concentrations exceed that of glutathione, nonoxidizable adducts can be formed in vitro. The S-(hydroxymethyl)glutathione adduct will be predominant in vivo, since circulating glutathione concentrations are reported to be 50 times that of formaldehyde in humans. Initial velocity, product inhibition, dead-end inhibition, and equilibrium binding studies indicate that the catalytic mechanism for oxidation of S-(hydroxymethyl)glutathione and 12-hydroxydodecanoic acid (12-HDDA) with NAD(+) is random bi-bi. Formation of an E.NADH.12-HDDA abortive complex was evident from equilibrium binding studies, but no substrate inhibition was seen with 12-HDDA. 12-Oxododecanoic acid (12-ODDA) exhibited substrate inhibition, which is consistent with a preferred pathway for substrate addition in the reductive reaction and formation of an abortive E.NAD(+).12-ODDA complex. The random mechanism is consistent with the published three-dimensional structure of the formaldehyde dehydrogenase.NAD(+) complex, which exhibits a unique semi-open coenzyme-catalytic domain conformation where substrates can bind or dissociate in any order. PMID:10978156

Sanghani, P C; Stone, C L; Ray, B D; Pindel, E V; Hurley, T D; Bosron, W F

2000-09-01

341

Regulation of bone mineral loss during lactation  

NASA Technical Reports Server (NTRS)

The effects of varyng dietary calcium and phosphorous levels, vitamin D deficiency, oophorectomy, adrenalectomy, and simultaneous pregnancy on bone mineral loss during lactation in rats are studied. The experimental procedures and evaluations are described. The femur ash weight of lactating and nonlactating rats are calculated. The data reveals that a decrease in dietary calcium of 0.02 percent results in an increased loss of bone mineral, an increase in calcium to 1.4 percent does not lessen bone mineral loss, and bone mineral loss in vitamin D deficient rats is independent of calcium levels. It is observed that changes in dietary phosphorous level, oophorectomy, adrenalectomy, and simultaneous pragnancy do not reduce bone mineral loss during lactation. The analysis of various hormones to determine the mechanism that triggers bone mineral loss during lactation is presented.

Brommage, R.; Deluca, H. F.

1985-01-01

342

Lactate shuttling and lactate use as fuel after traumatic brain injury: metabolic considerations.  

PubMed

Lactate is proposed to be generated by astrocytes during glutamatergic neurotransmission and shuttled to neurons as 'preferred' oxidative fuel. However, a large body of evidence demonstrates that metabolic changes during activation of living brain disprove essential components of the astrocyte-neuron lactate shuttle model. For example, some glutamate is oxidized to generate ATP after its uptake into astrocytes and neuronal glucose phosphorylation rises during activation and provides pyruvate for oxidation. Extension of the notion that lactate is a preferential fuel into the traumatic brain injury (TBI) field has important clinical implications, and the concept must, therefore, be carefully evaluated before implementation into patient care. Microdialysis studies in TBI patients demonstrate that lactate and pyruvate levels and lactate/pyruvate ratios, along with other data, have important diagnostic value to distinguish between ischemia and mitochondrial dysfunction. Results show that lactate release from human brain to blood predominates over its uptake after TBI, and strong evidence for lactate metabolism is lacking; mitochondrial dysfunction may inhibit lactate oxidation. Claims that exogenous lactate infusion is energetically beneficial for TBI patients are not based on metabolic assays and data are incorrectly interpreted. PMID:25204393

Dienel, Gerald A

2014-11-01

343

Not only students can express alcohol dehydrogenase: goldfish can too!  

PubMed

This article describes a novel approach to study the metabolic regulation of the respiratory system in vertebrates that suits physiology lessons for undergraduate students. It consists of an experimental demonstration of the goldfish's (Carassius auratus) adaptations to anoxia. The goldfish is one of the few vertebrates showing strong enzymatic plasticity for the expression of alcohol dehydrogenase (ADH), which allows it to survive long periods of severe anoxia. Therefore, we propose two simple laboratory exercises in which students are first asked to characterize the distribution of ADH isozymes in the goldfish by performing cellulose acetate electrophoresis. The second part of this laboratory lesson is the determination of liver glycogen. To further student comprehension, an interspecies comparative component is integrated, in which the same subjects are studied in an anoxia-sensitive species, the brook charr (Salvelinus fontinalis). ADH in goldfish is restricted to skeletal muscles, where it catalyzes alcoholic fermentation, permitting ethanol excretion through the gills and therefore preventing lactate acidosis caused by sustained glycolysis during anoxia. Electrophoresis also reveals the occurrence of a liver isozyme in the brook charr, which ADH catalyzes in the opposite pathway, allowing the usual ethanol degradation. As for the liver glycogen assay, it shows largely superior content in the goldfish liver compared with the brook charr, providing goldfish with a sustained energy supply during anoxia. The results of this laboratory exercise clearly demonstrate several physiological strategies developed by goldfish to cope with such a crucial environmental challenge as oxygen depletion. PMID:21098391

Chamberland, Valérie; Rioux, Pierre

2010-12-01

344

Not only students can express alcohol dehydrogenase: goldfish can too!  

NSDL National Science Digital Library

This article describes a novel approach to study the metabolic regulation of the respiratory system in vertebrates that suits physiology lessons for undergraduate students. It consists of an experimental demonstration of the goldfish's (Carassius auratus) adaptations to anoxia. The goldfish is one of the few vertebrates showing strong enzymatic plasticity for the expression of alcohol dehydrogenase (ADH), which allows it to survive long periods of severe anoxia. Therefore, we propose two simple laboratory exercises in which students are first asked to characterize the distribution of ADH isozymes in the goldfish by performing cellulose acetate electrophoresis. The second part of this laboratory lesson is the determination of liver glycogen. To further student comprehension, an interspecies comparative component is integrated, in which the same subjects are studied in an anoxia-sensitive species, the brook charr (Salvelinus fontinalis). ADH in goldfish is restricted to skeletal muscles, where it catalyzes alcoholic fermentation, permitting ethanol excretion through the gills and therefore preventing lactate acidosis caused by sustained glycolysis during anoxia. Electrophoresis also reveals the occurrence of a liver isozyme in the brook charr, which ADH catalyzes in the opposite pathway, allowing the usual ethanol degradation. As for the liver glycogen assay, it shows largely superior content in the goldfish liver compared with the brook charr, providing goldfish with a sustained energy supply during anoxia. The results of this laboratory exercise clearly demonstrate several physiological strategies developed by goldfish to cope with such a crucial environmental challenge as oxygen depletion.

