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Sample records for lactate dehydrogenase isoenzyme

  1. Peafowl lactate dehydrogenase: problem of isoenzyme identification.

    PubMed

    Rose, R G; Wilson, A C

    1966-09-16

    Peafowl, like other vertebrates, contain multiple forms of lactate dehydrogenase. The electrophoretic properties of the peafowl isoenzymes are unusual in that the isoenzyme from heart tissue can be either more or less anodic than that of muscle, depending on the pH. This finding focuses attention on the problem of isoenzyme identification. It is suggested that isoenzymes be identified on the basis of properties that are chemically and biologically more significant than electrophoretic mobility. PMID:5917779

  2. Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

    PubMed Central

    Kolk, A H; van Kuyk, L; Boettcher, B

    1978-01-01

    Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein. Images Fig. 2. Fig. 3. PMID:213050

  3. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1445 Lactate...

  4. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1445 Lactate...

  5. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1445 Lactate...

  6. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  7. 21 CFR 862.1445 - Lactate dehydrogenase isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lactate dehydrogenase isoenzymes test system. 862.1445 Section 862.1445 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  8. Separation of turkey lactate dehydrogenase isoenzymes using isoelectric focusing technique.

    PubMed

    Heinová, Dagmar; Kostecká, Zuzana; Csank, Tomáš

    2016-01-01

    Native polyacrylamide gel electrophoresis at pH 8.8 did not allow to separate lactate dehydrogenase (LDH) isoenzymes of turkey origin. Five electrophoretically distinguishable forms of the enzyme were detected in serum and tissues of turkey using IEF technique in a pH range of 3-9. Generally, three different groups were seen: (i) those having an anodic domination (heart, kidney, pancreas, and erythrocytes) with mainly LDH-1 fraction, (ii) those having a cathodic domination (breast muscle and serum) with prevalence of LDH-5, and (iii) those with a more uniform distribution (liver, spleen, lung, and brain). The specific enzyme activity was the highest in the breast muscle, followed by heart muscle, and brain. Low activities were detected in serum, kidney, and liver. PMID:26471476

  9. Lactate dehydrogenase isoenzyme patterns in blood cells from histiocytosis X children.

    PubMed

    Perlino, E; Marra, E; Maenza, S; de Terlizzi, M; Coppola, B C; Santostasi, T; Quagliariello, E

    1993-12-31

    To find a clinical assay for histiocytosis X (HX) diagnosis, measurements were made of both activity and isoenzyme distribution of lactate dehydrogenase (LDH; EC 1.1.1.27) from the blood cells of 6 acute phase and 9 remission patients. A significant increase in the LDH activity measured in the monocytes and lymphocytes isolated from the blood of the acute phase patients was found. The increased activity was due to an enhancement of the normal pattern of LDH isoenzymes in these cells and not to a change in isoenzyme distribution. No increase was found in monocyte LDH isoenzymes from the patients in remission. PMID:8143371

  10. Variability of intracellular lactate dehydrogenase isoenzymes in single human erythrocytes

    SciTech Connect

    Xue, Q.; Yeung, E.S. Iowa State Univ., Ames, IA )

    1994-04-01

    Trace amounts of enzymes within single human erythrocytes can be quantified by a combination of on-column reaction and capillary electrophoresis. A detection limit of 1.3 x 10[sup [minus]21] mol of LDH was achieved with laser-induced fluorescence by monitoring the product of the enzyme-catalyzed reaction between lactate and NAD[sup +]. Single erythrocyte analysis clearly isolates the major forms of LDH. The variation of total LDH activity in a population of cells from a single individual is large, but the relative activities of the isoenzymes LDH-1 and LDH-2 are fairly constant. This can be explained by the distribution of cell age in the population. A lower enzyme activity is indicative of senescence. The efficient separation of different LDH forms and the high detection sensitivity opens up the possibility of multiple-enzyme assays with a single mammalian cell. 41 refs., 5 figs.

  11. [Nervous regulation of glycogen concentration and the lactate dehydrogenase isoenzyme spectrum in the diaphragm of rats].

    PubMed

    Iakovlev, V F

    1981-01-01

    Content of glycogen, activity of lactate dehydrogenase (LDH) and its isoenzyme spectrum were studied in two cases of partial diaphragm denervation as well as in electro-stimulation of separate phrenic nerve branches. Dissimilar postdenervational alterations were observed in the content of glycogen and in the isozyme spectrum of LDH, which depended on the type of partial denervation. Stimulation of individual branches of the phrenic nerve showed that they separately affected the synthesis and consumption of glycogen. The data obtained suggest the nervous regulation of glycogensynthetic processes in muscle tissue. PMID:7467206

  12. Lactate dehydrogenase and its isoenzymes in serum from patients with multiple myeloma.

    PubMed

    Copur, S; Kus, S; Kars, A; Renda, N; Tekuzman, G; Firat, D

    1989-09-01

    Concentrations of total lactate dehydrogenase (LDH; EC 1.1.1.27) and LDH isoenzyme patterns were studied in serum of 19 patients with multiple myeloma and in 19 healthy controls. Patients were divided into three groups (pretreatment, nonresponders, and responders to treatment), based on their clinical status at the time of blood sampling for LDH. The LDH values were found to be significantly higher (P less than 0.05) in the pretreatment group and in the nonresponders than in the responders and the control group, the mean +/- SE values being 445 +/- 35 and 532 +/- 75 units/mL vs 349 +/- 75 and 190 +/- 7.1 units/mL, respectively. Compared with responders and healthy controls, newly diagnosed patients and nonresponders had slight diminutions in LDH-1 and LDH-2, but increased LDH-3. We conclude that determination of LDH and its isoenzymes in serum can be of value as prognostic factors in patients with multiple myeloma. PMID:2776328

  13. Synaptosomal lactate dehydrogenase isoenzyme composition is shifted toward aerobic forms in primate brain evolution.

    PubMed

    Duka, Tetyana; Anderson, Sarah M; Collins, Zachary; Raghanti, Mary Ann; Ely, John J; Hof, Patrick R; Wildman, Derek E; Goodman, Morris; Grossman, Lawrence I; Sherwood, Chet C

    2014-01-01

    With the evolution of a relatively large brain size in haplorhine primates (i.e. tarsiers, monkeys, apes, and humans), there have been associated changes in the molecular machinery that delivers energy to the neocortex. Here we investigated variation in lactate dehydrogenase (LDH) expression and isoenzyme composition of the neocortex and striatum in primates using quantitative Western blotting and isoenzyme analysis of total homogenates and synaptosomal fractions. Analysis of isoform expression revealed that LDH in synaptosomal fractions from both forebrain regions shifted towards a predominance of the heart-type, aerobic isoform LDH-B among haplorhines as compared to strepsirrhines (i.e. lorises and lemurs), while in the total homogenate of the neocortex and striatum there was no significant difference in LDH isoenzyme composition between the primate suborders. The largest increase occurred in synapse-associated LDH-B expression in the neocortex, with an especially remarkable elevation in the ratio of LDH-B/LDH-A in humans. The phylogenetic variation in the ratio of LDH-B/LDH-A was correlated with species-typical brain mass but not the encephalization quotient. A significant LDH-B increase in the subneuronal fraction from haplorhine neocortex and striatum suggests a relatively higher rate of aerobic glycolysis that is linked to synaptosomal mitochondrial metabolism. Our results indicate that there is a differential composition of LDH isoenzymes and metabolism in synaptic terminals that evolved in primates to meet increased energy requirements in association with brain enlargement. PMID:24686273

  14. SYNAPTOSOMAL LACTATE DEHYDROGENASE ISOENZYME COMPOSITION IS SHIFTED TOWARD AEROBIC FORMS IN PRIMATE BRAIN EVOLUTION

    PubMed Central

    Duka, Tetyana; Anderson, Sarah M.; Collins, Zachary; Raghanti, Mary Ann; Ely, John J.; Hof, Patrick R.; Wildman, Derek E.; Goodman, Morris; Grossman, Lawrence I.; Sherwood, Chet C.

    2014-01-01

    With the evolution of a relatively large brain size in haplorhine primates (i.e., tarsiers, monkeys, apes and humans), there have been associated changes in the molecular machinery that delivers energy to the neocortex. Here we investigated variation in lactate dehydrogenase (LDH) expression and isoenzyme composition of the neocortex and striatum in primates using quantitative Western blotting and isoenzyme analysis of total homogenates and synaptosomal fractions. Analysis of isoform expression revealed that LDH in the synaptosomal fraction from both forebrain regions shifted towards a predominance of the heart-type, aerobic isoforms, LDHB, among haplorhines as compared to strepsirrhines (i.e., lorises and lemurs), while in total homogenate of neocortex and striatum there was no significant difference in the LDH isoenzyme composition between the primate suborders. The largest increase occurred in synapse-associated LDH-B expression in the neocortex, displaying an especially remarkable elevation in the ratio of LDH-B to LDH-A in humans. The phylogenetic variation in LDH-B to LDH-A ratio was correlated with species typical brain mass, but not encephalization quotient. A significant LDHB increase in the sub-neuronal fraction from haplorhine neocortex and striatum suggests a relatively higher rate of aerobic glycolysis that is linked to synaptosomal mitochondrial metabolism. Our results indicate that there is differential composition of LDH isoenzymes and metabolism in synaptic terminals that evolved in primates to meet increased energy requirements in association with brain enlargement. PMID:24686273

  15. Microchip-based capillary electrophoresis for determination of lactate dehydrogenase isoenzymes.

    PubMed

    Zhuang, Gui-Sheng; Liu, Jing; Jia, Chun-Ping; Jin, Qing-Hui; Zhao, Jian-Long; Wang, Hui-min

    2007-06-01

    This article describes a novel microchip-based capillary electrophoresis and oncolumn enzymatic reaction analysis protocol for lactate dehydrogenase (LDH) isoenzymes with a home-made xenon lamp-induced fluorescence detection system. A microchip integrated with a temperature-control unit is designed and fabricated for low-temperature electrophoretic separation of LDH isoenzymes, optimal enzyme reaction temperature control, and product detection. A four-step operation and temperature control are employed for the determination of LDH activity by on-chip monitoring of the amount of incubation product of NADH during the fixed incubation period and at a fixed temperature. Experiments on the determination of LDH standard sample and serum LDH isoenzymes from a healthy adult donor are carried out. The results are comparable with those obtained by conventional CE. Shorter analysis times and a more stable and lower background baseline can be achieved. The efficient separation of different LDH forms indicates the potential of microfluidic devices for isoenzyme assay. PMID:17623478

  16. Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C4 of the Plateau Pika (Ochotona curzoniae)

    PubMed Central

    Wang, Yang; Wei, Lian; Wei, Dengbang; Li, Xiao; Xu, Lina; Wei, Linna

    2016-01-01

    Testis-specific lactate dehydrogenase (LDH-C4) is one of the lactate dehydrogenase (LDH) isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C4 in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae) belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000–5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C4 in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A4 (LDH-A4), Lactate Dehydrogenase B4 (LDH-B4), and LDH-C4 were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (PAGE). The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km) of LDH-C4 for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C4 for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki) of the LDH isoenzymes varied: LDH-A4 (Ki = 26.900 mmol/L), LDH-B4 (Ki = 23.800 mmol/L), and LDH-C4 (Ki = 65.500 mmol/L). These data suggest that inhibition of lactate by LDH-A4 and LDH-B4 were stronger than LDH-C4. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C4. PMID:26751442

  17. [Characteristics of the lactate dehydrogenase isoenzyme spectrum of the skin in the presence of thermal lesions].

    PubMed

    Nosova, I M; Zaets, T L; Kotkina, T I

    1977-09-01

    Experiments were conducted on rats; a study was made of the activity of lactic dehydrogenase (LDH) and its isoenzymes in the zone of affection and in the adjacent areas of the skin at various periods after the burn infliction; on the 1st--8th day there occurred a reduction of the sum total LDH activity in the scab zone and the underlying tissue by 70--80%, and in the margin and the intact skin--by 50%. These changes were accompanied by shifts in the isoenzymatic LDH spectrum in the affected tissue; the activity of fraction 5 displayed a sharp rise on the 1st day and that of fractions 2 and 3--a reduction, on the contrary, by the 8th day there was seen some diminution of fraction 5 activity and an elevation of fractions 2 and 3 activity. The following picture is observed on the 14th--22nd day after the burn; the sum total LDH activity remains low, the isoenzymatic LDH spectrum in the margin and the scab is largely normalized, whereas in the underlying tissue there persist changes in the LDH enzyme ratio (a reduction of fractions 1--3 activity, and a rise of fraction 5 activity). PMID:912082

  18. An atypical distribution of lactate dehydrogenase isoenzymes in the hooded seal (Cystophora cristata) brain may reflect a biochemical adaptation to diving.

    PubMed

    Hoff, Mariana Leivas Müller; Fabrizius, Andrej; Folkow, Lars P; Burmester, Thorsten

    2016-04-01

    The brains of some diving mammals can withstand periods of severe hypoxia without signs of deleterious effects. This may in part be due to an enhanced cerebral capacity for anaerobic energy production. Here, we have tested this hypothesis by comparing various parameters of the lactate dehydrogenase (LDH) in the brain of the hooded seal (Cystophora cristata) with those in the brains of the ferret (Mustela putorius furo) and mouse (Mus musculus). We found that mRNA and protein expression of lactate dehydrogenase a (LDHA) and lactate dehydrogenase b (LDHB), and also the LDH activity were significantly higher in the ferret brain than in brains of the hooded seal and the mouse (p < 0.0001). No conspicuous differences in the LDHA and LDHB sequences were observed. There was also no difference in the buffering capacities of the brains. Thus, an enhanced capacity for anaerobic energy production likely does not explain the higher hypoxia tolerance of the seal brain. However, the brain of the hooded seal had higher relative levels of LDHB isoenzymes (LDH1 and LDH2) compared to the non-diving mammals. Moreover, immunofluorescence studies showed more pronounced co-localization of LDHB and glial fibrillary acidic protein in the cortex of the hooded seal. Since LDHB isoenzymes primarily catalyze the conversion of lactate to pyruvate, this finding suggests that the contribution of astrocytes to the brain aerobic metabolism is higher in the hooded seal than in non-diving species. The cerebral tolerance of the hooded seal to hypoxia may therefore partly rely on different LDH isoenzymes distribution. PMID:26820264

  19. Adult-onset multiple acyl CoA dehydrogenation deficiency associated with an abnormal isoenzyme pattern of serum lactate dehydrogenase.

    PubMed

    Sugai, Fuminobu; Baba, Kousuke; Toyooka, Keiko; Liang, Wen-Chen; Nishino, Ichizo; Yamadera, Misaki; Sumi, Hisae; Fujimura, Harutoshi; Nishikawa, Yoshiro

    2012-02-01

    We report a case of a 37 year-old male with multiple acyl-CoA dehydrogenation deficiency (MADD). The patient had suffered from exercise intolerance in his hip and thigh muscles for one year. Then, restriction of carbohydrates for a diet made his symptoms rapidly deteriorate. Blood test revealed compound heterozygosity for two novel missense mutations in the electron transfer flavoprotein dehydrogenase gene (ETFDH), and an abnormal LDH isoenzyme pattern: LDH-1 (60.0%) and LDH-2 (26.0%) predominated with abnormally elevated LDH-1/LDH-2 ratio (2.3), compared with muscle-derived LDH-5 (4.0%). Oral riboflavin treatment significantly improved his exercise intolerance and the LDH profile: LDH-1 (34.4%), LDH-2 (34.9%), LDH-5 (6.9%) and LDH-1/LDH-2 ratio (1.0). The abnormal LDH isoenzyme pattern may be one feature of adult-onset MADD selectively affecting type I muscle fibers with relatively high LDH-1 content. PMID:21907580

  20. Lactate dehydrogenase test

    MedlinePlus

    ... injury Muscle weakness and loss of muscle tissue ( muscular dystrophy ) New abnormal tissue formation (usually cancer) Pancreatitis Stroke ... test LDH isoenzyme blood test Liver disease Mononucleosis Muscular dystrophy Pernicious anemia Stroke Vitamin B12 deficiency anemia Update ...