Dr. Pierre Rioux (Universite du Quebec a Rimouski Dept de Biologie Chimie et des Sciences de la Sante)

2010-10-01

345

Isocitrate Dehydrogenase Parameters of Enzyme Activity  

NSDL National Science Digital Library

One of four biology laboratories where students research the properties of a model enzyme, isocitrate dehydrogenase, by using scientifitic method, molecular biology enzyme assay techniques and data analysis using a computer graphing program.

John H. Williamson (Davidson College;); A. Malcolm Campbell (Davidson College;)

1999-01-01

346

Genetics Home Reference: Phosphoglycerate dehydrogenase deficiency  

MedlinePLUS

Phosphoglycerate dehydrogenase deficiency Related Gene(s) References Quick links to this topic MedlinePlus Health information Additional NIH Resources National Institutes of Health Educational resources Information pages Patient support ...

347

Comparative Genomics and Transcriptomics of Lactation  

Microsoft Academic Search

\\u000a Lactation is an important characteristic of mammalian reproduction sometimes referred to as the quintessence of mammals. Comparative\\u000a genomics and transcriptomics experiments are allowing a more in-depth molecular analysis of the evolution of lactation throughout\\u000a the mammalian kingdom and these recent results are reviewed here. Milk cell and mammary gland gene expression analysis with\\u000a sequencing methodology have started to reveal conserved

Christophe M. Lefèvre; Karensa Menzies; Julie A. Sharp; Kevin R. Nicholas

348

Drosophila Alcohol Dehydrogenase Polymorphism and Carbon-13 Fluxes: Opportunities for Epistasis and Natural Selection  

PubMed Central

The influence of genetic variations in Drosophila alcohol dehydrogenase (ADH) on steady-state metabolic fluxes was studied by means of (13)C NMR spectroscopy. Four pathways were found to be operative during 8 hr of ethanol degradation in third instar larvae of Drosophila. Seven strains differed by 18-25% in the ratio between two major pathway fluxes, i.e., into glutamate-glutamine-proline vs. lactate-alanine-trehalose. In general, Adh genotypes with higher ADH activity exhibit a twofold difference in relative carbon flux from malate into lactate and alanine vs. ?,?-trehalose compared to low ADH activity genotypes. Trehalose was degraded by the pentose-phosphate shunt. The pentose-phosphate shunt and malic enzyme could supply NADPH necessary for lipid synthesis from ethanol. Lactate and/or proline synthesis may maintain the NADH/NAD(+) balance during ethanol degradation. After 24 hr the flux into trehalose is increased, while the flux into lipids declines in Adh(F) larvae. In Adh(S) larvae the flux into lipids remains high. This co-ordinated nature of metabolism and the genotype-dependent differences in metabolic fluxes may form the basis for various epistatic interactions and ultimately for variations in organismal fitness. PMID:7982561

Freriksen, A.; de-Ruiter, BLA.; Scharloo, W.; Heinstra, PWH.

1994-01-01

349

Clinical use of lactate monitoring in critically ill patients  

PubMed Central

Increased blood lactate levels (hyperlactataemia) are common in critically ill patients. Although frequently used to diagnose inadequate tissue oxygenation, other processes not related to tissue oxygenation may increase lactate levels. Especially in critically ill patients, increased glycolysis may be an important cause of hyperlactataemia. Nevertheless, the presence of increased lactate levels has important implications for the morbidity and mortality of the hyperlactataemic patients. Although the term lactic acidosis is frequently used, a significant relationship between lactate and pH only exists at higher lactate levels. The term lactate associated acidosis is therefore more appropriate. Two recent studies have underscored the importance of monitoring lactate levels and adjust treatment to the change in lactate levels in early resuscitation. As lactate levels can be measured rapidly at the bedside from various sources, structured lactate measurements should be incorporated in resuscitation protocols. PMID:23663301

2013-01-01

350

Retinol dehydrogenases: Membrane-bound enzymes for the visual function.  

PubMed

Retinoid metabolism is important for many physiological functions, such as differenciation, growth, and vision. In the visual context, after the absorption of light in rod photoreceptors by the visual pigment rhodopsin, 11-cis retinal is isomerized to all-trans retinal. This retinoid subsequently undergoes a series of modifications during the visual cycle through a cascade of reactions occurring in photoreceptors and in the retinal pigment epithelium. Retinol dehydrogenases (RDHs) are enzymes responsible for crucial steps of this visual cycle. They belong to a large family of proteins designated as short-chain dehydrogenases/reductases. The structure of these RDHs has been predicted using modern bioinformatics tools, which allowed to propose models with similar structures including a common Rossman fold. These enzymes undergo oxidoreduction reactions, whose direction is dictated by the preference and concentration of their individual cofactor (NAD(H)/NADP(H)). This review presents the current state of knowledge on functional and structural features of RDHs involved in the visual cycle as well as knockout models. RDHs are described as integral or peripheral enzymes. A topology model of the membrane binding of these RDHs via their N- and (or) C-terminal domain has been proposed on the basis of their individual properties. Membrane binding is a crucial issue for these enzymes because of the high hydrophobicity of their retinoid substrates. PMID:25357265

Lhor, Mustapha; Salesse, Christian

2014-12-01

351

Thiol-catalyzed formation of lactate and glycerate from glyceraldehyde. [significance in molecular evolution  

NASA Technical Reports Server (NTRS)

The rate of lactate formation from glyceraldehyde, catalyzed by N-acetyl-cysteine at ambient temperature in aqueous sodium phosphate (pH 7.0), is more rapid at higher sodium phosphate concentrations and remains essentially the same in the presence and absence of oxygen. The dramatic increase in the rate of glycerate formation that is brought about by this thiol, N-acetylcysteine, is accompanied by commensurate decreases in the rates of glycolate and formate production. It is suggested that the thiol-dependent formation of lactate and glycerate occurs by way of their respective thioesters. Attention is given to the significance of these reactions in the context of molecular evolution.

Weber, A. L.

1983-01-01

352

IMP Dehydrogenase: Structural Schizophrenia and an Unusual Base  

SciTech Connect

Textbooks describe enzymes as relatively rigid templates for the transition state of a chemical reaction, and indeed an enzyme such as chymotrypsin, which catalyzes a relatively simple hydrolysis reaction, is reasonably well described by this model. Inosine monophosphate dehydrogenase (IMPDH) undergoes a remarkable array of conformational transitions in the course of a complicated catalytic cycle, offering a dramatic counterexample to this view. IMPDH displays several other unusual mechanistic features, including an Arg residue that may act as a general base catalyst and a dynamic monovalent cation site. Further, IMPDH appears to be involved in 'moon-lighting' functions that may require additional conformational states. How the balance between conformational states is maintained and how the various conformational states interconvert is only beginning to be understood.