  1. Lactate dehydrogenase-elevating virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This book chapter describes the taxonomic classification of Lactate dehydrogenase-elevating virus (LDV). Included are: host, genome, classification, morphology, physicochemical and physical properties, nucleic acid, proteins, lipids, carbohydrates, geographic range, phylogenetic properties, biologic...

  2. Lactate dehydrogenase (LD), alkaline phosphatase (ALP) isoenzymatic patterns in Iraqi children with visceral leishmaniasis before and after treatment with stibogluconate.

    PubMed

    Taher, Jasim Hameed; Al-Mulla Hummadi, Yassir Mustafa Kamal; Al-Bashir, Nada Muhammed Taha; Al-Araji, Ali Shaalan

    2016-06-01

    The mean levels of alkaline phosphatase (ALP), lactate dehydrogenase enzymes exhibited a significant elevation in visceral leishmaniasis (VL) patients compared to the control. There was no significant change in relation to the sex and age. ALP isoenzymes revealed three banding patterns which differ from the three zymodems which were obtained from control group. These differences may be due to isoenzymes activity of patients with VL before and after therapy. Lactate dehydrogenase (LD) isoenzymes revealed five banding patterns differ from the five normal zymodems. These differences mainly occurred due to LD isoenzymes activity in patients with VL before and after therapy. PMID:27413293

  3. Prognostic values of aldehyde dehydrogenase 1 isoenzymes in ovarian cancer

    PubMed Central

    Ma, Yu-mei; Zhao, Shan

    2016-01-01

    Aldehyde dehydrogenase 1 (ALDH1) activity has been used as a functional stem cell marker to isolate cancer stem cells in different cancer types, including ovarian cancer. However, which ALDH1’s isoenzymes are contributing to ALDH1 activity in ovarian cancer remains elusive. In addition, the prognostic value of an individual ALDH1 isoenzyme in ovarian cancer is not clear. Thus, we accessed the prognostic value of ALDH1 isoenzymes in ovarian cancer patients through the “Kaplan–Meier plotter” online database, which can be used to determine the effect of the genes on ovarian cancer prognosis. We found that high mRNA expression of five ALDH1 isoenzymes, such as ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, and ALDH1L1, was not correlated with overall survival (OS) for all 1,306 ovarian cancer patients. In addition, all five of the ALDH1 isoenzymes’ high mRNA expression was found to be uncorrelated with OS in serous cancer or endometrioid cancer patients. However, ALDH1A3’s high mRNA expression is associated with worse OS in grade II ovarian cancer patients, hazard ratio (HR) 1.53 (1.14–2.07), P=0.005. ALDH1A2’s high mRNA expression is significantly associated with worse OS in TP53 wild-type ovarian cancer patients, HR 2.86 (1.56–5.08), P=0.00036. In addition, ALDH1A3’s high mRNA expression is significantly associated with better OS in TP53 wild-type ovarian cancer patients, HR 0.56 (0.32–1.00), P=0.04. Our results indicate that although ALDH1 isoenzyme mRNA might not be a prognostic marker for overall ovarian cancer patients, some isoenzymes, such as ALDH1A2 and ALDH1A3, might be a good prognostic marker for some types of ovarian cancer patients. PMID:27110126

  4. Genetics Home Reference: lactate dehydrogenase deficiency

    MedlinePlus

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

  5. Isoenzymes.

    ERIC Educational Resources Information Center

    Daugherty, N. A.

    1979-01-01

    The uses of isoenzyme assays are reviewed as they relate to solutions of practical problems. Emphasizes their use of diagnostic tools in medicine, specifically in cardiac profiling. Suggests that principles of isoenzyme assays readily demonstrate molecular structure, acid-base equilibria, kinetics, and electrochemistry to general chemistry…

  6. Selective permeability of rat liver mitochondria to purified malate dehydrogenase isoenzymes in vitro.

    PubMed Central

    Passarella, S; Marra, E; Doonan, S; Quagliariello, E

    1980-01-01

    1. The mitochondrial malate dehydrogenase from rat liver has been purified to a state of homogeneity as judged by starch-gel electrophoresis and the cytoplasmic isoenzyme has been obtained in a partically purified state. 2. Inhibition of the isoenzymes by sulphite has been studied. 3. In mitochondria loaded with sulphite, the catalytic activity of the (partially inhibited) internal malate dehydrogenase has been measured by addition of oxaloacetate to the suspension medium and observation of the consequent decrease in fluorescence of NADH. 4. Addition of mitochondrial malate dehydrogenase to suspensions of mitochondria loaded with sulphite resulted in an increase in the level of intramitochondrial enzymic activity as measured by the above technique. Addition of the cytoplasmic isoenzyme did not result in such an increase. 5. These results show that mitochondria in suspension are permeable to the mitochondrial malate dehydrogenase but not to the cytoplasmic isoenzyme. 6. This conclusion has been confirmed by direct measurement of a decrease of enzyme activity in solution and an increase inside the mitochondria after incubation of organelles in solutions containing mitochondrial malate dehydrogenase. No such effect was observed with the cytoplasmic isoenzyme. 7. Some features of the permeation process have been studied. PMID:7236231

  7. Role of pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during starvation

    PubMed Central

    Jeoung, Nam Ho; Wu, Pengfei; Joshi, Mandar A.; Jaskiewicz, Jerzy; Bock, Cheryl B.; Depaoli-Roach, Anna A.; Harris, Robert A.

    2006-01-01

    The PDC (pyruvate dehydrogenase complex) is strongly inhibited by phosphorylation during starvation to conserve substrates for gluconeogenesis. The role of PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) in regulation of PDC by this mechanism was investigated with PDHK4−/− mice (homozygous PDHK4 knockout mice). Starvation lowers blood glucose more in mice lacking PDHK4 than in wild-type mice. The activity state of PDC (percentage dephosphorylated and active) is greater in kidney, gastrocnemius muscle, diaphragm and heart but not in the liver of starved PDHK4−/− mice. Intermediates of the gluconeogenic pathway are lower in concentration in the liver of starved PDHK4−/− mice, consistent with a lower rate of gluconeogenesis due to a substrate supply limitation. The concentration of gluconeogenic substrates is lower in the blood of starved PDHK4−/− mice, consistent with reduced formation in peripheral tissues. Isolated diaphragms from starved PDHK4−/− mice accumulate less lactate and pyruvate because of a faster rate of pyruvate oxidation and a reduced rate of glycolysis. BCAAs (branched chain amino acids) are higher in the blood in starved PDHK4−/− mice, consistent with lower blood alanine levels and the importance of BCAAs as a source of amino groups for alanine formation. Non-esterified fatty acids are also elevated more in the blood of starved PDHK4−/− mice, consistent with lower rates of fatty acid oxidation due to increased rates of glucose and pyruvate oxidation due to greater PDC activity. Up-regulation of PDHK4 in tissues other than the liver is clearly important during starvation for regulation of PDC activity and glucose homoeostasis. PMID:16606348

  8. The alcohol dehydrogenase isoenzyme alcohol dehydrogenase IV as a candidate marker of Helicobacter pylori infection

    PubMed Central

    Laniewska-Dunaj, Magdalena; Strumnik, Anna; Szmitkowski, Maciej

    2014-01-01

    Introduction Helicobacter pylori infection is associated with decreased alcohol dehydrogenase (ADH) activity in the gastric mucosa. The decrease in gastric ADH activity depends on the severity of inflammation and mucosal injury. This damage can be a reason of the release of enzyme from gastric mucosa and leads to the increase of the ADH activity in the sera of patients with H. pylori infection. Material and methods Serum samples were taken from 140 patients with H. pylori infection. Total ADH activity was measured by photometric method with p-nitrosodimethylaniline as a substrate and ALDH activity by the fluorometric method with 6-methoxy-2-naphtaldehyde. For the measurement of the activity of class I and II isoenzymes we employed the fluorometric methods, with class-specific fluorogenic substrates. The activity of class III ADH was measured by the photometric method with n-octanol and class IV with m-nitrobenzaldehyde as a substrate. Results The activity of ADH IV in the serum of patients with H. pylori infection increased about 42% (7.86 mU/l) in the comparison to the control level (4.52 mU/l). Total activity of ADH was 1105 mU/l in patients group and 682 mU/l in control. The diagnostic sensitivity for ADH IV was 88%, specificity 90%, positive and negative predictive values were 91% and 84% respectively. Area under ROC curve for ADH IV was 0.84. Conclusions Helicobacter pylori infection of gastric mucosa is reflected in the serum by significant increase of class IV and total ADH activity. The results suggest a potential role for ADH IV as a marker of H. pylori infection. PMID:25395946

  9. Lactate dehydrogenase in sickle cell disease.

    PubMed

    Stankovic Stojanovic, Katia; Lionnet, François

    2016-07-01

    Lactate dehydrogenase (LDH) activity is elevated in many pathological states. Interest in LDH activity in sickle cell disease (SCD) has developed out of an increased comprehension of the pathophysiological process and the clinical course of the disease. Elevated LDH activity in SCD comes from various mechanisms, especially intravascular hemolysis, as well as ischemia-reperfusion damage and tissular necrosis. Intravascular hemolysis is associated with vasoconstriction, platelet activation, endothelial damage, and vascular complications. LDH has been used as a diagnostic and prognostic factor of acute and chronic complications. In this review we have evaluated the literature where LDH activity was examined during steady-state or acute conditions in SCD. PMID:27138446

  10. Subcellular localization of branched-chain amino acid aminotransferase and lactate dehydrogenase C4 in rat and mouse spermatozoa.

    PubMed Central

    Montamat, E E; Vermouth, N T; Blanco, A

    1988-01-01

    Spermatozoa isolated from rat and mouse epididymes show a relatively high branched-chain amino acid aminotransferase (leucine aminotransferase, EC 2.6.1.6) activity. There is a significant reduction of leucine aminotransferase and of the isoenzyme C4 of lactate dehydrogenase (EC 1.1.1.27) in the gametes during their epididymal transit. Studies of patterns of liberation of the leucine aminotransferase and of the lactate dehydrogenase C4 from intact spermatozoa, treated with increasing concentrations of digitonin, indicate that both enzymes have the same dual subcellular location, i.e. in the cytosol and in the mitochondria. PMID:3214422

  11. Subcellular localization of branched-chain amino acid aminotransferase and lactate dehydrogenase C4 in rat and mouse spermatozoa.

    PubMed

    Montamat, E E; Vermouth, N T; Blanco, A

    1988-11-01

    Spermatozoa isolated from rat and mouse epididymes show a relatively high branched-chain amino acid aminotransferase (leucine aminotransferase, EC 2.6.1.6) activity. There is a significant reduction of leucine aminotransferase and of the isoenzyme C4 of lactate dehydrogenase (EC 1.1.1.27) in the gametes during their epididymal transit. Studies of patterns of liberation of the leucine aminotransferase and of the lactate dehydrogenase C4 from intact spermatozoa, treated with increasing concentrations of digitonin, indicate that both enzymes have the same dual subcellular location, i.e. in the cytosol and in the mitochondria. PMID:3214422

  12. Catecholamine regulation of lactate dehydrogenase in rat brain cell culture

    SciTech Connect

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

    1980-03-25

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

  13. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lactate dehydrogenase test system. 862.1440 Section 862.1440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1440 Lactate dehydrogenase...

  14. Human liver alcohol dehydrogenase. 1. The primary structure of the beta 1 beta 1 isoenzyme.

    PubMed

    Hempel, J; Bühler, R; Kaiser, R; Holmquist, B; de Zalenski, C; von Wartburg, J P; Vallee, B; Jörnvall, H

    1984-12-17

    Determination of the amino acid sequence of the beta 1 subunit from the class I (pyrazole-sensitive) human liver alcohol dehydrogenase isoenzyme beta 1 beta 1 revealed a 373-residue structure differing at 48 positions (including a gap) from that of the subunit of the well studied horse liver alcohol dehydrogenase EE isoenzyme. The structure deduced is compatible with known differences in composition, ultraviolet absorbance, electrophoretic mobility and catalytic properties between the horse and human enzymes. All zinc-liganding residues of the horse E subunit are strictly conserved in the human beta 1 subunit, despite an earlier report of a mutation involving Cys-46. This residue therefore remains conserved in all known alcohol dehydrogenase structures. However, the total cysteine content of the beta 1 structure is raised from 14 in the subunit of the horse enzyme to 15 by a Tyr----Cys exchange. Most exchanges are on the surface of the molecule and of a well conserved nature. Substitutions close to the catalytic centre are of interest to explain the altered substrate specificity and different catalytic activity of the beta 1 homodimer. Functionally, a Ser----Thr exchange at position 48 appears to be of special importance, since Thr-48 in beta 1 instead of Ser-48 in the horse enzyme can restrict available space. Four other substitutions also line the active-site pocket, and appear to constitute partly compensated exchanges. PMID:6391920

  15. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes. PMID:25542170

  16. Monoterpene alcohol metabolism: identification, purification, and characterization of two geraniol dehydrogenase isoenzymes from Polygonum minus leaves.

    PubMed

    Hassan, Maizom; Maarof, Nur Diyana; Ali, Zainon Mohd; Noor, Normah Mohd; Othman, Roohaida; Mori, Nobuhiro

    2012-01-01

    NADP(+)-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glycerol, 2-mercaptoethanol, thiourea, and phenylmethylsulfonylfluoride in tricine-NaOH buffer (pH 7.5). The enzyme preparation was separated into two activity peaks, geraniol-DH I and II, by DEAE-Toyopearl 650M column chromatography at pH 7.5. Both isoenzymes were purified to homogeneity in three chromatographic steps. The geraniol-DH isoenzymes were similar in molecular mass, optimal temperature, and pH, but the isoelectric point, substrate specificity, and kinetic parameters were different. The K(m) values for geraniol of geraniol-DH I and II appeared to be 0.4 mM and 0.185 mM respectively. P. minus geraniol-DHs are unusual among geraniol-DHs in view of their thermal stability and optimal temperatures, and also their high specificity for allylic alcohols and NADP(+). PMID:22878188

  17. S-Nitrosoglutathione is a substrate for rat alcohol dehydrogenase class III isoenzyme.

    PubMed

    Jensen, D E; Belka, G K; Du Bois, G C

    1998-04-15

    An enzyme isolated from rat liver cytosol (native molecular mass 78. 3 kDa; polypeptide molecular mass 42.5 kDa) is capable of catalysing the NADH/NADPH-dependent degradation of S-nitrosoglutathione (GSNO). The activity utilizes 1 mol of coenzyme per mol of GSNO processed. The isolated enzyme has, as well, several characteristics that are unique to alcohol dehydrogenase (ADH) class III isoenzyme: it is capable of catalysing the NAD+-dependent oxidations of octanol (insensitive to inhibition by 4-methylpyrazole), methylcrotyl alcohol (stimulated by added pentanoate) and 12-hydroxydodecanoic acid, and also the NADH/NADPH-dependent reduction of octanal. Methanol and ethanol oxidation activity is minimal. The enzyme has formaldehyde dehydrogenase activity in that it is capable of catalysing the NAD+/NADP+-dependent oxidation of S-hydroxymethylglutathione. Treatment with the arginine-specific reagent phenylglyoxal prevents the pentanoate stimulation of methylcrotyl alcohol oxidation and markedly diminishes the enzymic activity towards octanol, 12-hydroxydodecanoic acid and S-hydroxymethylglutathione; the capacity to catalyse GSNO degradation is also checked. Additionally, limited peptide sequencing indicates 100% correspondence with known ADH class III isoenzyme sequences. Kinetic studies demonstrate that GSNO is an exceptionally active substrate for this enzyme. S-Nitroso-N-acetylpenicillamine and S-nitrosated human serum albumin are not substrates; the activity towards S-nitrosated glutathione mono- and di-ethyl esters is minimal. Product analysis suggests that glutathione sulphinamide is the major stable product of enzymic GSNO processing, with minor yields of GSSG and NH3; GSH, hydroxylamine, nitrite, nitrate and nitric oxide accumulations are minimal. Inclusion of GSH in the reaction mix decreases the yield of the supposed glutathione sulphinamide in favor of GSSG and hydroxylamine. PMID:9531510