Hedstrom,L.; Gan, L.

2006-01-01

353

Maternal prolactin inhibition during lactation affects physical performance evaluated by acute exhaustive swimming exercise in adult rat offspring.  

PubMed

Maternal prolactin inhibition at the end of lactation programs for metabolic syndrome and hypothyroidism in adult offspring, which could negatively affect exercise performance. We evaluated the effects of maternal hypoprolactinemia in late lactation on physical performance in adult progeny. Lactating Wistar rats were treated with bromocriptine (BRO, 1?mg per day) or saline on days 19, 20, and 21 of lactation and offspring were followed until 180 days old. Physical performance was recorded in untrained rats at 90 and 180 days by an acute exhaustive swimming test (exercise group-Ex). At day 90, BRO offspring showed higher visceral fat mass, higher plasma thiobarbituric acid reactive substances, lower total antioxidant capacity, higher liver glycogen, lower glycemia, and normal insulinemia. Although thyroid hormones (TH) levels were unchanged, mitochondrial glycerol phosphate dehydrogenase (mGPD) activity was lower in muscle and in brown adipose tissue (BAT). At this age, BRO-Ex offspring showed higher exercise capacity, lower blood lactate, higher serum T3, and higher muscle and BAT mGPD activities. At day 180, BRO offspring showed central obesity, hypothyroidism, insulin resistance, and lower EDL (extensor digitorum longus) muscle glycogen with unaltered plasma oxidative stress markers. This group showed no alteration of exercise capacity or blood lactate. After exercise, EDL and liver glycogen were lower, while T3 levels, BAT and muscle mGPD activities were normalized. Liver glycogen seem to be related with higher exercise capacity in younger BRO offspring, while the loss of this temporary advantage maybe related to the hypothyroidism and insulin resistance developed with age. PMID:22314333

Casimiro-Lopes, G; Lisboa, P C; Koury, J C; Boaventura, G; Passos, M C F; Moura, E G

2012-02-01

354

Metabolism in 1,3-propanediol fed-batch fermentation by a D-lactate deficient mutant of Klebsiella pneumoniae.  

PubMed

Klebsiella pneumoniae HR526, a new isolated 1,3-propanediol (1,3-PD) producer, exhibited great productivity. However, the accumulation of lactate in the late-exponential phase remained an obstacle of 1,3-PD industrial scale production. Hereby, mutants lacking D-lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D-lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed-batch fermentation. In experiments using pure glycerol as feedstock, the 1,3-PD concentrations, conversion, and productivity increased from 95.39 g L(-1), 0.48 and 1.98 g L(-1) h(-1) to 102. 06 g L(-1), 0.52 mol mol(-1) and 2.13 g L(-1) h(-1), respectively. The diol (1,3-PD and 2,3-butanediol) conversion increased from 0.55 mol mol(-1) to a maximum of 0.65 mol mol(-1). Lactate would not accumulate until 1,3-PD exceeded 84 g L(-1), and the final lactate concentration decreased dramatically from more than 40 g L(-1) to <3 g L(-1). Enzymic measurements showed LDH activity decreased by 89-98% during fed-batch fermentation, and other related enzyme activities were not affected. NADH/NAD(+) enhanced more than 50% in the late-exponential phase as the D-lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3-PD industrial production. PMID:19572314

Xu, Yun-Zhen; Guo, Ni-Ni; Zheng, Zong-Ming; Ou, Xian-Jin; Liu, Hong-Juan; Liu, De-Hua

2009-12-01

355

Highly specific micromethod for the enzymatic determination of radioactive ( sup 14 C)lactate  

SciTech Connect

By collecting released {sup 14}CO{sub 2} following the enzymatic decarboxylation of radiolabeled lactate, picomoles of the latter can be precisely, easily, and reproducibly measured in small biological fluids. This radioactive ({sup 14}C)lactate microassay does not require a neutralization step, nor does it require chemical extractions and partioning procedures, ion exchange, or pyruvate derivatization. Under our specified conditions this simple reaction goes to completion in 90 min. Using this assay in porous adipose cells, with the cell number logarithmically less than that found in other literature methods, the measured glycolytic flux rates were consistent with those previously reported. In these studies, glycolysis was initiated with (U-{sup 14}C)glucose 6-phosphate. This microradioactive lactate assay is useful when dealing with minute tissue samples and/or microliter aliquots of biological fluids.

McCormick, K.L.; Shetler, M.; Mick, G.J. (State Univ. of New York, Syracuse (USA))

1991-02-01

356

Lactation performance of mid-Lactation Dairy Cows Fed ruminally Degradable protein at Concentrations Lower  

E-print Network

Lactation performance of mid-Lactation Dairy Cows Fed ruminally Degradable protein at Concentrations Lower than national research Council recommendations1 J. Cyriac,* a. G. rius,* m. L. mcGilliard,* r. e. pearson,* B. J. Bequette, and m. D. Hanigan*2 *Department of Dairy Science, Virginia

Bequette, Brian J.

357

The metabolism and action of insulin and glucagon in lactating and non-lactating goats  

E-print Network

The metabolism and action of insulin and glucagon in lactating and non-lactating goats J. GRIZARD. N. R. A., Theix, 63122 Ceyrat, France. Introduction. Insulin and glucagon may be involved are not well understood. The aim of the present study was to analyse the metabolism of insulin and glucagon

Paris-Sud XI, Université de

358

Expression and activities of class IV alcohol dehydrogenase and class III aldehyde dehydrogenase in human mouth  

Microsoft Academic Search

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the principal enzymes responsible for the oxidation of ingested ethanol in humans. To study these two enzymes in surgical specimens of attached gingiva and tongue, we have examined the isozyme patterns by agarose isoelectric focusing and determined the enzyme activities. Class IV ?-ADH, class III ?-ADH, and class III ALDH3 were detected

Yuan-Jang Dong; Tzi-Kang Peng; Shih-Jiun Yin

1996-01-01

359

Proline dehydrogenase (oxidase) in cancer.  

PubMed

Proline dehydrogenase (oxidase, PRODH/POX), the first enzyme in the proline degradative pathway, plays a special role in tumorigenesis and tumor development. Proline metabolism catalyzed by PRODH/POX is closely linked with the tricarboxylic acid (TCA) cycle and urea cycle. The proline cycle formed by the interconversion of proline and ?(1) -pyrroline-5-carboxylate (P5C) between mitochondria and cytosol interlocks with pentose phosphate pathway. Importantly, by catalyzing proline to P5C, PRODH/POX donates electrons into the electron transport chain to generate ROS or ATP. In earlier studies, we found that PRODH/POX functions as a tumor suppressor to initiate apoptosis, inhibit tumor growth, and block the cell cycle, all by ROS signaling. It also suppresses hypoxia inducible factor signaling by increasing ?-ketoglutarate. During tumor progression, PRODH/POX is under the control of various tumor-associated factors, such as tumor suppressor p53, inflammatory factor peroxisome proliferator-activated receptor gamma (PPAR?), onco-miRNA miR-23b*, and oncogenic transcription factor c-MYC. Recent studies revealed the two-sided features of PRODH/POX-mediated regulation. Under metabolic stress such as oxygen and glucose deprivation, PRODH/POX can be induced to serve as a tumor survival factor through ATP production or ROS-induced autophagy. The paradoxical roles of PRODH/POX can be understood considering the temporal and spatial context of the tumor. Further studies will provide additional insights into this protein and on its metabolic effects in tumors, which may lead to new therapeutic strategies. PMID:22886911

Liu, Wei; Phang, James M

2012-01-01

360

Lactate stimulates vasculogenic stem cells via the thioredoxin system and engages an autocrine activation loop involving hypoxia-inducible factor 1.  

PubMed

The recruitment and differentiation of circulating stem/progenitor cells (SPCs) in subcutaneous Matrigel in mice was assessed. There were over one million CD34(+) SPCs per Matrigel plug 18 h after Matrigel implantation, and including a polymer to elevate the lactate concentration increased the number of SPCs by 3.6-fold. Intricate CD34(+) cell-lined channels were linked to the systemic circulation, and lactate accelerated cell differentiation as evaluated based on surface marker expression and cell cycle entry. CD34(+) SPCs from lactate-supplemented Matrigel exhibited significantly higher concentrations of thioredoxin 1 (Trx1) and hypoxia-inducible factor 1 (HIF-1) than cells from unsupplemented Matrigel, whereas Trx1 and HIF-1 in CD45(+) leukocytes were not elevated by lactate. Results obtained using small inhibitory RNA (siRNA) specific to HIF-1 and mice with conditionally HIF-1 null myeloid cells indicated that SPC recruitment and lactate-mediated effects were dependent on HIF-1. Cells from lactate-supplemented Matrigel had higher concentrations of phosphorylated extracellular signal-regulated kinases 1 and 2, Trx1, Trx reductase (TrxR), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1) than cells from unsupplemented Matrigel. SPC recruitment and protein changes were inhibited by siRNA specific to lactate dehydrogenase, TrxR, or HIF-1 and by oxamate, apocynin, U0126, N-acetylcysteine, dithioerythritol, and antibodies to VEGF or SDF-1. Oxidative stress from lactate metabolism by SPCs accelerated further SPC recruitment and differentiation through Trx1-mediated elevations in HIF-1 levels and the subsequent synthesis of HIF-1-dependent growth factors. PMID:18710947

Milovanova, Tatyana N; Bhopale, Veena M; Sorokina, Elena M; Moore, Jonni S; Hunt, Thomas K; Hauer-Jensen, Martin; Velazquez, Omaida C; Thom, Stephen R

2008-10-01

361

Lactate Stimulates Vasculogenic Stem Cells via the Thioredoxin System and Engages an Autocrine Activation Loop Involving Hypoxia-Inducible Factor 1?  

PubMed Central

The recruitment and differentiation of circulating stem/progenitor cells (SPCs) in subcutaneous Matrigel in mice was assessed. There were over one million CD34+ SPCs per Matrigel plug 18 h after Matrigel implantation, and including a polymer to elevate the lactate concentration increased the number of SPCs by 3.6-fold. Intricate CD34+ cell-lined channels were linked to the systemic circulation, and lactate accelerated cell differentiation as evaluated based on surface marker expression and cell cycle entry. CD34+ SPCs from lactate-supplemented Matrigel exhibited significantly higher concentrations of thioredoxin 1 (Trx1) and hypoxia-inducible factor 1 (HIF-1) than cells from unsupplemented Matrigel, whereas Trx1 and HIF-1 in CD45+ leukocytes were not elevated by lactate. Results obtained using small inhibitory RNA (siRNA) specific to HIF-1 and mice with conditionally HIF-1 null myeloid cells indicated that SPC recruitment and lactate-mediated effects were dependent on HIF-1. Cells from lactate-supplemented Matrigel had higher concentrations of phosphorylated extracellular signal-regulated kinases 1 and 2, Trx1, Trx reductase (TrxR), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1) than cells from unsupplemented Matrigel. SPC recruitment and protein changes were inhibited by siRNA specific to lactate dehydrogenase, TrxR, or HIF-1 and by oxamate, apocynin, U0126, N-acetylcysteine, dithioerythritol, and antibodies to VEGF or SDF-1. Oxidative stress from lactate metabolism by SPCs accelerated further SPC recruitment and differentiation through Trx1-mediated elevations in HIF-1 levels and the subsequent synthesis of HIF-1-dependent growth factors. PMID:18710947

Milovanova, Tatyana N.; Bhopale, Veena M.; Sorokina, Elena M.; Moore, Jonni S.; Hunt, Thomas K.; Hauer-Jensen, Martin; Velazquez, Omaida C.; Thom, Stephen R.