  18. Characterization of 17beta-hydroxysteroid dehydrogenase isoenzyme expression in benign and malignant human prostate.

    PubMed

    Elo, J P; Akinola, L A; Poutanen, M; Vihko, P; Kyllönen, A P; Lukkarinen, O; Vihko, R

    1996-03-28

    In the present study, expressions of 17beta-hydroxysteroid dehydrogenase (17HSD) types 1, 2, and 3, 5alpha-reductase type 2 and human androgen receptor mRNAs were determined in 12 benign prostatic hyperplasia and 17 prostatic carcinoma specimens. 17HSD type 2 was found to be the principle isoenzyme expressed in the prostate. Significantly higher expressions of 17HSD type 2 and 5alpha-reductase type 2 were detected in benign prostatic hyperplasia compared with the carcinoma specimens. Expression of the androgen receptor in the 2 groups was not significantly different. 17HSD type 3 mRNA was not detected in any of the specimens investigated. Only low constructive expression of the 2.3 kb mRNA of 17HSD type 1 was seen. Immunohistochemical analysis indicated that this did not lead to significant enzyme expression, only faint staining for the enzyme protein being detected, mainly in uroepithelial cells. No significant correlation was found between any of the mRNAs analysed, but the data on 5alpha-reductase type 2 mRNA support the presence of an increased proportion of 5alpha-dihydrotesterone in the hyperplastic prostate. In cultured PC-3 prostatic cancer cells and in the transiently transfected human embryonic kidney 293 cells, 17HSD type 2 was found exclusively to convert 5alpha-dihydrotestosterone and testosterone into the less potent 17-keto compounds 5alpha-androstanedione and 4-androstenedione, respectively. We suggest that the 17HSD type 2 isoenzyme plays a part in the metabolic pathway, resulting in the inactivation of testosterone and 5alpha-dihydrotestosterone locally in the prostate. The enzyme expressed in the prostate could, therefore, protect cells from excessive androgen action. PMID:8608963

  19. Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase.

    PubMed Central

    Mat-Jan, F; Alam, K Y; Clark, D P

    1989-01-01

    Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion. PMID:2644194

  20. Effect of Intramuscular or Intrahepatic Injections of Clostridium perfringens on Rabbit Serum Lactate Dehydrogenase 1

    PubMed Central

    Thomason, Dwayne

    1970-01-01

    Serum lactate dehydrogenase (LDH) activity, LDH isoenzyme pattern, phospholipase C activity, phosphorous level, hemoglobin, and erythrocyte osmotic fragility were followed in rabbits after intramuscular (IM) or intrahepatic (IH) injections of Clostridium perfringens. On the first day after IM injection, there was a drop in LDH activity; this was followed by an increase of LDH activity on the third and sixth day. On the seventh day, LDH activity began to decline, and by the ninth day it had almost returned to normal. On the sixth day after IM injection, there was an increase in serum LDH isoenzyme 5, hemoglobin, and erythrocyte osmotic fragility, but the increase of erythrocyte osmotic fragility and serum hemoglobin could not be attributed to phospholipase C activity since that enzyme was not detected nor was there an increase in serum phosphorus. C. perfringens was recovered by culturing the wound of IM-injected rabbits but not recovered from IH-injected rabbits. Rabbits injected IH showed no change from normal values in any of the tests performed. PMID:16557808

  1. Major Role of NAD-Dependent Lactate Dehydrogenases in Aerobic Lactate Utilization in Lactobacillus plantarum during Early Stationary Phase

    PubMed Central

    Goffin, Philippe; Lorquet, Frédérique; Kleerebezem, Michiel; Hols, Pascal

    2004-01-01

    NAD-independent lactate dehydrogenases are commonly thought to be responsible for lactate utilization during the stationary phase of aerobic growth in Lactobacillus plantarum. To substantiate this view, we constructed single and double knockout mutants for the corresponding genes, loxD and loxL. Lactate-to-acetate conversion was not impaired in these strains, while it was completely blocked in mutants deficient in NAD-dependent lactate dehydrogenase activities, encoded by the ldhD and ldhL genes. We conclude that NAD-dependent but not NAD-independent lactate dehydrogenases are involved in this process. PMID:15375150

  2. Reappraisal of the regulation of lactococcal L-lactate dehydrogenase.

    PubMed

    van Niel, Ed W J; Palmfeldt, Johan; Martin, Rani; Paese, Marco; Hahn-Hägerdal, Bärbel

    2004-03-01

    Lactococcal lactate dehydrogenases (LDHs) are coregulated at the substrate level by at least two mechanisms: the fructose-1,6-biphosphate/phosphate ratio and the NADH/NAD ratio. Among the Lactococcus lactis species, there are strains that are predominantly regulated by the first mechanism (e.g., strain 65.1) or by the second mechanism (e.g., strain NCDO 2118). A more complete model of the kinetics of the regulation of lactococcal LDH is discussed. PMID:15006814

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

    PubMed

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

    2011-11-22

    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 μmoles 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

  4. NADP+-Preferring d-Lactate Dehydrogenase from Sporolactobacillus inulinus

    PubMed Central

    Zhu, Lingfeng; Xu, Xiaoling; Wang, Limin; Ma, Yanhe

    2015-01-01

    Hydroxy acid dehydrogenases, including l- and d-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD+ as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a d-lactate dehydrogenase from a Sporolactobacillus inulinus strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and in vivo and in vitro enzymatic activity analyses. The residue Asn174 was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases. PMID:26150461

  5. Lactate dehydrogenase X, malate dehydrogenase and total protein in rat spermatozoa during epididymal transit.

    PubMed

    Vermouth, N T; Carriazo, C S; Ponce, R H; Blanco, A

    1986-01-01

    Lactate dehydrogenase isozyme X (LDH X), malate dehydrogenase (MDH) and total soluble protein have been determined in lysates of spermatozoa isolated from caput, corpus and cauda of rat epididymis. Transit of spermatozoa through epididymis is accompanied by a reduction of LDH X, MDH and total protein per cell in sexually rested animals. The profiles of reduction along epididymal segments are different for the three variables studied. Mating with receptive females during the 5 days prior to determinations increases significantly the levels of MDH in spermatozoa from all sections of epididymis and produces increase of total soluble protein in the cells contained in cauda. PMID:3956158

  6. Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling

    PubMed Central

    Kim, Jin Man; Kwon, So Hyun; Lee, Seoung Hoon; Lee, Soo Young; Jeong, Daewon

    2016-01-01

    Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A2B2 isotype during osteoclast differentiation as well as the LDH A1B3 and B4 isotypes during osteoclast maturation after pre-osteoclast formation. Glucose consumption and lactate production in growth media were accelerated during osteoclast differentiation, together with enhanced expression of H+-lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consumption via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabolism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stimulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast formation via osteoclast precursor fusion and NFATc1 signaling. PMID:27077737

  7. Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals

    PubMed Central

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

    2014-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.03640.001 PMID:25247702

  8. Yeast cell-based analysis of human lactate dehydrogenase isoforms.

    PubMed

    Mohamed, Lulu Ahmed; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

    2015-12-01

    Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae, with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc/LDH strains are expected to be of use for versatile analyses of human LDH. PMID:26126931

  9. Temporal changes in the involvement of pyruvate dehydrogenase complex in muscle lactate accumulation during lipopolysaccharide infusion in rats

    PubMed Central

    Alamdari, N; Constantin-Teodosiu, D; Murton, A J; Gardiner, S M; Bennett, T; Layfield, R; Greenhaff, P L

    2008-01-01

    A characteristic manifestation of sepsis is muscle lactate accumulation. This study examined any putative (causative) association between pyruvate dehydrogenase complex (PDC) inhibition and lactate accumulation in the extensor digitorum longus (EDL) muscle of rats infused with lipopolysaccharide (LPS), and explored the involvement of increased transcription of muscle-specific pyruvate dehydrogenase kinase (PDK) isoenzymes. Conscious, male Sprague–Dawley rats were infused i.v. with saline (0.4 ml h−1, control) or LPS (150 μg kg−1 h−1) for 2 h, 6 h or 24 h (n = 6–8). Muscle lactate concentration was elevated after 2, 6 and 24 h LPS infusion. Muscle PDC activity was the same at 2 h and 6 h, but was 65% lower after 24 h of LPS infusion (P < 0.01), when there was a 47% decrease in acetylcarnitine concentration (P < 0.05), and a 24-fold increase in PDK4 mRNA expression (P < 0.001). These changes were preceded by marked increases in tumour necrosis factor-α and interleukin-6 mRNA expression at 2 h. The findings indicate that the early (2 and 6 h) elevation in muscle lactate concentration during LPS infusion was not attributable to limited muscle oxygen availability or ATP production (evidenced by unchanged ATP and phosphocreatine (PCr) concentrations) or to PDC inhibition, whereas after 24 h, muscle lactate accumulation appears to have resulted from PDC activation status limiting pyruvate flux, most probably due to cytokine-mediated up-regulation of PDK4 transcription. PMID:18218678

  10. Dissociation and rate constants of some human liver alcohol dehydrogenase isoenzymes.

    PubMed

    Pietruszko, R; de Zalenski, C; Theorell, H

    1976-01-01

    ADH from human liver forms binary complexes with NADH, associated with a blue shift of the peak of the fluorescence emission of NADH. The wavelength shift is the same for all isoenzymes but the accompanying intensification of the fluorescence is different. The fluorescence is further increased by the formation of the very tight ternary enzyme-NADH-isobutyramide complexes. These properties are similar to those for the horse liver ADH, as well as the molecular weight of E=40 000 per active site of the dimer molecule (EE). "Stopped-flow" determined velocity constants (ER in equilibrium E+R) were found to be in good agreement with ethanol activity constants previously determined by activity measurement, confirming the validity of the ordered ternary complex mechanism also for the human ADH. No single isoenzyme activity as high as that reported by Mourad and Woronick or Drum has been found. PMID:184631

  11. Purification and Characterization of Chloroplastic NADP-Isocitrate Dehydrogenase from Mixotrophic Tobacco Cells (Comparison with the Cytosolic Isoenzyme).

    PubMed Central

    Galvez, S.; Bismuth, E.; Sarda, C.; Gadal, P.

    1994-01-01

    Green, mixotrophic tobacco (Nicotiana tabacum) cell cultures in the exponential growth phase were found to have two clearly distinguishable NADP-isocitrate dehydrogenase (ICDH; EC 1.1.1.42) isoenzymes. Their elution behavior during anion-exchange column chromatography was similar to that described previously for the cytosolic (ICDH1) and chloroplastic (ICDH2) enzymes from pea (Pisum sativum) leaves. ICDH2 was absent in etiolated tobacco cell suspensions and appeared during the greening process. Both isoforms were purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation and anion-exchange and affinity chromatography. The isoenzymes were separated on a DEAE-Sephacel column, but the most effective step was a Matrex Red-A column, which enabled an overall purification of 833- and 1328-fold for ICDH1 and ICDH2, respectively. Polyclonal antibodies were raised against each isoform. The ICDH2-specific antibody was used to localize tobacco leaf ICDH2 in situ by an immunogold labeling technique. The enzyme was found largely, if not exclusively, in the chloroplasts of green leaves. ICDH1 and ICDH2 were shown to have apparent native molecular weights of 117,000 and 136,000, respectively, and to consist of identical, 48.5-kD subunits. Similar apparent Km values for NADP, D(+)isocitrate, and Mg2+ were found for the two enzymes when assayed with Mg2+ as the metal cofactor. PMID:12232226

  12. Human Lactate Dehydrogenase A Inhibitors: A Molecular Dynamics Investigation

    PubMed Central

    Shi, Yun; Pinto, B. Mario

    2014-01-01

    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

  13. Circadian rhythm of lactate dehydrogenase in rat testis.

    PubMed

    Vermouth, N T; Ponce, R H; Carriazo, C S; Blanco, A

    1984-01-01

    Activity of total lactate dehydrogenase (LDH) and of the isozyme X (LDH X or C4) have been determined at 2 hr intervals during 24 hr cycles in testis of adult rats maintained since birth in a photoperiod of 14 hr light: 10 hr dark. LDH X activity of epididymal sections (caput, corpus and cauda) from the same animals was also determined. Total LDH and LDH X activities in testis exhibited circadian rhythms with different timing. LDH X in the three portions of epididymis showed diurnal variations similar to those in testis. Rats subjected to constant light or constant dark presented marked modifications of LDH X profiles, indicating that the photoperiod plays a synchronizer role. While total soluble proteins did not show variations in testis of rats exposed to the photoperiod, a circadian rhythm was demonstrated in animals maintained in constant light or dark. PMID:6467917

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

    PubMed

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

    2013-11-01

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

  15. Lactate Dehydrogenase in Hepatocellular Carcinoma: Something Old, Something New

    PubMed Central

    Faloppi, Luca; Bianconi, Maristella; Memeo, Riccardo; Casadei Gardini, Andrea; Giampieri, Riccardo; Bittoni, Alessandro; Andrikou, Kalliopi; Del Prete, Michela; Cascinu, Stefano; Scartozzi, Mario

    2016-01-01

    Hepatocellular carcinoma (HCC) is the most common primary liver tumour (80–90%) and represents more than 5.7% of all cancers. Although in recent years the therapeutic options for these patients have increased, clinical results are yet unsatisfactory and the prognosis remains dismal. Clinical or molecular criteria allowing a more accurate selection of patients are in fact largely lacking. Lactic dehydrogenase (LDH) is a glycolytic key enzyme in the conversion of pyruvate to lactate under anaerobic conditions. In preclinical models, upregulation of LDH has been suggested to ensure both an efficient anaerobic/glycolytic metabolism and a reduced dependence on oxygen under hypoxic conditions in tumour cells. Data from several analyses on different tumour types seem to suggest that LDH levels may be a significant prognostic factor. The role of LDH in HCC has been investigated by different authors in heterogeneous populations of patients. It has been tested as a potential biomarker in retrospective, small, and nonfocused studies in patients undergoing surgery, transarterial chemoembolization (TACE), and systemic therapy. In the major part of these studies, high LDH serum levels seem to predict a poorer outcome. We have reviewed literature in this setting trying to resume basis for future studies validating the role of LDH in this disease. PMID:27314036

  16. Expression of Plasmodium falciparum lactate dehydrogenase in Escherichia coli.

    PubMed

    Bzik, D J; Fox, B A; Gonyer, K

    1993-05-01

    A Plasmodium falciparum gene is described which encodes lactate dehydrogenase activity (P. falciparum LDH). The P. falciparum LDH gene contains no introns and is present in a single copy on chromosome 13. P. falciparum LDH was expressed in all asexual blood stages as a 1.6-kb mRNA. The predicted 316 amino acid protein coding region of P. falciparum LDH was inserted into the prokaryotic expression vector pKK223-3 and a 33-kDa protein having LDH activity was synthesized in Escherichia coli. P. falciparum LDH primary structure displays high amino acid similarity (50-57%) to vertebrate and bacterial LDH, but lacks the amino terminal extension observed in all vertebrate LDH. The majority of amino acid residues implicated in substrate and coenzyme binding and catalysis of other LDH are well conserved in P. falciparum LDH. However, several notable differences in amino acid composition were observed. P. falciparum LDH contained several distinctive single amino acid insertions and deletions compared to other LDH enzymes, and most remarkably, it contained a novel insertion of 5 amino acids within the conserved mobile loop region near arginine residue 109, a residue which is known to make contact with pyruvate in the ternary complex of other LDH. These results suggest that novel features of P. falciparum LDH primary structure may be correlated with previously characterized and distinctive kinetic, biochemical, immunochemical, and electrophoretic properties of P. falciparum LDH. PMID:8515777

  17. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    SciTech Connect

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D.

    2014-11-07

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.

  18. SERUM VALUES OF ALKALINE PHOSPHATASE AND LACTATE DEHYDROGENASE IN OSTEOSARCOMA

    PubMed Central

    ZUMÁRRAGA, JUAN PABLO; BAPTISTA, ANDRÉ MATHIAS; ROSA, LUIS PABLO DE LA; CAIERO, MARCELO TADEU; CAMARGO, OLAVO PIRES DE

    2016-01-01

    ABSTRACT Objective: To study the relationship between the pre and post chemotherapy (CT) serum levels of alkaline phosphatase (AP) and lactate dehydrogenase (LDH), and the percentage of tumor necrosis (TN) found in specimens after the pre surgical CT in patients with osteosarcoma. Methods: Series of cases with retrospective evaluation of patients diagnosed with osteosarcoma. Participants were divided into two groups according to serum values of both enzymes. The values of AP and LDH were obtained before and after preoperative CT. The percentage of tumor necrosis (TN) of surgical specimens of each patient was also included. Results: One hundred and thirty seven medical records were included from 1990 to 2013. Both the AP as LDH decreased in the patients studied, being the higher in pre CT than post CT. The average LHD decrease was 795.12U/L and AP decrease was 437.40 U/L. The average TN was 34.10 %. There was no statistically significant correlation between the serums values and the percentage of tumoral necrosis. Conclusion: The serum levels values of AP and LDH are not good predictors for the chemotherapy-induced necrosis in patients with osteosarcoma. Level of Evidence IV, Case Series. PMID:27217815

  19. Acetate Utilization in Lactococcus lactis Deficient in Lactate Dehydrogenase: a Rescue Pathway for Maintaining Redox Balance

    PubMed Central

    Hols, Pascal; Ramos, Ana; Hugenholtz, Jeroen; Delcour, Jean; de Vos, Willem M.; Santos, Helena; Kleerebezem, Michiel

    1999-01-01

    Acetate was shown to improve glucose fermentation in Lactococcus lactis deficient in lactate dehydrogenase. 13C and 1H nuclear magnetic resonance studies using [2-13C]glucose and [2-13C]acetate as substrates demonstrated that acetate was exclusively converted to ethanol. This novel pathway provides an alternative route for NAD+ regeneration in the absence of lactate dehydrogenase. PMID:10464231

  20. Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases.

    PubMed

    Zhou, Meixia; Crawford, Yongping; Ng, Domingos; Tung, Jack; Pynn, Abigail F J; Meier, Angela; Yuk, Inn H; Vijayasankaran, Natarajan; Leach, Kimberly; Joly, John; Snedecor, Bradley; Shen, Amy

    2011-04-20

    Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors. PMID:21392546

  1. Purification and Partial Kinetic and Physical Characterization of Two Chloroplast-Localized NADP-Specific Glutamate Dehydrogenase Isoenzymes and Their Preferential Accumulation in Chlorella sorokiniana Cells Cultured at Low or High Ammonium Levels 1

    PubMed Central

    Bascomb, Newell F.; Schmidt, Robert R.

    1987-01-01

    Two ammonium-inducible, chloroplast-localized NADP-specific glutamate dehydrogenase isoenzymes were purified to homogeneity from Chlorella sorokiniana. These isoenzymes were homopolymers of either α- or β-subunits with molecular weights of 55,500 or 53,000, respectively. The α-isoenzyme was preferentially induced at low ammonium concentrations (2 millimolar or lower), whereas only the β-isoenzyme accumulated after cells were fully induced (120 minutes) at high ammonium concentrations (29 millimolar). Purification of isoenzymes was achieved by (NH4)2SO4 fractionation, gel-filtration, anion-exchange fast protein liquid chromatography, and affinity chromatography. The α- and β-isoenzymes were separated by their differential binding to Type 4 nicotinamide adenine dinucleotide phosphate-Sepharose. Both isoenzymes bound to an antibody affinity column to which purified antibody (prepared against β-isoenzyme) was covalently attached. Peptide mapping of the subunits showed them to have a high degree of sequence homology. Both subunits were synthesized in vitro from precursor protein(s) with a molecular weight of 58,500. Although the subunits have similar chemical, physical, and antigenic properties, their holoenzymes have strikingly different ammonium Km values. The ammonium Km of the β-isoenzyme remained constant at approximately 75 millimolar, whereas this Km of the α-isoenzyme ranged from 0.02 to 3.5 millimolar, depending upon nicotinamide adenine dinucleotide phosphate concentration. Images Fig. 1 Fig. 2 Fig. 7 Fig. 8 PMID:16665219

  2. Mechanism of Thermal Adaptation in the Lactate Dehydrogenases.

    PubMed

    Peng, Huo-Lei; Egawa, Tsuyoshi; Chang, Eric; Deng, Hua; Callender, Robert

    2015-12-10

    The mechanism of thermal adaptation of enzyme function at the molecular level is poorly understood but is thought to lie within the structure of the protein or its dynamics. Our previous work on pig heart lactate dehydrogenase (phLDH) has determined very high resolution structures of the active site, via isotope edited IR studies, and has characterized its dynamical nature, via laser-induced temperature jump (T-jump) relaxation spectroscopy on the Michaelis complex. These particular probes are quite powerful at getting at the interplay between structure and dynamics in adaptation. Hence, we extend these studies to the psychrophilic protein cgLDH (Champsocephalus gunnari; 0 °C) and the extreme thermophile tmLDH (Thermotoga maritima LDH; 80 °C) for comparison to the mesophile phLDH (38-39 °C). Instead of the native substrate pyruvate, we utilize oxamate as a nonreactive substrate mimic for experimental reasons. Using isotope edited IR spectroscopy, we find small differences in the substate composition that arise from the detailed bonding patterns of oxamate within the active site of the three proteins; however, we find these differences insufficient to explain the mechanism of thermal adaptation. On the other hand, T-jump studies of reduced β-nicotinamide adenine dinucleotide (NADH) emission reveal that the most important parameter affecting thermal adaptation appears to be enzyme control of the specific kinetics and dynamics of protein motions that lie along the catalytic pathway. The relaxation rate of the motions scale as cgLDH > phLDH > tmLDH in a way that faithfully matches kcat of the three isozymes. PMID:26556099

  3. Phylogenetic analysis of vertebrate lactate dehydrogenase (LDH) multigene families.

    PubMed

    Li, Yi-Ju; Tsoi, Stephen C-M; Mannen, Hideyuka; Shoei-lung Li, Steven

    2002-05-01

    In this paper we analyzed 49 lactate dehydrogenase (LDH) sequences, mostly from vertebrates. The amino acid sequence differences were found to be larger for a human-killifish pair than a human-lamprey pair. This indicates that some protein sequence convergence may occur and reduce the sequence differences in distantly related species. We also examined transitions and transversions separately for several species pairs and found that the transitions tend to be saturated in the distantly related species pair, while transversions are increasing. We conclude that transversions maintain a conservative rate through the evolutionary time. Kimura's two-parameter model for multiple-hit correction on transversions only was used to derive a distance measure and then construct a neighbor-joining (NJ) tree. Three findings were revealed from the NJ tree: (i) the branching order of the tree is consistent with the common branch pattern of major vertebrates; (ii) Ldh-A and Ldh-B genes were duplicated near the origin of vertebrates; and (iii) Ldh-C and Ldh-A in mammals were produced by an independent gene duplication in early mammalian history. Furthermore, a relative rate test showed that mammalian Ldh-C evolved more rapidly than mammalian Ldh-A. Under a two-rate model, this duplication event was calibrated to be approximately 247 million years ago (mya), dating back to the Triassic period. Other gene duplication events were also discovered in Xenopus, the first duplication occurring approximately 60-70 mya in both Ldh-A and Ldh-B, followed by another recent gene duplication event, approximately 20 mya, in Ldh-B. PMID:11965434

  4. Age-related responses of right ventricle in swim-trained rats: changes in lactate and pyruvate contents and lactate dehydrogenase activity.

    PubMed

    Anitha, V; Asha Devi, S

    1996-09-18

    Age related changes in carbohydrate substrates such as, glucose, glycogen, pyruvic acid and lactic acid and the activity of lactate dehydrogenase (LDH) and LDH isoenzyme profile were evaluated in the right ventricle (RV) of swim-trained rats of 6- (adult), 12- (middle-aged) and 18- (old) months-of-age. Moderate hypertrophy was seen in the heart and RV in response to training in all age groups with the 12 months exhibiting a significant increase. While resting levels of pyruvate and glucose in the RV showed small elevations in adult and middle-aged rats, lactic acid showed reductions in all ages. Glycogen supercompensation was seen in the RV of trained animals. These age-related alterations in RV were associated with decreases in blood lactic acid and glucose in the trained rats belonging to all ages. Total protein of the RV decreased with age and exercise increased the content. Total LDH and M4-LDH activities decreased with age. However, training increased their activities in all ages. These changes in the RV suggests that swimming activity produces adaptations (e.g. increased LDH and M4) in all age groups. Considering the degree of adaptations, it can be suggested that adult and middle-aged are suitable for initiating swim-training programs, but not in old age. PMID:8869911

  5. Plasma Lactate Dehydrogenase Levels Predict Mortality in Acute Aortic Syndromes

    PubMed Central

    Morello, Fulvio; Ravetti, Anna; Nazerian, Peiman; Liedl, Giovanni; Veglio, Maria Grazia; Battista, Stefania; Vanni, Simone; Pivetta, Emanuele; Montrucchio, Giuseppe; Mengozzi, Giulio; Rinaldi, Mauro; Moiraghi, Corrado; Lupia, Enrico

    2016-01-01

    Abstract In acute aortic syndromes (AAS), organ malperfusion represents a key event impacting both on diagnosis and outcome. Increased levels of plasma lactate dehydrogenase (LDH), a biomarker of malperfusion, have been reported in AAS, but the performance of LDH for the diagnosis of AAS and the relation of LDH with outcome in AAS have not been evaluated so far. This was a bi-centric prospective diagnostic accuracy study and a cohort outcome study. From 2008 to 2014, patients from 2 Emergency Departments suspected of having AAS underwent LDH assay at presentation. A final diagnosis was obtained by aortic imaging. Patients diagnosed with AAS were followed-up for in-hospital mortality. One thousand five hundred seventy-eight consecutive patients were clinically eligible, and 999 patients were included in the study. The final diagnosis was AAS in 201 (20.1%) patients. Median LDH was 424 U/L (interquartile range [IQR] 367–557) in patients with AAS and 383 U/L (IQR 331–460) in patients with alternative diagnoses (P < 0.001). Using a cutoff of 450 U/L, the sensitivity of LDH for AAS was 44% (95% confidence interval [CI] 37–51) and the specificity was 73% (95% CI 69–76). Overall in-hospital mortality for AAS was 23.8%. Mortality was 32.6% in patients with LDH ≥ 450 U/L and 16.8% in patients with LDH < 450 U/L (P = 0.006). Following stratification according to LDH quartiles, in-hospital mortality was 12% in the first (lowest) quartile, 18.4% in the second quartile, 23.5% in the third quartile, and 38% in the fourth (highest) quartile (P = 0.01). LDH ≥ 450 U/L was further identified as an independent predictor of death in AAS both in univariate and in stepwise logistic regression analyses (odds ratio 2.28, 95% CI 1.11–4.66; P = 0.025), in addition to well-established risk markers such as advanced age and hypotension. Subgroup analysis showed excess mortality in association with LDH ≥ 450 U/L in elderly, hemodynamically stable

  6. Lactate dehydrogenase from autotrophic and heterotrophic cells of the marine diatom Cylindrotheca fusiformis Reimann & Lewin.

    PubMed

    Darley, W M; Smiley, R H

    1976-10-01

    Cultures of Cylindrotheca furisormis grown either autotrohpically or heterotrophically on lactate contained significant amounts of NAD-dependent L(+)-lactate dehydrogenase (EC 1.1.1.27). Polyacylamide gel electrophoresis of crude enzyme extracts revealed a single band which was indistinguishable between autotrohpic and heterotrohpic cells. The Km for lactate of partially purified preparations was lower under heterotrophic conditions. The specific activity in crude extracts was higher under autotrophic than heterotrophic conditions; it dropped precipitously when autotrophic cells were transferred to the dark, increasing again only in the presence of lactate. These and related observations suggest that this enzyme has at most only a minor role in the assimilation of lactate during heterotrophic growth on lactate. PMID:184899

  7. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 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...

  8. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 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...

  9. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 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...

  10. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 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...

  11. Functional Replacement of the Escherichia coli d-(−)-Lactate Dehydrogenase Gene (ldhA) with the l-(+)-Lactate Dehydrogenase Gene (ldhL) from Pediococcus acidilactici†

    PubMed Central

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

    2003-01-01

    The microbial production of l-(+)-lactic acid is rapidly expanding to allow increased production of polylactic 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 with biocatalysis (complex nutrients, antibiotics, aeration, product purification, and waste disposal), a recombinant derivative of Escherichia coli W3110 was developed that contains five chromosomal deletions (focA-pflB frdBC adhE ackA ldhA). This strain was constructed from a d-(−)-lactic acid-producing strain, SZ63 (focA-pflB frdBC adhE ackA), by replacing part of the chromosomal ldhA coding region with Pediococcus acidilactici ldhL encoding an l-lactate dehydrogenase. Although the initial strain (SZ79) grew and fermented poorly, a mutant (SZ85) was readily isolated by selecting for improved growth. SZ85 exhibited a 30-fold increase in l-lactate dehydrogenase activity in comparison to SZ79, functionally replacing the native d-lactate dehydrogenase activity. Sequencing revealed mutations in the upstream, coding, and terminator regions of ldhL in SZ85, which are presumed to be responsible for increased l-lactate dehydrogenase activity. SZ85 produced l-lactic acid in M9 mineral salts medium containing glucose or xylose with a yield of 93 to 95%, a purity of 98% (based on total fermentation products), and an optical purity greater than 99%. Unlike other recombinant biocatalysts for l-lactic acid, SZ85 remained prototrophic and is devoid of plasmids and antibiotic resistance genes. PMID:12676706

  12. Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production

    PubMed Central

    Dave, Khyati K.; Punekar, Narayan S.

    2015-01-01

    Different engineered organisms have been used to produce L-lactate. Poor yields of lactate at low pH and expensive downstream processing remain as bottlenecks. Aspergillus niger is a prolific citrate producer and a remarkably acid tolerant fungus. Neither a functional lactate dehydrogenase (LDH) from nor lactate production by A. niger is reported. Its genome was also investigated for the presence of a functional ldh. The endogenous A. niger citrate synthase promoter relevant to A. niger acidogenic metabolism was employed to drive constitutive expression of mouse lactate dehydrogenase (mldhA). An appraisal of different branches of the A. niger pyruvate node guided the choice of mldhA for heterologous expression. A high copy number transformant C12 strain, displaying highest LDH specific activity, was analyzed under different growth conditions. The C12 strain produced 7.7 g/l of extracellular L-lactate from 60 g/l of glucose, in non-neutralizing minimal media. Significantly, lactate and citrate accumulated under two different growth conditions. Already an established acidogenic platform, A. niger now promises to be a valuable host for lactate production. PMID:26683313

  13. Molecular cloning of two human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzymes that are identical with chlordecone reductase and bile-acid binder.