2008-01-01

362

Cumulative lactate and hospital mortality in ICU patients  

PubMed Central

Background Both hyperlactatemia and persistence of hyperlactatemia have been associated with bad outcome. We compared lactate and lactate-derived variables in outcome prediction. Methods Retrospective observational study. Case records from 2,251 consecutive intensive care unit (ICU) patients admitted between 2001 and 2007 were analyzed. Baseline characteristics, all lactate measurements, and in-hospital mortality were recorded. The time integral of arterial blood lactate levels above the upper normal threshold of 2.2 mmol/L (lactate-time-integral), maximum lactate (max-lactate), and time-to-first-normalization were calculated. Survivors and nonsurvivors were compared and receiver operating characteristic (ROC) analysis were applied. Results A total of 20,755 lactate measurements were analyzed. Data are srpehown as median [interquartile range]. In nonsurvivors (n = 405) lactate-time-integral (192 [0–1881] min·mmol/L) and time-to-first normalization (44.0 [0–427] min) were higher than in hospital survivors (n = 1846; 0 [0–134] min·mmol/L and 0 [0–75] min, respectively; all p < 0.001). Normalization of lactate <6 hours after ICU admission revealed better survival compared with normalization of lactate >6 hours (mortality 16.6% vs. 24.4%; p < 0.001). AUC of ROC curves to predict in-hospital mortality was the largest for max-lactate, whereas it was not different among all other lactate derived variables (all p > 0.05). The area under the ROC curves for admission lactate and lactate-time-integral was not different (p = 0.36). Conclusions Hyperlactatemia is associated with in-hospital mortality in a heterogeneous ICU population. In our patients, lactate peak values predicted in-hospital mortality equally well as lactate-time-integral of arterial blood lactate levels above the upper normal threshold. PMID:23446002

2013-01-01

363

Benzene toxicity: emphasis on cytosolic dihydrodiol dehydrogenases  

SciTech Connect

Blood dyscrasias such as leukopenia and anemia have been clearly identified as consequences of chronic benzene exposure. The metabolites, phenol, catechol, and hydroquinone produced inhibition of /sup 59/Fe uptake in mice which followed the same time course as that produced by benzene. The inhibitor of benzene oxidation, 3-amino-1,2,4-triazole, mitigated the inhibitory effects of benzene and phenol only. These data support the contention that benzene toxicity is mediated by a metabolite and suggest that the toxicity of phenol is a consequence of its metabolism to hydroquinone and that the route of metabolism to catechol may also contribute to the production of toxic metabolite(s). The properties of mouse liver cytosolic dihydrodiol dehydrogenases were examined. These enzymes catalyze the NADP/sup +/-dependent oxidation of trans-1,2-dihydro-1,2-dihydroxybenzene (BDD) to catechol, a possible toxic metabolite of benzene produced via this metabolic route. Four distinct dihydrodiol dehydrogenases (DD1, DD2, DD3, and DD4) were purified to apparent homogeneity as judged by SDS polyacrylamide gel electrophoresis and isoelectric focusing. DD1 appeared to be identical to the major ketone reductase and 17..beta..-hydroxysteroid dehydrogenase activity in the liver. DD2 exhibited aldehyde reductase activity. DD3 and DD4 oxidized 17..beta..-hydroxysteroids, but no carbonyl reductase activity was detected. These relationships between BDD dehydrogenases and carbonyl reductase and/or 17..beta..-hydroxysteroid dehydrogenase activities were supported by several lines of evidence.

Bolcsak, L.E.

1982-01-01

364

NAD + -dependent Formate Dehydrogenase from Plants  

PubMed Central

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) widely occurs in nature. FDH consists of two identical subunits and contains neither prosthetic groups nor metal ions. This type of FDH was found in different microorganisms (including pathogenic ones), such as bacteria, yeasts, fungi, and plants. As opposed to microbiological FDHs functioning in cytoplasm, plant FDHs localize in mitochondria. Formate dehydrogenase activity was first discovered as early as in 1921 in plant; however, until the past decade FDHs from plants had been considerably less studied than the enzymes from microorganisms. This review summarizes the recent results on studying the physiological role, properties, structure, and protein engineering of plant formate dehydrogenases. PMID:22649703

Alekseeva, A.A.; Savin, S.S.; Tishkov, V.I.

2011-01-01

365

Breast diseases during pregnancy and lactation  

PubMed Central

Breast is a typical female sexual physiologic organ that is influenced by steroid hormone from menarche until menopause. Therefore various diseases can be developed by continuous action of estrogen and progesterone. Breast diseases are mainly categorized as benign and malignant. It is very important to distinguish the malignancy from breast diseases. However, it is very difficult to diagnose malignancy in pregnant and lactating women even though the same breast diseases took place. Therefore, we will review breast diseases such as breast carcinoma during pregnancy and lactation. PMID:24327995

Yu, Ji Hoon; Kim, Min Jeong; Cho, Hyonil; Liu, Hyun Ju; Han, Sei-Jun

2013-01-01

366

21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.  

Code of Federal Regulations, 2011 CFR

... (a) Identification. A 6-phosphogluconate dehydrogenase test system is a device intended to measure the activity of the enzyme 6-phosphogluconate dehydrogenase (6 PGD) in serum and erythrocytes. Measurements of...

2011-04-01

367

21 CFR 862.1565 - 6-Phosphogluconate dehydrogenase test system.  

Code of Federal Regulations, 2013 CFR

... (a) Identification. A 6-phosphogluconate dehydrogenase test system is a device intended to measure the activity of the enzyme 6-phosphogluconate dehydrogenase (6 PGD) in serum and erythrocytes. Measurements of...

2013-04-01

368

Genetics Home Reference: 17-beta hydroxysteroid dehydrogenase 3 deficiency  

MedlinePLUS

... PubMed Recent literature OMIM Genetic disorder catalog Conditions > 17-beta hydroxysteroid dehydrogenase 3 deficiency On this page: ... names Glossary definitions Reviewed November 2008 What is 17-beta hydroxysteroid dehydrogenase 3 deficiency? 17-beta hydroxysteroid ...

369

Genetics Home Reference: 3-beta-hydroxysteroid dehydrogenase deficiency  

MedlinePLUS

... PubMed Recent literature OMIM Genetic disorder catalog Conditions > 3-beta-hydroxysteroid dehydrogenase deficiency On this page: Description ... names Glossary definitions Reviewed February 2010 What is 3-beta-hydroxysteroid dehydrogenase deficiency? 3-beta (?)-hydroxysteroid ...

370

Aerobic Production and Utilization of Lactate Satisfy Increased Energy Demands Upon Neuronal Activation in Hippocampal Slices and Provide Neuroprotection Against Oxidative Stress  

PubMed Central

Ever since it was shown for the first time that lactate can support neuronal function in vitro as a sole oxidative energy substrate, investigators in the field of neuroenergetics have been debating the role, if any, of this glycolytic product in cerebral energy metabolism. Our experiments employed the rat hippocampal slice preparation with electrophysiological and biochemical methodologies. The data generated by these experiments (a) support the hypothesis that lactate, not pyruvate, is the end-product of cerebral aerobic glycolysis; (b) indicate that lactate plays a major and crucial role in affording neural tissue to respond adequately to glutamate excitation and to recover unscathed post-excitation; (c) suggest that neural tissue activation is accompanied by aerobic lactate and NADH production, the latter being produced when the former is converted to pyruvate by mitochondrial lactate dehydrogenase (mLDH); (d) imply that NADH can be utilized as an endogenous scavenger of reactive oxygen species (ROS) to provide neuroprotection against ROS-induced neuronal damage. PMID:22275901

Schurr, Avital; Gozal, Evelyne

2012-01-01

371

Surgeon General's Workshop on Breastfeeding and Human Lactation.  