    PubMed Central

    Deyashiki, Y; Ogasawara, A; Nakayama, T; Nakanishi, M; Miyabe, Y; Sato, K; Hara, A

    1994-01-01

    Human liver contains two dihydrodiol dehydrogenases, DD2 and DD4, associated with 3 alpha-hydroxysteroid dehydrogenase activity. We have raised polyclonal antibodies that cross-reacted with the two enzymes and isolated two 1.2 kb cDNA clones (C9 and C11) for the two enzymes from a human liver cDNA library using the antibodies. The clones of C9 and C11 contained coding sequences corresponding to 306 and 321 amino acid residues respectively, but lacked 5'-coding regions around the initiation codon. Sequence analyses of several peptides obtained by enzymic and chemical cleavages of the two purified enzymes verified that the C9 and C11 clones encoded DD2 and DD4 respectively, and further indicated that the sequence of DD2 had at least additional 16 residues upward from the N-terminal sequence deduced from the cDNA. There was 82% amino acid sequence identity between the two enzymes, indicating that the enzymes are genetic isoenzymes. A computer-based comparison of the cDNAs of the isoenzymes with the DNA sequence database revealed that the nucleotide and amino acid sequences of DD2 and DD4 are virtually identical with those of human bile-acid binder and human chlordecone reductase cDNAs respectively. Images Figure 1 PMID:8172617

  14. Modification of Rhizopus lactate dehydrogenase for improved resistance to fructose 1,6-bisphosphate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhizopus oryzae is frequently used for fermentative production of lactic acid. We determined that one of the key enzymes, lactate dehydrogenase (LDH), involved in synthesis of lactic acid by R. oryzae was significantly inhibited by fructose 1,6-bisphosphate (FBP) at physiological concentrations. Thi...

  15. Relationship of lactate dehydrogenase activity to body measurements of Angus x Charolais cows and calves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives were to examine 1) relationships between lactate dehydrogenase (LDH) activity and body measurements of grazing beef cows, and 2) the association between maternal LDH activity in late gestation and subsequent calf birth weight (BRW), hip height (HH) at weaning, and adjusted weaning weight ...

  16. Relationship of lactate dehydrogenase activity with body measeurements of Angus x Charolais cows and calves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Angus x Charolais cows (n = 87) and their Angus-sired, spring-born calves (n = 86) were utilized to examine relationships between lactate dehydrogenase (LDH) activity and body measurements of beef cows; and the relationship between maternal LDH activity in late gestation and subsequent calf birth we...

  17. Increasing the heme-dependent respiratory efficiency of Lactococcus lactis by inhibition of lactate dehydrogenase.

    PubMed

    Arioli, Stefania; Zambelli, Daniele; Guglielmetti, Simone; De Noni, Ivano; Pedersen, Martin B; Pedersen, Per Dedenroth; Dal Bello, Fabio; Mora, Diego

    2013-01-01

    The discovery of heme-induced respiration in Lactococcus lactis has radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity of L. lactis during growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism. PMID:23064338

  18. Membrane-bound, pyridine nucleotide-independent L-lactate dehydrogenase of Rhodopseudomonas sphaeroides.

    PubMed Central

    Markwell, J P; Lascelles, J

    1978-01-01

    Rhodopseudomonas sphaeroides has a pyridine nucleotide-independent L-lactate dehydrogenase associated with the membrane fraction of cells grown either aerobically or phototrophically. The dehydrogenase is present in cells grown on a variety of carbon sources, but at levels less than 20% of that found in cells grown with DL-lactate. The dehydrogenase has been purified 45-fold from membranes of strain L-57, a non-photosynthetic mutant, by steps involving solubilization with lauryl dimethylamine oxide and three anion-exchange chromatography steps. The purified enzyme was specific for the L-isomer of lactate. The Km of the purified enzyme for L-lactate is 1.4 mM, whereas that of the membrane-associated enzyme is 0.5 mM. The enzyme activity was inhibited competitively by D-lactate and non-competitively by oxalate and oxamate. Quinacrine, a flavin analog, also inhibited the activity. The inducible enzyme may serve as a marker of membrane protein in studies of membrane development. PMID:304854

  19. Inhibition of stress mediated cell death by human lactate dehydrogenase B in yeast.

    PubMed

    Sheibani, Sara; Jones, Natalie K; Eid, Rawan; Gharib, Nada; Arab, Nagla T T; Titorenko, Vladimir; Vali, Hojatollah; Young, Paul A; Greenwood, Michael T

    2015-08-01

    We report the identification of human L- lactate dehydrogenase B (LDHB) as a novel Bax suppressor. Yeast heterologously expressing LDHB is also resistant to the lethal effects of copper indicating that it is a general suppressor of stress mediated cell death. To identify potential LDHB targets, LDHB was expressed in yeast mutants defective in apoptosis, necrosis and autophagy. The absence of functional PCD regulators including MCA1, YBH3, cyclophilin (CPR3) and VMA3, as well as the absence of the pro-survival autophagic pathway (ATG1,7) did not interfere with the LDHB mediated protection against copper indicating that LDHB functions independently of known PCD regulators or by simply blocking or stimulating a common PCD promoting or inhibitory pathway. Measurements of lactate levels revealed that short-term copper stress (1.6 mM, 4 h), does not increase intracellular levels of lactate, instead a three-fold increase in extracellular lactate was observed. Thus, yeast cells resemble mammalian cells where different stresses are known to lead to increased lactate production leading to lactic acidosis. In agreement with this, we found that the addition of exogenous lactic acid to growth media was sufficient to induce cell death that could be inhibited by the expression of LDHB. Taken together our results suggest that lactate dehydrogenase is a general suppressor of PCD in yeast. PMID:26032856

  20. Assessment of freshness and freeze-thawing of sea bream fillets (Sparus aurata) by a cytosolic enzyme: Lactate dehydrogenase.

    PubMed

    Diop, Mamadou; Watier, Denis; Masson, Pierre-Yves; Diouf, Amadou; Amara, Rachid; Grard, Thierry; Lencel, Philippe

    2016-11-01

    The evaluation of freshness and freeze-thawing of fish fillets was carried out by assessment of autolysis of cells using a cytosolic enzyme lactate dehydrogenase. Autolysis plays an important role in spoilage of fish and postmortem changes in fish tissue are due to the breakdown of the cellular structures and release of cytoplasmic contents. The outflow of a cytosolic enzyme, lactate dehydrogenase, was studied in sea bream fillets and the Sparus aurata fibroblasts (SAF-1) cell-line during an 8day storage period at +4°C. A significant increase of lactate dehydrogenase release was observed, especially after 5days of storage. The ratio between the free and the total lactate dehydrogenase activity is a promising predictive marker to measure the quality of fresh fish fillets. The effect of freeze-thawing on cytosolic lactate dehydrogenase and lysosomal α-d-glucosidase activities was also tested. Despite the protecting effect of the tissue compared to the cell-line, a loss of lactate dehydrogenase activity, but not of α-d-glucosidase, was observed. In conclusion, lactate dehydrogenase may be used as a marker to both assess freshness of fish and distinguish between fresh and frozen-thawed fish fillets. PMID:27211667

  1. Metabolic Imaging: A link between Lactate Dehydrogenase A, Lactate and Tumor Phenotype

    PubMed Central

    Thakur, Sunitha B.; Vider, Jelena; Russell, James; Blasberg, Ronald; Koutcher, Jason A.

    2014-01-01

    Purpose We compared the metabolic profiles and the association between LDH-A expression and lactate production in two isogenic murine breast cancer cell lines and tumors (67NR and 4T1). These cell lines were derived from a single mammary tumor and have different growth and metabolic phenotypes. Experimental Design LDH-A expression, lactate concentration, glucose utilization and oxygen consumption were measured in cells, and the potential relationship between tumor lactate levels (measured by magnetic resonance spectroscopic imaging (MRSI)) and tumor glucose utilization (measured by [18F] 2-deoxy-2-fluoro-D-glucose positron emission tomography ([18F]FDG-PET)) was assessed in orthotopic breast tumors derived from these cell lines. Results We show a substantial difference in LDH-A expression between 67NR and 4T1 cells under normoxia and hypoxia. We also show that small orthotopic 4T1 tumors generate tenfold more lactate than corresponding 67NR tumors. The high lactate levels in small primary 4T1 tumors are associated with intense pimonidazole staining (a hypoxia indicator). Less intense hypoxia staining was observed in the larger 67NR tumors, and is consistent with the gradual increase and plateau of lactate concentration in enlarging 67NR tumors. Conclusions Lactate-MRSI has a greater dynamic range than [18F]FDG-PET and may be a more sensitive measure with which to evaluate the aggressive and metastatic potential of primary breast tumors. PMID:21844011

  2. Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus lateralis muscle

    PubMed Central

    Rasmussen, Hans N; van Hall, Gerrit; Rasmussen, Ulla F

    2002-01-01

    The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation procedure were assayed for marker enzymes and lactate dehydrogenase (LDH). The mitochondrial fraction contained no LDH activity (detection limit ∼0.05 % of the tissue activity) and the distribution of LDH activity among the fractions paralleled that of pyruvate kinase, i.e. LDH was fractionated as a cytoplasmic enzyme. Respiratory experiments with the mitochondrial fraction also indicated the absence of LDH. Lactate did not cause respiration, nor did it affect the respiration of pyruvate + malate. The major part of the native cytochrome c was retained in the isolated mitochondria, which, furthermore, showed high specific rates of state 3 respiration. This excluded artificial loss from the mitochondria of all activity of a possible LDH. It was concluded that skeletal muscle mitochondria are devoid of LDH and unable to metabolize lactate. PMID:12042361

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

    PubMed

    Singh, R P; Sastry, K V H; Pandey, N K; Shit, N; Agrawal, R; Singh, K B; Mohan, Jag; Saxena, V K; Moudgal, R P

    2011-02-01

    Lactate dehydrogenase enzyme present in quail seminal plasma has been characterized. Polyacrylamide gel electrophoresis and subsequently with LDH specific staining of seminal plasma revealed a single isozyme in quail semen. Studies on substrate inhibition, pH for optimum activity and inhibitor (urea) indicated the isozyme present in the quail semen has catalytic properties like LDH-1 viz. H-type. Furthermore, unlike other mammalian species, electrophoretic and kinetic investigations did not support the existence of semen specific LDH-X isozyme in quail semen. The effect of exogenous lactate and pyruvate on sperm metabolic activity was also studied. The addition of 1 mM lactate or pyruvate to quail semen increased sperm metabolic activity. Our results suggested that both pyruvate and lactate could be used by quail spermatozoa to maintain their basic functions. Since the H-type isozyme is important for conversion of lactate to pyruvate under anaerobic conditions it was postulated that exogenous lactate being converted into pyruvate via LDH present in semen may be used by sperm mitochondria to generate ATP. During conversion of lactate to pyruvate NADH is being generated that may be useful for maintaining sperm mitochondrial membrane potential. PMID:21074838

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

    PubMed Central

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

    1968-01-01

    1. The preparation of lactate dehydrogenase covalently attached to anion-exchange cellulose particles and sheets by use of a dichloro-sym-triazinyl dyestuff, Procion brilliant orange MGS, is described. 2. The stability and kinetic properties of these preparations were investigated. 3. An equation is derived to describe the change in concentration of a substrate when passed through a uniform bed of a substrate-inhibited enzyme. A number of theoretical curves are shown to illustrate the system. 4. A titrimetric assay for lactate dehydrogenase is described, and shown to be stoicheiometric over the range pH5·0–9·2. 5. The results are discussed in relation to previous work, and the effects of charged groups on the support, and of the diffusion film surrounding any particle in suspension, are treated qualitatively. PMID:5673529

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

    PubMed Central

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

    2013-01-01

    DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2:1 protein:aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer:protein complex crystal structure was solved at 2.1-Å resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson–Crick base-paired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics. PMID:24043813

  6. Cloning and nucleotide sequence of the Lactobacillus casei lactate dehydrogenase gene.

    PubMed Central

    Kim, S F; Baek, S J; Pack, M Y

    1991-01-01

    An allosteric L-(+)-lactate dehydrogenase gene of Lactobacillus casei ATCC 393 was cloned in Escherichia coli, and the nucleotide sequence of the gene was determined. The gene was composed of an open reading frame of 981 bp, starting with a GTG codon and ending with a TAA codon. The sequences for the promoter and ribosome binding site were identified, and a sequence for a structure resembling a rho-independent transcription terminator was also found. Images PMID:1768113

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

    PubMed Central

    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.

    2014-01-01

    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

  8. Clinical research on neuroblastoma based on serum lactate dehydrogenase.

    PubMed

    Pang, Q M; Li, K; Ma, L J; Sun, R P

    2015-01-01

    In recent years, more and more scholars tend to study neuroblastoma (NB) since it possesses increasing morbidity, but lack of effective treatment. This paper aims to investigate variation and clinical significance of the neuron-specific enolase (NSE) and lactic dehydrogenase (LDH) level in serum of children with NB before and after Auto Peripheral Blood Stem Cell Transplantation (APBSCT). A total of 90 children with NB from various hospitals were included in this research, and we analyzed the relationship between levels of NSE and LDH and the change of disease by comparing the two levels before and after APBSCT treatment. The results indicated that the positive rate of NSE in serum was high before treatment, and the levels of NSE and LDH were remarkably higher than those when the treatment was valid; after comprehensive treatment of chemotherapy, excision and radiotherapy, there was a significant difference of NSE and LDH levels in serum between children with complete remission (CR) and those with partial remission (PR); however, no significant differences of NSE and LDH levels were found among children in progressive stage compared to before treatment. It is believed that NSE and LDH levels are associated to the recurrence and treatment effect of NB, proving that both can reflect tumor load, therefore they can be taken as the auxiliary indicators for monitoring curative effects of NB treatment. PMID:25864749

  9. Cloning, nucleotide sequence, and transcriptional analysis of the Pediococcus acidilactici L-(+)-lactate dehydrogenase gene.

    PubMed Central

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

    1995-01-01

    Recombinant plasmids containing the Pediococcus acidilactici L-(+)-lactate dehydrogenase gene (ldhL) were isolated by complementing for growth under anaerobiosis of an Escherichia coli lactate dehydrogenase-pyruvate formate lyase double mutant. The nucleotide sequence of the ldhL gene predicted a protein of 323 amino acids showing significant similarity with other bacterial L-(+)-lactate dehydrogenases and especially with that of Lactobacillus plantarum. The ldhL transcription start points in P. acidilactici were defined by primer extension, and the promoter sequence was identified as TCAAT-(17 bp)-TATAAT. This sequence is closely related to the consensus sequence of vegetative promoters from gram-positive bacteria as well as from E. coli. Northern analysis of P. acidilactici RNA showed a 1.1-kb ldhL transcript whose abundance is growth rate regulated. These data, together with the presence of a putative rho-independent transcriptional terminator, suggest that ldhL is expressed as a monocistronic transcript in P. acidilactici. PMID:7887607

  10. The Archaeoglobus fulgidus d-Lactate Dehydrogenase Is a Zn2+ Flavoprotein

    PubMed Central

    Reed, David W.; Hartzell, Patricia L.