National Technical Information Service (NTIS)

The Surgeon General's Workshop on Breastfeeding and Human Lactation represents a milestone in our continuing efforts to improve the health of our nation's mothers and infants. Research findings have documented the benefits of human milk and lactation for ...

1984-01-01

372

LactMed: New NLM Database on Drugs and Lactation  

MedlinePLUS

... Current Issue Past Issues Research News From NIH LactMed: New NLM Database on Drugs and Lactation Past ... milk, infant levels in blood, potential effects in breast-feeding infants and on lactation itself. The American Academy ...

373

Enhanced stress tolerance in Escherichia coli and Nicotiana tabacum expressing a betaine aldehyde dehydrogenase/choline dehydrogenase fusion protein.  

PubMed

In Escherichia coli the osmoprotective compound glycine betaine is produced from choline by two enzymes; choline dehydrogenase (CDH) oxidizes choline to betaine aldehyde and then further on to glycine betaine, while betaine aldehyde dehydrogenase (BADH) facilitates the conversion of betaine aldehyde to glycine betaine. To evaluate the importance of BADH, a BADH/CDH fusion enzyme was constructed and expressed in E. coli and in Nicotiana tabacum. The fusion enzyme displayed both enzyme activities, and a coupled reaction could be measured. The enzyme was characterized regarding molecular weight and the dependence of the enzyme activities on environmental factors (salt, pH, and poly(ethylene glycol) addition). At high choline concentrations, E. coli cells expressing BADH/CDH were able to grow to higher final densities and to accumulate more glycine betaine than cells expressing CDH only. The intracellular glycine betaine levels were almost 5-fold higher for BADH/CDH when product concentration was related to CDH activity. Also, after culturing the cells at high NaCl concentrations, more glycine betaine was accumulated. On medium containing 20 mM choline, transgenic tobacco plants expressing BADH/CDH grew considerably faster than vector-transformed control plants. PMID:12467448

Yilmaz, Jenny Lindberg; Bülow, Leif

2002-01-01

374

Increase of energy balance significantly alters major lipogenic gene expression in lactation ewes.  

PubMed

The objective of the present study was to examine changes observed in the expression of cytosolic NADP isocitrate dehydrogenase (ICDH) and glucose 6-phosphate dehydrogenase (G6PD) genes, the major implicated genes in ruminant lipogenesis in terms of produce NADPH, during the early post-weaning period in dairy ewes in respect to energy intake, and to further correlate the noted changes with their respective enzymatic activities. A total of 21 subcutaneous adipose tissue samples were obtained from seven lactating (2nd lactation period) dairy ewes of the Chios breed. Adipose tissue samples were taken from the tail head region at weeks 1, 2, and 4 after weaning (45 days after parturition). Dairy ewes were in negative energy balance during weeks 1 and 2 after weaning and they moved into a strong positive energy balance at week 4 after weaning. Expression of ICDH and G6PD genes and their respective enzymatic activity was determined. Results showed that both genes' expression and enzymatic activities were significantly minimal at week 1 after weaning, reaching a maximum level at week 4 after weaning (P < 0.05). A 3.5-fold and a 5-fold increase of G6PD and ICDH mRNA levels were observed, respectively. Concerning their respective enzymatic activities, a 5.5-fold and 2-fold increase was noted, respectively. A positive correlation was found between ICDH and G6PD gene expression (P < 0.001) indicating a synchronized response to energy intake changes. Almost similar changes were observed for enzymatic activities, rendering these enzymes as potential biochemical markers of ovine lipogenesis. PMID:22292701

Laliotis, George P; Bizelis, Iosif; Vitsa, Alkistis; Rogdakis, Emmanuel

2012-01-01

375

Bacterial contamination of commercially available ethacridine lactate (acrinol) products  

Microsoft Academic Search

Bacterial contamination of commercially available ethacridine lactate (acrinol) solutions and cotton gauze soaked in ethacridine lactate solution was investigated. Of 56 samples from ethacridine lactate solutions (eight products, seven manufacturers), seven samples (12·5%) of two products (two manufacturers) were contaminated with 101-104 colony forming units (cfu)\\/mL of Burkholderia pickettii. Of 67 samples obtained from gauze soaked in ethacridine lactate solution

S. Oie; A. Kamiya

1996-01-01

376

Microbial production of lactate-containing polyesters.  

PubMed

Due to our increasing concerns on environmental problems and limited fossil resources, biobased production of chemicals and materials through biorefinery has been attracting much attention. Optimization of the metabolic performance of microorganisms, the key biocatalysts for the efficient production of the desired target bioproducts, has been achieved by metabolic engineering. Metabolic engineering allowed more efficient production of polyhydroxyalkanoates, a family of microbial polyesters. More recently, non-natural polyesters containing lactate as a monomer have also been produced by one-step fermentation of engineered bacteria. Systems metabolic engineering integrating traditional metabolic engineering with systems biology, synthetic biology, protein/enzyme engineering through directed evolution and structural design, and evolutionary engineering, enabled microorganisms to efficiently produce natural and non-natural products. Here, we review the strategies for the metabolic engineering of microorganisms for the in vivo biosynthesis of lactate-containing polyesters and for the optimization of whole cell metabolism to efficiently produce lactate-containing polyesters. Also, major problems to be solved to further enhance the production of lactate-containing polyesters are discussed. PMID:23718266

Yang, Jung Eun; Choi, So Young; Shin, Jae Ho; Park, Si Jae; Lee, Sang Yup

2013-11-01

377

Vaginal rings for contraception in lactating women  

Microsoft Academic Search

Contraceptive methods for breastfeeding women should be safe for the mother and infant and should not interfere with lactation. Progestin-only methods meet these conditions and can be used from the sixth week postpartum. Because all progestins are excreted in milk, those that are insufficiently active by the oral route are preferable to avoid any possible effect on the baby. These

Rebeca Massai; Soledad D??az; Ted Jackanicz; Horacio B Croxatto

2000-01-01

378

[Dermatologic steroid therapy during pregnancy and lactation].  