    1999-01-01

    Archaeoglobus fulgidus, a hyperthermophilic, archaeal sulfate reducer, is one of the few organisms that can utilize d-lactate as a sole source for both carbon and electrons. The A. fulgidus open reading frame, AF0394, which is predicted to encode a d-(−)-lactate dehydrogenase (Dld), was cloned, and its product was expressed in Escherichia coli as a fusion with the maltose binding protein (MBP). The 90-kDa MBP-Dld fusion protein was more efficiently expressed in E. coli when coexpressed with the E. coli dnaY gene, encoding the arginyl tRNA for the codons AGA and AGG. When cleaved from the fusion protein by treatment with factor Xa, the recombinant Dld (rDld) has an apparent molecular mass of 50 kDa, similar to that of the native A. fulgidus Dld enzyme. Both the purified MBP-Dld fusion protein and its rDld cleavage fragment have lactate dehydrogenase activities specific for d-lactate, are stable at 80°C, and retain activity after exposure to oxygen. The flavin cofactor FAD, which binds rDld apoprotein with a 1:1 stoichiometry, is essential for activity. PMID:10601217

  11. Identification of lactate dehydrogenase as a mammalian pyrroloquinoline quinone (PQQ)-binding protein

    PubMed Central

    Akagawa, Mitsugu; Minematsu, Kenji; Shibata, Takahiro; Kondo, Tatsuhiko; Ishii, Takeshi; Uchida, Koji

    2016-01-01

    Pyrroloquinoline quinone (PQQ), a redox-active o-quinone, is an important nutrient involved in numerous physiological and biochemical processes in mammals. Despite such beneficial functions, the underlying molecular mechanisms remain to be established. In the present study, using PQQ-immobilized Sepharose beads as a probe, we examined the presence of protein(s) that are capable of binding PQQ in mouse NIH/3T3 fibroblasts and identified five cellular proteins, including l-lactate dehydrogenase (LDH) A chain, as potential mammalian PQQ-binding proteins. In vitro studies using a purified rabbit muscle LDH show that PQQ inhibits the formation of lactate from pyruvate in the presence of NADH (forward reaction), whereas it enhances the conversion of lactate to pyruvate in the presence of NAD+ (reverse reaction). The molecular mechanism underlying PQQ-mediated regulation of LDH activity is attributed to the oxidation of NADH to NAD+ by PQQ. Indeed, the PQQ-bound LDH oxidizes NADH, generating NAD+, and significantly catalyzes the conversion of lactate to pyruvate. Furthermore, PQQ attenuates cellular lactate release and increases intracellular ATP levels in the NIH/3T3 fibroblasts. Our results suggest that PQQ, modulating LDH activity to facilitate pyruvate formation through its redox-cycling activity, may be involved in the enhanced energy production via mitochondrial TCA cycle and oxidative phosphorylation. PMID:27230956

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

    PubMed

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

    2014-12-01

    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

  13. Lactate Dehydrogenase Is the Key Enzyme for Pneumococcal Pyruvate Metabolism and Pneumococcal Survival in Blood

    PubMed Central

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

    2014-01-01

    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

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

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

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

  15. LDH isoenzyme blood test

    MedlinePlus

    ... such as hepatitis Lung tissue death Muscle injury Muscular dystrophy Pancreatitis Lung tissue death Stroke Risks Veins and ... attack Hemolytic anemia Hepatitis Lactate dehydrogenase test Mononucleosis Muscular dystrophy Stroke Update Date 1/31/2015 Updated by: ...

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

    PubMed

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

    2014-01-15

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

  17. Identification of 3,6-disubstituted dihydropyrones as inhibitors of human lactate dehydrogenase.

    PubMed

    Fauber, Benjamin P; Dragovich, Peter S; Chen, Jinhua; Corson, Laura B; Ding, Charles Z; Eigenbrot, Charles; Labadie, Sharada; Malek, Shiva; Peterson, David; Purkey, Hans E; Robarge, Kirk; Sideris, Steve; Ultsch, Mark; Wei, BinQing; Yen, Ivana; Yue, Qin; Zhou, Aihe

    2014-12-15

    A series of 3,6-disubstituted dihydropyrones were identified as inhibitors of human lactate dehydrogenase (LDH)-A. Structure activity relationships were explored and a series of 6,6-spiro analogs led to improvements in LDHA potency (IC50 <350 nM). An X-ray crystal structure of an improved compound bound to human LDHA was obtained and it illustrated additional opportunities to enhance the potency of these compounds, resulting in the identification of 51 (IC50=30 nM). PMID:25467161

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

    PubMed Central

    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

    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 μM, while at 2.5 μM, 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

  19. Different rates of synthesis and degradation of two chloroplastic ammonium-inducible NADP-specific glutamate dehydrogenase isoenzymes during induction and deinduction in Chlorella sorokiniana cells

    SciTech Connect

    Bascomb, N.F.; Prunkard, D.E.; Schmidt, R.R.

    1987-01-01

    The kinetics of accumulation (per milliliter of culture) of the ..cap alpha..- and ..beta..-subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The ..beta..-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the ..cap alpha..-subunit holoenzyme(s). After 120 minutes, the ..cap alpha..-subunit ceased accumulating and thereafter remained at a constant level. From pulse-chase experiments, using /sup 35/SO/sub 4/ and immunochemical procedures, the rate of synthesis of the ..cap alpha..-subunit was shown to be greater than the ..beta..-subunit during the first 80 minutes of induction. The ..cap alpha..- and ..beta..-subunits had different rates of degradation during the induction period (t/sub 1/2/ = 50 versus 150 minutes, respectively) and during the deinduction period (t/sub 1/2/ = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed.

  20. Prostate cancer cells metabolize d-lactate inside mitochondria via a D-lactate dehydrogenase which is more active and highly expressed than in normal cells.

    PubMed

    de Bari, Lidia; Moro, Loredana; Passarella, Salvatore

    2013-03-01

    Although D-lactate metabolism has been shown to occur in a variety of mitochondria, the metabolic fate of D-lactate in cancer cells has never been investigated, as it is believed to be exported to the extracellular phase. We show that mitochondria from both cancer (PC-3) and normal (PNT1A) prostate cells can metabolize D-lactate in an energy competent manner. This is due to the mitochondrial D-lactate dehydrogenase, a membrane flavoprotein, the activity and protein level of which are higher in PC-3 than in PNT1A cells, as detected by both kinetic and immunological analysis. D-Lactate can enter prostate mitochondria and cause the export of newly synthesized malate in a carrier-mediated manner, with the rate of malate efflux from mitochondria twofold higher in cancer. PMID:23333299

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

    PubMed

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

    2014-07-01

    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 2.60Å 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

  2. Novel biohybrids of layered double hydroxide and lactate dehydrogenase enzyme: Synthesis, characterization and catalytic activity studies

    NASA Astrophysics Data System (ADS)

    Djebbi, Mohamed Amine; Braiek, Mohamed; Hidouri, Slah; Namour, Philippe; Jaffrezic-Renault, Nicole; Ben Haj Amara, Abdesslem

    2016-02-01

    The present work introduces new biohybrid materials involving layered double hydroxides (LDH) and biomolecule such as enzyme to produce bioinorganic system. Lactate dehydrogenase (Lac Deh) has been chosen as a model enzyme, being immobilized onto MgAl and ZnAl LDH materials via direct ion-exchange (adsorption) and co-precipitation methods. The immobilization efficiency was largely dependent upon the immobilization methods. A comparative study shows that the co-precipitation method favors the immobilization of great and tunable amount of enzyme. The structural behavior, chemical bonding composition and morphology of the resulting biohybrids were determined by X-ray diffraction (XRD) study, Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM), respectively. The free and immobilized enzyme activity and kinetic parameters were also reported using UV-Visible spectroscopy. However, the modified LDH materials showed a decrease in crystallinity as compared to the unmodified LDH. The change in activity of the immobilized lactate dehydrogenase was considered to be due, to the reduced accessibility of substrate molecules to the active sites of the enzyme and the partial conformational change of the Lac Deh molecules as a result of the immobilization way. Finally, it was proven that there is a correlation between structure/microstructure and enzyme activity dependent on the immobilization process.

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

    PubMed

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

    1988-12-01

    Lactate dehydrogenase-1 (EC 1.1.1.27), 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 125I-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 131I-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. PMID:3197286

  4. Multiplex Fluorescent Immunoassay for Detection of Mice Infected with Lactate Dehydrogenase Elevating Virus

    PubMed Central

    Adams, Veronica; Myles, Matthew H

    2013-01-01

    Commercially available diagnostic tools for the detection of lactate dehydrogenase elevating virus (LDV) infection have been restricted to measurement of serum lactate dehydrogenase (LDH) activity levels and detection of the viral genome by RT-PCR assays. Serologic diagnosis of LDV infection has not been widely adopted due to the belief that the formation of antigen–antibody complexes and B-cell polyclonal activation may confound interpretation of results. In the current study, we inoculated BALB/c, C57BL/6, and Swiss Webster mice with LDV to compare the diagnostic reliability of a commercially available multiplex fluorescent immunoassay for the detection of antiLDV antibodies with that of the LDH enzyme assay. The serologic assay was vastly more sensitive and specific than was the LDH enzyme assay. Moreover, the serologic assay detected antiviral antibodies throughout the 3-mo time course of this study. These results suggest that antigen–antibody complex formation and polyclonal B-cell activation had little effect on assay performance. PMID:23849407

  5. Regulation of lactate dehydrogenase and change of fermentation products in streptococci.

    PubMed Central

    Yamada, T; Carlsson, J

    1975-01-01

    Streptococcus mutans JC 2 produced mainly lactate as a fermentation product when grown in nitrogen-limited continuous culture in the presence of an excess of glucose and produced formate, acetate, and ethanol, but no lactate, under glucose-limited conditions. The levels of lactate dehydrogenase (LDH) in these cultures were of the same order of magnitude, and the activity of LDH was completely dependent on fructose-1,6-diphosphate (FDP). The intracellular level of FDP was high and the level of phosphoenolpyruvate (PEP) was low under the glucose-excess conditions. In the glucose-limited cultures, all glycolytic intermediates studied, except PEP, were low. S. mutans FIL, which had an FDP-independent LDH and similar levels of glycolytic intermediates as S. mutans JC2, produced mainly lactate under glucose-excess or under glucose-limited conditions. LDH of Streptococcus bovis ATCC 9809 was dependent on FDP for activity at a low concentration of pyruvate but had a significant activity without FDP at a high concentration of pyruvate. This strain also produced mainly lactate both under glucose-excess and glucose-limited conditions. The levels and characteristics of these LDHs were not changed by the culture conditions. These results indicate that changes in the intracellular level of FDP regulate LDH activity, which in turn influences the type of fermentation products produced by streptococci. PEP, adenosine 5'-monophosphate, adenosine 5'-diphosphate, and inorganic phosphate significantly inhibited LDH activity from S. mutans JC 2 and may also participate in the regulation of LDH activity in other streptococci. PMID:1176435

  6. 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

    Zammit, V A; Newsholme, E A

    1976-01-01

    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

  7. Myristica fragrans Suppresses Tumor Growth and Metabolism by Inhibiting Lactate Dehydrogenase A.

    PubMed

    Kim, Eun-Yeong; Choi, Hee-Jung; Park, Mi-Ju; Jung, Yeon-Seop; Lee, Syng-Ook; Kim, Keuk-Jun; Choi, Jung-Hye; Chung, Tae-Wook; Ha, Ki-Tae

    2016-01-01

    Most cancer cells predominantly produce ATP by maintaining a high rate of lactate fermentation, rather than by maintaining a comparatively low rate of tricarboxylic acid cycle, i.e., Warburg's effect. In the pathway, the pyruvate produced by glycolysis is converted to lactic acid by lactate dehydrogenase (LDH). Here, we demonstrated that water extracts from the seeds of Myristica fragrans Houtt. (MF) inhibit the in vitro enzymatic activity of LDH. MF effectively suppressed cell growth and the overall Warburg effect in HT29 human colon cancer cells. Although the expression of LDH-A was not changed by MF, both lactate production and LDH activity were decreased in MF-treated cells under both normoxic and hypoxic conditions. In addition, intracellular ATP levels were also decreased by MF treatment, and the uptake of glucose was also reduced by MF treatment. Furthermore, the experiment on tumor growth in the in vivo mice model revealed that MF effectively reduced the growth of allotransplanted Lewis lung carcinoma cells. Taken together, these results suggest that MF effectively inhibits cancer growth and metabolism by inhibiting the activity of LDH, a major enzyme responsible for regulating cancer metabolism. These results implicate MF as a potential candidate for development into a novel drug against cancer through inhibition of LDH activity. PMID:27430914

  8. A detailed investigation of the properties of lactate dehydrogenase in which the 'Essential' cysteine-165 is modified by thioalkylation.

    PubMed Central

    Bloxham, D P; Sharma, R P; Wilton, D C

    1979-01-01

    The reaction of pig heart lactate dehydrogenase with methyl methanethiosulphonate resulted in the modification of one thiol group per protomer, and this was located at cysteine-165 in the enzyme sequence. On reduction, both the thiomethylation of cysteine-165 and any changes in kinetic properties of the enzyme were completely reversed. Cysteine-165 has been considered essential for catalytic activity; however, cysteine-165-thiomethylated dehydrogenase possessed full catalytic activity, although the affinity of the enzyme for carbonyl-or hydroxy-containing substrates was markedly decreased. The nicotinamide nucleotide-binding capacity was unaffected, as judged by the formation of fluorescent complexes with NADH. The enzyme-mediated activation of NAD+, as judged by sulphite addition, was unaffected in thiomethylated lactate dehydrogenase. However, the affinity of oxamate for the enzyme--NADH complex was decreased by 100-fold and it was calculated that this constituted a net increase of 10.4 kJ/mol in the activation energy for binding. Thiomethylated lactate dehydrogenase was able to form an abortive adduct between NAD+ and fluoropyruvate. However, the equilibrium constant for adduct formation between pyruvate and NAD+ was too low to demonstrate this complex at reasonable pyruvate concentrations. A conformational change in the protein structure on selective thiomethylation was revealed by the decreased thermostability of the modified enzyme. The alteration of lactate dehydrogenase catalytic properties on modification depended on the bulk of the reagent used, since thioethylation resulted in an increase in Km for pyruvate (13.5 +/- 3.5 mm) and an 85% decrease in maximum catalytic activity. The implications of all these findings for the catalytic mechanism of lactate dehydrogenase are discussed. PMID:36072

  9. Diammonium phosphate stimulates transcription of L-lactate dehydrogenase leading to increased L-lactate production in the thermotolerant Bacillus coagulans strain.

    PubMed

    Sun, Lifan; Li, Yanfeng; Wang, Limin; Wang, Yanping; Yu, Bo

    2016-08-01

    Exploration of cost-effective fermentation substrates for efficient lactate production is an important economic objective. Although some organic nitrogen sources are also cheaper, inorganic nitrogen salts for lactate fermentation have additional advantages in facilitating downstream procedures and significantly improving the commercial competitiveness of lactate production. In this study, we first established an application of diammonium phosphate to replace yeast extract with a reduced 90 % nitrogen cost for a thermotolerant Bacillus coagulans strain. In vivo enzymatic and transcriptional analyses demonstrated that diammonium phosphate stimulates the gene expression of L-lactate dehydrogenase, thus providing higher specific enzyme activity in vivo and increasing L-lactic acid production. This new information provides a foundation for establishing a cost-effective process for polymer-grade L-lactic acid production in an industrial setting. PMID:26883345

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

    PubMed Central

    2010-01-01

    Aims As one of the five Lactate dehydrogenase (LDH) isoenzymes, LDH5 has the highest efficiency to catalyze pyruvate transformation to lactate. LDH5 overexpression in cancer cells induces an upregulated glycolytic metabolism and reduced dependence on the presence of oxygen. Here we analyzed LDH5 protein expression in a well characterized large cohort of primary lung cancers in correlation to clinico-pathological data and its possible impact on patient survival. Methods Primary lung cancers (n = 269) and non neoplastic lung tissue (n = 35) were tested for LDH5 expression by immunohistochemistry using a polyclonal LDH5 antibody (ab53010). The results of LDH5 expression were correlated to clinico-pathological data as well as to patient's survival. In addition, the results of the previously tested Transketolase like 1 protein (TKTL1) expression were correlated to LDH5 expression. Results 89.5% (n = 238) of NSCLC revealed LDH5 expression whereas LDH5 expression was not detected in non neoplastic lung tissues (n = 34) (p < 0.0001). LDH5 overexpression was associated with histological type (adenocarcinoma = 57%, squamous cell carcinoma = 45%, large cell carcinoma = 46%, p = 0.006). No significant correlation could be detected with regard to TNM-stage, grading or survival. A two sided correlation between the expression of TKTL1 and LDH5 could be shown (p = 0.002) within the overall cohort as well as for each grading and pN group. A significant correlation between LDH5 and TKTL1 within each histologic tumortype could not be revealed. Conclusions LDH5 is overexpressed in NSCLC and could hence serve as an additional marker for malignancy. Furthermore, LDH5 correlates positively with the prognostic marker TKTL1. Our results confirm a close link between the two metabolic enzymes and indicate an alteration in the glucose metabolism in the process of malignant transformation. PMID:20385008

  11. Lactate dehydrogenase as a marker of Plasmodium infection in malaria vector Anopheles.

    PubMed

    Riandey, M F; Sannier, C; Peltre, G; Monteny, N; Cavaleyra, M

    1996-06-01

    Lactate dehydrogenase (Ldh) electrophoresis showed the presence of Plasmodium yoelii yoelii in Anopheles stephensi and An. gambiae. The Ldh appeared as an additional band (pLdh) whose activity was more intense with 3-acetyl pyridine adenine dinucleotide as coenzyme than with beta nicotin-amide adenine dinucleotide. Several allelic forms occurred both in the vector and the host. The isoelectric point of Ldh, similar in the vector and host, differed from those of Ldh from mosquito and mouse. The presence of pLdh was detected from the 2nd to the 28th day of infection. The pLdh appeared to be proportional to the number of sporozoites present in infected salivary glands. However, pLdh was not found in salivary glands or midguts, but it was detected in the rest of the corresponding mosquito. The origin and use of pLdh as a marker of Plasmodium in its vector is discussed. PMID:8827592

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

    PubMed

    Dragovich, Peter S; Fauber, Benjamin P; Boggs, Jason; Chen, Jinhua; Corson, Laura B; Ding, Charles Z; Eigenbrot, Charles; Ge, HongXiu; Giannetti, Anthony M; Hunsaker, Thomas; Labadie, Sharada; Li, Chiho; Liu, Yichin; Liu, Yingchun; Ma, Shuguang; Malek, Shiva; Peterson, David; Pitts, Keith E; Purkey, Hans E; Robarge, Kirk; Salphati, Laurent; Sideris, Steve; Ultsch, Mark; VanderPorten, Erica; Wang, Jing; Wei, BinQing; Xu, Qing; Yen, Ivana; Yue, Qin; Zhang, Huihui; Zhang, Xuying; Zhou, Aihe

    2014-08-15

    A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC50=1.7 μM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.18 μM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure-activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F=45%). PMID:25037916

  13. Targeting a Rate-Promoting Vibration with an Allosteric Mediator in Lactate Dehydrogenase.

    PubMed

    Dzierlenga, Michael W; Schwartz, Steven D

    2016-07-01

    We present a new type of allosteric modulation in which a molecule bound outside the active site modifies the chemistry of an enzymatic reaction through rapid protein dynamics. As a test case for this type of allostery, we chose an enzyme with a well-characterized rate-promoting vibration, lactate dehydrogenase; identified a suitable small molecule for binding; and used transition path sampling to obtain ensembles of reactive trajectories. We found that the small molecule significantly affected the reaction by changing the position of the transition state and, through applying committor distribution analysis, showed that it removed the protein component from the reaction coordinate. The ability of a small-molecule to disrupt enzymatic reactions through alteration of subpicosecond protein motion opens the door for new experimental studies on protein motion coupled to enzymatic reactions and possibly the design of drugs to target these enzymes. PMID:27327209

  14. [THE LEVEL OF LACTATE DEHYDROGENASE AS A MARKER OF RENAL DYSFUNCTION IN NEONATES WITH ASPHYXIA].

    PubMed

    Loboda, A M

    2015-01-01

    The article examines the possibility of determining the level of lactate dehydrogenase (LD) in biological fluids as a marker of renal dysfunction and energy supply in neonates with asphyxia. Investigation included 200 full-term newborns with disturbance kidney function: 100 infants who had severe asphyxia, and 100--with moderate asphyxia. LD activity was determined by kinetic spectrophotometric method. Determination of the activity of LD in the urine in the early neonatal period it is advisable to use as a non-invasive marker for the diagnosis of renal dysfunction in neonates with asphyxia. The content of LD in the blood serum can be used as one of the early markers of kidney damage in newborns with asphyxia. PMID:27491157

  15. Kinetic resolution of 2-hydroxybutanoate racemic mixtures by NAD-independent L-lactate dehydrogenase.

    PubMed

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

    2011-04-01

    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 (0.197 M) 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 at a high concentration (0.193 M). Using a simple ion exchange process with the macroporous anion exchange resin D301, D-2-hydroxybutanoate was separated from the biotransformation system with a high recovery of 84.7%. PMID:21295977

  16. Development of L-lactate dehydrogenase biosensor based on porous silicon resonant microcavities as fluorescence enhancers.

    PubMed

    Jenie, S N Aisyiyah; Prieto-Simon, Beatriz; Voelcker, Nicolas H

    2015-12-15

    The up-regulation of L-lactate dehydrogenase (LDH), an intracellular enzyme present in most of all body tissues, is indicative of several pathological conditions and cellular death. Herein, we demonstrate LDH detection using porous silicon (pSi) microcavities as a luminescence-enhancing optical biosensing platform. Non-fluorescent resazurin was covalently attached onto the pSi surface via thermal hydrocarbonisation, thermal hydrosylilation and acylation. Each surface modification step was confirmed by means of FTIR and the optical shifts of the resonance wavelength of the microcavity. Thermal hydrocarbonisation also afforded excellent surface stability, ensuring that the resazurin was not reduced on the pSi surface. Using a pSi microcavity biosensor, the fluorescence signal upon detection of LDH was amplified by 10 and 5-fold compared to that of a single layer and a detuned microcavity, respectively, giving a limit of detection of 0.08 U/ml. The biosensor showed a linear response between 0.16 and 6.5 U/ml, covering the concentration range of LDH in normal as well as damaged tissues. The biosensor was selective for LDH and did not produce a signal upon incubation with another NAD-dependant enzyme L-glutamic dehydrogenase. The use of the pSi microcavity as a sensing platform reduced reagent usage by 30% and analysis time threefold compared to the standard LDH assay in solution. PMID:26201980

  17. Surface modification of silicon dioxide, silicon nitride and titanium oxynitride for lactate dehydrogenase immobilization.

    PubMed

    Saengdee, Pawasuth; Chaisriratanakul, Woraphan; Bunjongpru, Win; Sripumkhai, Witsaroot; Srisuwan, Awirut; Jeamsaksiri, Wutthinan; Hruanun, Charndet; Poyai, Amporn; Promptmas, Chamras

    2015-05-15

    Three different types of surface, silicon dioxide (SiO2), silicon nitride (Si3N4), and titanium oxynitride (TiON) were modified for lactate dehydrogenase (LDH) immobilization using (3-aminopropyl)triethoxysilane (APTES) to obtain an amino layer on each surface. The APTES modified surfaces can directly react with LDH via physical attachment. LDH can be chemically immobilized on those surfaces after incorporation with glutaraldehyde (GA) to obtain aldehyde layers of APTES-GA modified surfaces. The wetting properties, chemical bonding composition, and morphology of the modified surface were determined by contact angle (CA) measurement, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM), respectively. In this experiment, the immobilized protein content and LDH activity on each modified surface was used as an indicator of surface modification achievement. The results revealed that both the APTES and APTES-GA treatments successfully link the LDH molecule to those surfaces while retaining its activity. All types of tested surfaces modified with APTES-GA gave better LDH immobilizing efficiency than APTES, especially the SiO2 surface. In addition, the SiO2 surface offered the highest LDH immobilization among tested surfaces, with both APTES and APTES-GA modification. However, TiON and Si3N4 surfaces could be used as alternative candidate materials in the preparation of ion-sensitive field-effect transistor (ISFET) based biosensors, including lactate sensors using immobilized LDH on the ISFET surface. PMID:25108848

  18. Epitopes of human testis-specific lactate dehydrogenase deduced from a cDNA sequence

    SciTech Connect

    Millan, J.L.; Driscoll, C.E.; LeVan, K.M.; Goldberg, E.

    1987-08-01

    The sequence and structure of human testis-specific L-lactate dehydrogenase (LDHC/sub 4/, LDHX; (L)-lactate:NAD/sup +/ oxidoreductase, EC 1.1.1.27) has been derived from analysis of a complementary DNA (cDNA) clone comprising the complete protein coding region of the enzyme. From the deduced amino acid sequence, human LDHC/sub 4/ is as different from rodent LDHC/sub 4/ (73% homology) as it is from human LDHA/sub 4/ (76% homology) and porcine LDHB/sub 4/ (68% homology). Subunit homologies are consistent with the conclusion that the LDHC gene arose by at least two independent duplication events. Furthermore, the lower degree of homology between mouse and human LDHC/sub 4/ and the appearance of this isozyme late in evolution suggests a higher rate of mutation in the mammalian LDHC genes than in the LDHA and -B genes. Comparison of exposed amino acid residues of discrete anti-genic determinants of mouse and human LDHC/sub 4/ reveals significant differences. Knowledge of the human LDHC/sub 4/ sequence will help design human-specific peptides useful in the development of a contraceptive vaccine.

  19. Theoretical site-directed mutagenesis: Asp168Ala mutant of lactate dehydrogenase

    PubMed Central

    Ferrer, Silvia; Tuñón, Iñaki; Moliner, Vicent; Williams, Ian H.

    2008-01-01

    Molecular simulations based on the use of hybrid quantum mechanics/molecular mechanics methods are able to provide detailed information about the complex enzymatic reactions and the consequences of specific mutations on the activity of the enzyme. In this work, the reduction of pyruvate to lactate catalysed by wild-type and Asp168Ala mutant lactate dehydrogenase (LDH) has been studied by means of simulations using a very flexible molecular model consisting of the full tetramer of the enzyme, together with the cofactor NADH, the substrate and solvent water molecules. Our results indicate that the Asp168Ala mutation provokes a shift in the pKa value of Glu199 that becomes unprotonated at neutral pH in the mutant enzyme. This change compensates the loss of the negative charge of Asp168, rendering a still active enzyme. Thus, our methodology gives a calculated barrier height for the Asp168Ala mutant 3 kcal mol−1 higher than that for wild-type LDH, which is in very good agreement with the experiment. The computed potential energy surfaces reveal the reaction pathways and transition structures for the wild-type and mutant enzymes. Hydride transfer is less advanced and the proton transfer is more advanced in the Asp168Ala mutant than in the wild type. This approach provides a very powerful tool for the analysis of the roles of key active-site residues. PMID:18682365

  20. Lactate dehydrogenase-A inhibition induces human glioblastoma multiforme stem cell differentiation and death

    PubMed Central

    Daniele, Simona; Giacomelli, Chiara; Zappelli, Elisa; Granchi, Carlotta; Trincavelli, Maria Letizia; Minutolo, Filippo; Martini, Claudia

    2015-01-01

    Therapies that target the signal transduction and metabolic pathways of cancer stem cells (CSCs) are innovative strategies to effectively reduce the recurrence and significantly improve the outcome of glioblastoma multiforme (GBM). CSCs exhibit an increased rate of glycolysis, thus rendering them intrinsically more sensitive to prospective therapeutic strategies based on the inhibition of the glycolytic pathway. The enzyme lactate dehydrogenase-A (LDH-A), which catalyses the interconversion of pyruvate and lactate, is up-regulated in human cancers, including GBM. Although several papers have explored the benefits of targeting cancer metabolism in GBM, the effects of direct LDH-A inhibition in glial tumours have not yet been investigated, particularly in the stem cell subpopulation. Here, two representative LDH-A inhibitors (NHI-1 and NHI-2) were studied in GBM-derived CSCs and compared to differentiated tumour cells. LDH-A inhibition was particularly effective in CSCs isolated from different GBM cell lines, where the two compounds blocked CSC formation and elicited long-lasting effects by triggering both apoptosis and cellular differentiation. These data demonstrate that GBM, particularly the stem cell subpopulation, is sensitive to glycolytic inhibition and shed light on the therapeutic potential of LDH-A inhibitors in this tumour type. PMID:26494310

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

    PubMed Central

    2015-01-01

    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

  2. Free energy landscape of the Michaelis complex of lactate dehydrogenase: A network analysis of atomistic simulations

    NASA Astrophysics Data System (ADS)

    Pan, Xiaoliang; Schwartz, Steven

    2015-03-01

    It has long been recognized that the structure of a protein is a hierarchy of conformations interconverting on multiple time scales. However, the conformational heterogeneity is rarely considered in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD+). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they are catalytic competent at different reaction rates. In this study, millisecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network the Michaelis complex and the structures of the substates at atomistic scale. It also shed some light on understanding the complete picture of the catalytic mechanism of LDH.

  3. Addressable self-immobilization of lactate dehydrogenase across multiple length scales.

    PubMed

    Cetinel, Sibel; Caliskan, H Burak; Yucesoy, Deniz T; Donatan, A Senem; Yuca, Esra; Urgen, Mustafa; Karaguler, Nevin G; Tamerler, Candan

    2013-02-01

    Successful nanobiotechnology implementation largely depends on control over the interfaces between inorganic materials and biological molecules. Controlling the orientations of biomolecules and their spatial arrangements on the surface may transform many technologies including sensors, to energy. Here, we demonstrate the self-organization of L-lactate dehydrogenase (LDH), which exhibits enhanced enzymatic activity and stability on a variety of gold surfaces ranging from nanoparticles to electrodes, by incorporating a gold-binding peptide tag (AuBP2) as the fusion partner for Bacillus stearothermophilus LDH (bsLDH). Binding kinetics and enzymatic assays verified orientation control of the enzyme on the gold surface through the genetically incorporated peptide tag. Finally, redox catalysis efficiency of the immobilized enzyme was detected using cyclic voltammetry analysis in enzyme-based biosensors for lactate detection as well as in biofuel cell energy systems as the anodic counterpart. Our results demonstrate that the LDH enzyme can be self-immobilized onto different gold substrates using the short peptide tag under a biologically friendly environment. Depending on the desired inorganic surface, the proposed peptide-mediated path could be extended to any surface to achieve single-step oriented enzyme immobilization for a wide range of applications. PMID:23386458

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

    PubMed

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

    2013-06-01

    The objective of the present study was to study the characteristics of lactate dehydrogenase (LDH) from Hypoderma sinense larva. H. sinense larvae were collected from yak (Bos grunniens) and identified by a PCR-RFLP method. Analysis of LDH activity showed that the total LDH activity in H. sinense larva was negatively correlated with the length of larva. Polyacrylamide gel electrophoresis of the extracts of H. sinense larvae revealed one band of LDH, which was then purified by affinity chromatography and gel filtration. This enzyme showed an approximately 36 kDa band on SDS-gel under both reducing and non-reducing conditions, in addition, size exclusion chromatography analysis showed that its molecular weight was smaller than bovine serum albumin (67 kDa), indicating that it contains only one subunit. Michaelis constants (Km) values assay revealed that LDH from H. sinense larva showed significantly lower Km for lactate than other animals. LDH of H. sinense larva was stable at 60 °C for 15 min, and also exhibited high catalytic efficiency in a wide range of pH. HgCl₂ at the concentration of 0.1mM significantly decreased the activity of LDH from H. sinense larva but not at the concentration of 0.01 mM. The results of the present study demonstrate that LDH from H. sinense larva is a thermal stable and pH insensitive enzyme suitable for catalyzing both forward and reverse reactions. PMID:23474203

  5. Energy Landscape of the Michaelis Complex of Lactate Dehydrogenase: Relationship to Catalytic Mechanism

    PubMed Central

    2015-01-01

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

  6. The contribution of lactic acid to acidification of tumours: studies of variant cells lacking lactate dehydrogenase.

    PubMed Central

    Yamagata, M.; Hasuda, K.; Stamato, T.; Tannock, I. F.