PubMed

Dermatological therapy during pregnancy and lactation should be scrutinized regarding possible side effects for mother or child. Glucocorticoids are a group of agents that have been employed for many years and with appropriate indications and careful monitoring can be used safely and effectively in pregnancy. PMID:11757466

Pfeiffer, C

2001-11-01

379

Fermentative biohydrogen production from lactate and acetate.  

PubMed

In this study, a continuous-flow stirred tank reactor (CSTR) fed with lactate and acetate was operated to enrich hydrogen-producing bacteria. By varying the influent substrate concentrations and hydraulic retention times (HRT), the volumetric loading rate (VLR) of 55.64 kg-COD/m(3)/day seemed to be optimum for this enriched culture for fermentative hydrogen production from lactate and acetate. The results of batch experiments confirmed that the enriched culture tended to fulfill the e(-) equiv requirement for cell growth at a lower VLR condition (21.77 kg-COD/m(3)/day), while it could largely distribute the e(-) equiv for hydrogen production at a higher VLR condition. However, a maximum lactate/acetate concentration allowed for enriching this culture existed, especially at a lower HRT condition in which wash-out can be an issue for this enriched culture. Finally, the results of cloning and sequencing indicated that Clostridium tyrobutyricum was considered the major hydrogen-producing bacteria in the CSTR fed with lactate and acetate. PMID:22318084

Wu, Chao-Wei; Whang, Liang-Ming; Cheng, Hai-Hsuan; Chan, Kan-Chi

2012-06-01

380

The Quaternary Structure of the Saccharomyces cerevisiae Succinate Dehydrogenase  

Microsoft Academic Search

Succinate dehydrogenases and fumarate reductases are complex mitochondrial or bacterial respiratory chain proteins with remarkably similar structures and functions. Succinate dehydrogenase oxidizes succinate and reduces ubiquinone using a flavin adenine dinucle- otide cofactor and iron-sulfur clusters to transport elec- trons. A model of the quaternary structure of the tetrameric Saccharomyces cerevisiae succinate dehydrogenase was constructed based on the crystal structures

Kayode S. Oyedotun; Bernard D. Lemire

381

Genetics Home Reference: 3-hydroxyacyl-CoA dehydrogenase deficiency  

MedlinePLUS

... CoA dehydrogenase deficiency, are known as fatty acid oxidation disorders. Read more about the HADH gene. How do ... Deficiency of 3-hydroxyacyl-CoA dehydrogenase United Mitochondrial ... information on the diagnosis or management of 3-hydroxyacyl-CoA dehydrogenase deficiency ...

382

Dichloroacetate enhances apoptotic cell death via oxidative damage and attenuates lactate production in metformin-treated breast cancer cells.  

PubMed

The unique metabolism of breast cancer cells provides interest in exploiting this phenomenon therapeutically. Metformin, a promising breast cancer therapeutic, targets complex I of the electron transport chain leading to an accumulation of reactive oxygen species (ROS) that eventually lead to cell death. Inhibition of complex I leads to lactate production, a metabolic byproduct already highly produced by reprogrammed cancer cells and associated with a poor prognosis. While metformin remains a promising cancer therapeutic, we sought a complementary agent to increase apoptotic promoting effects of metformin while attenuating lactate production possibly leading to greatly improved efficacy. Dichloroacetate (DCA) is a well-established drug used in the treatment of lactic acidosis which functions through inhibition of pyruvate dehydrogenase kinase (PDK) promoting mitochondrial metabolism. Our purpose was to examine the synergy and mechanisms by which these two drugs kill breast cancer cells. Cell lines were subjected to the indicated treatments and analyzed for cell death and various aspects of metabolism. Cell death and ROS production were analyzed using flow cytometry, Western blot analysis, and cell counting methods. Images of cells were taken with phase contrast microscopy or confocal microscopy. Metabolism of cells was analyzed using the Seahorse XF24 analyzer, lactate assays, and pH analysis. We show that when DCA and metformin are used in combination, synergistic induction of apoptosis of breast cancer cells occurs. Metformin-induced oxidative damage is enhanced by DCA through PDK1 inhibition which also diminishes metformin promoted lactate production. We demonstrate that DCA and metformin combine to synergistically induce caspase-dependent apoptosis involving oxidative damage with simultaneous attenuation of metformin promoted lactate production. Innovative combinations such as metformin and DCA show promise in expanding breast cancer therapies. PMID:25212175

Haugrud, Allison B; Zhuang, Yongxian; Coppock, Joseph D; Miskimins, W Keith

2014-10-01

383

Purification and some properties of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans.  

PubMed

Thiosulfate dehydrogenase was purified from Acidithiobacillus ferrooxidans using three purification steps. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography, and gel permeation chromatography. Specific activity of the purified enzyme (after IEC) was 3.26 nkat/mg, and yield of the enzyme was 78%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is a tetramer composed of four probably identical subunits of relative molecular weight 45,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 3.0. The isoelectric point of the enzyme was 8.3. Enzyme activity was found to be particularly sensitive to the histidine-selective reagent diethylpyrocarbonate. Reagents selective for arginine, cysteine, and tryptophane had no effect on enzyme activity. PMID:17454821

Janiczek, O; Zemanova, J; Mandl, M

2007-01-01

384

Mechanistic enzymology of CO dehydrogenase from Clostridium thermoaceticum  

SciTech Connect

The final steps in acetyl-CoA biosynthesis by anaerobic bacteria are performed by carbon monoxide dehydrogenase (CODH), a nickel/iron-sulfur protein. An important achievement was to establish conditions under which acetyl-CoA synthesis by purified enzymes equals the in vivo rate of acetate synthesis. Under these optimized conditions we established that the rate limiting step in the synthesis of acetyl-CoA from methyl-H[sub 4]folate, CO and CoA is likely to be the methylation of CODH by the methylated corrinoid/iron-sulfur protein. We then focused on stopped flow studies of this rate limiting transmethylation reaction and established its mechanism. We have studied the carbonylation of CODH by infrared and resonance Raman spectroscopy and determined that the [Ni-Fe[sup 3-4]S[sub 4

Ragsdale, S.W.

1992-01-01

385

Protein moonlighting in iron metabolism: glyceraldehyde-3-phosphate dehydrogenase (GAPDH).  

PubMed

Iron is essential for the survival of both prokaryotic and eukaryotic organisms. It functions as a cofactor for several vital enzymes and iron deprivation is fatal to cells. However, at the same time, excess amounts of iron are also toxic to cells due to the formation of free radicals via the Fenton reaction. As a consequence of its double-edged behaviour, the uptake and regulation of iron involves an intricate balance of acquisition, trafficking, recycling and shuffling between various tissues and organs. This is accomplished by differential regulation of genes involving numerous proteins and enzymes. Several of the proteins identified in these processes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aconitase and lactoferrin (Lf), possess multiple functions within the cell. Such proteins are referred to as moonlighting or multifunctional proteins, whereby proteins initially thought to possess a single well-established function have subsequently been discovered to exhibit alternative functions. In many cases, these multiple functions are conserved across species. PMID:25399609

Boradia, Vishant Mahendra; Raje, Manoj; Raje, Chaaya Iyengar

2014-12-01

386

Effects of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity.  

PubMed

Various alcoholic beverages containing different concentrations of ethanol are widely consumed, and excessive alcohol consumption may result in serious health problems. The consumption of alcoholic beverages is often accompanied by non-alcoholic beverages, such as herbal infusions, tea and carbonated beverages to relieve drunk symptoms. The aim of this study was to supply new information on the effects of these beverages on alcohol metabolism for nutritionists and the general public, in order to reduce problems associated with excessive alcohol consumption. The effects of 57 kinds of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity were evaluated. Generally, the effects of these beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity are very different. The results suggested that some beverages should not be drank after excessive alcohol consumption, and several beverages may be potential dietary supplements for the prevention and treatment of problems related to excessive alcohol consumption. PMID:24162728

Li, Sha; Gan, Li-Qin; Li, Shu-Ke; Zheng, Jie-Cong; Xu, Dong-Ping; Li, Hua-Bin

2014-01-01

387

Glucocorticoids, 11?-hydroxysteroid dehydrogenase, and fetal programming  

Microsoft Academic Search

Glucocorticoids, 11?-hydroxysteroid dehydrogenase, and fetal programming. Epidemiological studies in many distinct human populations have associated low weight or thinness at birth with a substantially increased risk of cardiovascular and metabolic disorders, including hypertension and insulin resistance\\/type 2 diabetes, in adult life. The concept of fetal “programming” has been advanced to explain this phenomenon. Prenatal glucocorticoid therapy reduces birthweight, and steroids

Jonathan R Seckl; Mark Cleasby; Moffat J Nyirenda

2000-01-01

388

Genetics Home Reference: Succinic semialdehyde dehydrogenase deficiency  

MedlinePLUS

... The ALDH5A1 gene provides instructions for producing the succinic semialdehyde dehydrogenase enzyme. This enzyme is involved in the breakdown of a chemical that transmits signals in the brain (neurotransmitter) called gamma-amino butyric acid (GABA). The primary role of GABA is to ...

389

Serum Dihydrolipoamide Dehydrogenase Is a Labile Enzyme  

PubMed Central

Dihydrolipoamide dehydrogenase (DLDH) is a multifunctional oxidoreductase and is well known as an essential component of four mammalian mitochondrial multienzyme complexes: pyruvate dehydrogenase, ?-ketoglutarate dehydrogenase, branched chain ?-keto acid dehydrogenase, and the glycine cleavage system. However, existence of extracellular DLDH in mammals, if any, has not been clearly defined. The present article reports identification and biochemical characterization of serum DLDH. Proteomic analysis of rat serum using blue native polyacrylamide gel electrophoresis (BN-PAGE) and mass spectrometry peptide sequencing led to generation of 6 tryptic peptides in one band that matched to mitochondrial DLDH, indicating the existence of DLDH in rat serum. Measurement of enzymatic activity also indicated the existence of DLDH in human and mouse serum. Further biochemical analysis of rat serum DLDH revealed that this enzyme lacked diaphorase activity and could not be detected on Western blots probed with antibodies that recognized mitochondrial DLDH. Moreover, both ammonium sulfate fractioning and gel filtration of serum samples rendered a great loss in DLDH activity, indicating that the enzyme activity of this serum protein, unlike that of mitochondrial DLDH, is very labile. When DTT was supplemented in the buffer used for gel filtration, DLDH activity was found to be largely preserved; indicating that serum DLDH is susceptible to air-implicated inactivation. Results of the present study indicate that serum DLDH differs from mitochondrial DLDH in that it is a very labile enzyme. PMID:23646291

Yan, Liang-Jun; Thangthaeng, Nopporn; Sumien, Nathalie; Forster, Michael J.

2013-01-01

390

Glucose6-phosphate dehydrogenase deficiency in Spain  

Microsoft Academic Search

Examination of 2520 male subjects in Spain by the Dye Reduction Test revealed five cases of Glucose-6-phosphate dehydrogenase deficiency, all originating from the east coast of Spain and the Balearic Islands. In these areas the enzyme deficiency seems to occur focally at low frequencies near 1%.

G. Flatz; Ria Düren

1967-01-01

391

The effect of inspiratory muscle training upon maximum lactate steady-state and blood lactate concentration  

Microsoft Academic Search

Several studies have reported that improvements in endurance performance following respiratory muscle training (RMT) are associated with a decrease in blood lactate concentration ([Lac]B). The present study examined whether pressure threshold inspiratory muscle training (IMT) elicits an increase in the cycling power output corresponding to the maximum lactate steady state (MLSS). Using a double-blind, placebo-controlled design, 12 healthy, non-endurance-trained male

Alison K. McConnell; Graham R. Sharpe

2005-01-01

392

The physiological role of liver alcohol dehydrogenase  

PubMed Central

1. Yeast alcohol dehydrogenase was used to determine ethanol in the portal and hepatic veins and in the contents of the alimentary canal of rats given a diet free from ethanol. Measurable amounts of a substance behaving like ethanol were found. Its rate of interaction with yeast alcohol dehydrogenase and its volatility indicate that the substance measured was in fact ethanol. 2. The mean alcohol concentration in the portal blood of normal rats was 0.045mm. In the hepatic vein, inferior vena cava and aorta it was about 15 times lower. 3. The contents of all sections of the alimentary canal contained measurable amounts of ethanol. The highest values (average 3.7mm) were found in the stomach. 4. Infusion of pyrazole (an inhibitor of alcohol dehydrogenase) raised the alcohol concentration in the portal vein 10-fold and almost removed the difference between portal and hepatic venous blood. 5. Addition of antibiotics to the food diminished the ethanol concentration of the portal blood to less than one-quarter and that of the stomach contents to less than one-fortieth. 6. The concentration of alcohol in the alimentary canal and in the por