    1998-01-01

    Solid tumours develop an acidic extracellular environment with high concentration of lactic acid, and lactic acid produced by glycolysis has been assumed to be the major cause of tumour acidity. Experiments using lactate dehydrogenase (LDH)-deficient ras-transfected Chinese hamster ovarian cells have been undertaken to address directly the hypothesis that lactic acid production is responsible for tumour acidification. The variant cells produce negligible quantities of lactic acid and consume minimal amounts of glucose compared with parental cells. Lactate-producing parental cells acidified lightly-buffered medium but variant cells did not. Tumours derived from parental and variant cells implanted into nude mice were found to have mean values of extracellular pH (pHe) of 7.03 +/- 0.03 and 7.03 +/- 0.05, respectively, both of which were significantly lower than that of normal muscle (pHe = 7.43 +/- 0.03; P < 0.001). Lactic acid concentration in variant tumours (450 +/- 90 microg g(-1) wet weight) was much lower than that in parental tumours (1880 +/- 140 microg/g(-1)) and similar to that in serum (400 +/- 35 microg/g(-1)). These data show discordance between mean levels of pHe and lactate content in tumours; the results support those of Newell et al (1993) and suggest that the production of lactic acid via glycolysis causes acidification of culture medium, but is not the only mechanism, and is probably not the major mechanism responsible for the development of an acidic environment within solid tumours. PMID:9667639

  7. Development of an enzymatic assay system of D-lactate using D-lactate dehydrogenase and a UV-LED fluorescent spectrometer.

    PubMed

    Chen, Chien-Ming; Chen, Shih-Ming; Chien, Po-Jen; Yu, Han-Yin

    2015-12-10

    In this study, we aimed to develop a new enzymatic assay system of d-lactate with good precision, accuracy, and sensitivity for the determination of D-lactate concentrations in rat serum. D-Lactate dehydrogenase (D-LDH) was utilized to catalyze D-lactate and NAD(+) to pyruvate and NADH, respectively. The generated NADH was excited by using a 340-nm UV-light-emitting diode (LED), and the fluorescence at 491 nm was detected to determine the concentration of D-lactate in rat serum. The optics, consisting of the sample cuvette, were set on three-dimensional stages to receive the most intensive fluorescence signal into the spectrometer. The optimal conditions of the D-LDH reaction were pH 8.5 and 25 °C for 90 min. The results showed that the new D-lactate assay system had good linearity (R(2)=0.9964) in the calibration range from 5 to 150 μM. Intra-day and inter-day accuracies were in the range of 103.96-109.09% and 102.84-104.59%, respectively, and the intra-day and inter-day precision was 4.28-6.82% and 4.04-12.40%, respectively. Finally, serum D-lactate concentrations determined by the proposed enzymatic assay system were compared with those obtained by a conventional HPLC method. The newly developed D-lactate assay system could detect 10-15 samples in 90 min, whereas the HPLC method could detect only one sample over the same time period. PMID:26265307

  8. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

    SciTech Connect

    Fenglei Li

    2006-08-09

    The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties. Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed in this system to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme has capability of eliminating common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening. Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. In this crystallization method, a gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. In addition

  9. Prognostic significance of pretreated serum lactate dehydrogenase level in nasopharyngeal carcinoma among Chinese population

    PubMed Central

    Zhang, Mingwei; Wei, Shushan; Su, Li; Lv, Wenlong; Hong, Jinsheng

    2016-01-01

    Abstract Background: A large number of studies have investigated the prognostic value of pretreated lactate dehydrogenase (LDH) level in nasopharyngeal carcinoma (NPC) patients while the role of it was inconsistent and inconclusive. Hence, the aim of the current study was to conduct a meta-analysis of all published studies to quantify the prognostic impact of pretreated serum LDH in NPC for Chinese population. Objectives: The aim of the current study was to conduct a meta-analysis of all published studies to quantify the prognostic impact of pretreated serum lactate dehydrogenase (LDH) in nasopharyngeal carcinoma (NPC) for Chinese population. Methods: The PubMed, Medline, Embase, and Web of Science databases were searched for studies that assessed survival outcome and LDH in NPC. Overall survival (OS) was the primary survival outcome. Distant metastasis-free survival (DMFS) and disease-free survival (DFS) were secondary outcomes. The pooled hazard ratios (HRs), associated with 95% confidence intervals (95% CIs), were combined to calculate overall effects. The Cochran Q and I2 statistics were used to assess heterogeneity. When apparent heterogeneity was observed, sensitivity and meta-regression analyses were performed to explore its origin. Results: Sixteen studies, which included 14,803 patients, were enrolled in the current meta-analysis to yield statistics. Overall, the pooled HR for OS in 11 eligible studies with high LDH level was 1.79 (95% CI = 1.47–2.12), and the pooled HR for DMFS in 9 eligible studies with high LDH level was 1.85 (95% CI = 1.48–2.22). Meanwhile, the pooled HR for DFS in 5 eligible studies with high LDH level was 1.63 (95% CI = 1.34–1.91). Egger test and funnel plots revealed that the publication bias in the current meta-analysis was insignificant. Conclusions: The present meta-analysis demonstrated that high pretreated LDH level is significantly associated with poorer OS, DMFS, and DFS, suggesting that pretreated LDH could

  10. The drs tumor suppressor regulates glucose metabolism via lactate dehydrogenase-B.

    PubMed

    Tambe, Yukihiro; Hasebe, Masahiro; Kim, Chul Jang; Yamamoto, Akitsugu; Inoue, Hirokazu

    2016-01-01

    Previously, we showed that drs contributes to suppression of malignant tumor formation in drs-knockout (KO) mice. In this study, we demonstrate the regulation of glucose metabolism by drs using comparisons of drs-KO and wild-type (WT) mouse embryonic fibroblasts (MEFs). Extracellular acidification, lactate concentration, and glucose consumption in drs-KO cells were significantly greater than those in WT cells. Metabolomic analyses also confirmed enhanced glycolysis in drs-KO cells. Among glycolysis-regulating proteins, expression of lactate dehydrogenase (LDH)-B was upregulated at the post-transcriptional level in drs-KO cells and increased LDH-B expression, LDH activity, and acidification of culture medium in drs-KO cells were suppressed by retroviral rescue of drs, indicating that LDH-B plays a critical role for glycolysis regulation mediated by drs. In WT cells transformed by activated K-ras, expression of endogenous drs mRNA was markedly suppressed and LDH-B expression was increased. In human cancer cell lines with low drs expression, LDH-B expression was increased. Database analyses also showed the correlation between downregulation of drs and upregulation of LDH-B in human colorectal cancer and lung adenocarcinoma tissues. Furthermore, an LDH inhibitor suppressed anchorage-independent growth of human cancer cells and MEF cells transformed by activated K-ras. These results indicate that drs regulates glucose metabolism via LDH-B. Downregulating drs may contribute to the Warburg effect, which is closely associated with malignant progression of cancer cells. PMID:25620379

  11. Hypoxically inducible barley lactate dehydrogenase: cDNA cloning and molecular analysis

    SciTech Connect

    Hondred, D. ); Hanson, A.D. Univ. de Montreal, Quebec )

    1990-09-01

    In the roots of barley and other cereals, hypoxia induces a set of five isozymes of L-lactate dehydrogenase (LDH; (S)-lactate:NADH oxidoreductase, EC 1.1.1.27). Biochemical and genetic data indicate that the five LDH isozymes are tetramers that arise from random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH were isolated from a {lambda}gt11 cDNA library derived from hypoxically treated barley roots. The library was screened with antiserum raised against barley LDH purified {approx}3,000-fold by an improved three-step procedure. Immunopositive clones were rescreened with a cDNA probe synthesized by the polymerase chain reaction using primers modeled from the amino acid sequences of two tryptic LDH peptides. Two types of LDH clones were found. Nucleotide sequence analysis of one representative insert of each type (respectively, 1,305 and 1,166 base pairs) revealed open reading framed encoding 10 peptide fragments of LDH. The 1,305-base-pair insert included the entire coding region of a 356-residue LDH monomer. The nucleotide sequences of the two LDH cDNAs were 92% identical in the coding region, but highly divergent in the 3{prime} noncoding region, and thus probably correspond to the two postulated Ldh loci. The deduced amino acid sequences of the two barley LDHs were 96% identical to each other and very similar to those from vertebrate and bacterial LDHs. RNA blot hybridization showed a single mRNA band of 1.5 kilobases whose level rose about 8-fold in roots during hypoxic induction, as did the level of translatable LDH message.

  12. Gradual neofunctionalization in the convergent evolution of trichomonad lactate and malate dehydrogenases

    PubMed Central

    Steindel, Phillip A.; Chen, Emily H.; Wirth, Jacob D.

    2016-01-01

    Abstract Lactate and malate dehydrogenases (LDH and MDH) are homologous, core metabolic enzymes common to nearly all living organisms. LDHs have evolved convergently from MDHs at least four times, achieving altered substrate specificity by a different mechanism each time. For instance, the LDH of anaerobic trichomonad parasites recently evolved independently from an ancestral trichomonad MDH by gene duplication. LDH plays a central role in trichomonad metabolism by catalyzing the reduction of pyruvate to lactate, thereby regenerating the NAD+ required for glycolysis. Using ancestral reconstruction methods, we identified the biochemical and evolutionary mechanisms responsible for this convergent event. The last common ancestor of these enzymes was a highly specific MDH, similar to modern trichomonad MDHs. In contrast, the LDH lineage evolved promiscuous activity by relaxing specificity in a gradual process of neofunctionalization involving one highly detrimental substitution at the “specificity residue” (R91L) and many additional mutations of small effect. L91 has different functional consequences in LDHs and in MDHs, indicating a prominent role for epistasis. Crystal structures of modern‐day and ancestral enzymes show that the evolution of substrate specificity paralleled structural changes in dimerization and α‐helix orientation. The relatively small “specificity residue” of the trichomonad LDHs can accommodate a range of substrate sizes and may permit solvent to access the active site, both of which promote substrate promiscuity. The trichomonad LDHs present a multi‐faceted counterpoint to the independent evolution of LDHs in other organisms and illustrate the diverse mechanisms by which protein function, structure, and stability coevolve. PMID:26889885

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

    PubMed

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

    2014-09-01

    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

  14. Purification and determination of the binding site of lactate dehydrogenase from chicken breast muscle on immobilized ferric ions.

    PubMed

    Chaga, G; Andersson, L; Porath, J

    1992-12-25

    Lactate dehydrogenase from chicken breast muscle was purified to homogeneity in one step by immobilized metal ion affinity chromatography. The purified enzyme was used to localize the binding site to immobilized Fe(III) ions. After cyanogen bromide degradation and digestion with trypsin, small enzyme fragments capable of binding to immobilized Fe(III) ions were obtained. It is proposed that several histidyl groups are involved in the binding. PMID:1487526

  15. Common and rare variants associating with serum levels of creatine kinase and lactate dehydrogenase

    PubMed Central

    Kristjansson, Ragnar P.; Oddsson, Asmundur; Helgason, Hannes; Sveinbjornsson, Gardar; Arnadottir, Gudny A.; Jensson, Brynjar O.; Jonasdottir, Aslaug; Jonasdottir, Adalbjorg; Bragi Walters, G.; Sulem, Gerald; Oskarsdottir, Arna; Benonisdottir, Stefania; Davidsson, Olafur B.; Masson, Gisli; Th Magnusson, Olafur; Holm, Hilma; Sigurdardottir, Olof; Jonsdottir, Ingileif; Eyjolfsson, Gudmundur I.; Olafsson, Isleifur; Gudbjartsson, Daniel F.; Thorsteinsdottir, Unnur; Sulem, Patrick; Stefansson, Kari

    2016-01-01

    Creatine kinase (CK) and lactate dehydrogenase (LDH) are widely used markers of tissue damage. To search for sequence variants influencing serum levels of CK and LDH, 28.3 million sequence variants identified through whole-genome sequencing of 2,636 Icelanders were imputed into 63,159 and 98,585 people with CK and LDH measurements, respectively. Here we describe 13 variants associating with serum CK and 16 with LDH levels, including four that associate with both. Among those, 15 are non-synonymous variants and 12 have a minor allele frequency below 5%. We report sequence variants in genes encoding the enzymes being measured (CKM and LDHA), as well as in genes linked to muscular (ANO5) and immune/inflammatory function (CD163/CD163L1, CSF1, CFH, HLA-DQB1, LILRB5, NINJ1 and STAB1). A number of the genes are linked to the mononuclear/phagocyte system and clearance of enzymes from the serum. This highlights the variety in the sources of normal diversity in serum levels of enzymes. PMID:26838040

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

    PubMed

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

    2014-08-01

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

  17. Evaluation of antibody-dependent cell cytotoxicity using lactate dehydrogenase (LDH) measurement.

    PubMed

    Broussas, Matthieu; Broyer, Lucile; Goetsch, Liliane

    2013-01-01

    In order to improve therapeutic antibodies efficacy in cancer patients, several strategies were developed. One of these strategies consists in the enhancement of effector functions. Antibody-dependent cellular cytotoxicity (ADCC) was shown to mediate the activity of several therapeutic antibodies through interaction of the constant fragment (Fc) with immune cells. The interactions of Fc fragment can be modulated by engineering through modifications of the carbohydrate moieties or through modifications of some critical amino acids for its binding. Such modifications have to be studied in an in vitro assay to evaluate their impact on the regulation of effector functions. Here, we described a method to evaluate ADCC using a nonradioactive assay based on the measurement of lactate dehydrogenase (LDH) release. NK cells were purified by negative immunomagnetic selection and used as effector cells to trigger ADCC against specific target tumor cells. The LDH release measurement from lysed cells is performed after 4 h incubation. This method can replace the (51)Cr release assay since it is less restrictive and highly sensitive. PMID:23475728

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

    PubMed Central

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

    2014-01-01

    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

  19. Evaluation on the inhibition of pyrrol-2-yl ethanone derivatives to lactate dehydrogenase and anticancer activities

    NASA Astrophysics Data System (ADS)

    Lu, Na-Na; Weng, Zhao-Yue; Chen, Qiu-Yun; Boison, Daniel; Xiao, Xin-Xin; Gao, Jing

    2016-08-01

    Lactate dehydrogenase A (LDH-A) is a potentially important metabolic target for the inhibition of the highly activated glycolysis pathway in cancer cells. In order to develop bifunctional compounds as inhibitor of LDH-A and anticancer agents, two pyrrol-2-yl methanone (or ethanone) derivatives (PM1 and PM2) were synthesized and evaluated as inhibitors of LDH-A based on the enzyme assay and cell assay by spectroscopy analysis. Fluorescence and CD spectra results demonstrated that both the change of second structure of LDH-A and the affinity interaction for compounds to LDH-A gave great effect on the activity of LDH-A. In particular, low concentration of compounds (1 μμ-25 μμ) could change the level of pyruvate in cancer cells. Moreover, the in vitro assay results demonstrated that pyrrol-2-yl ethanone derivatives can inhibit the proliferation of cancer cells. Therefore, pyrrol-2-yl ethanone derivatives (PM2) can be both LDH-A inhibitor and anticancer agents.

  20. Lactate Dehydrogenase Is an Important Prognostic Indicator for Hepatocellular Carcinoma after Partial Hepatectomy12

    PubMed Central

    Zhang, Jing-Ping; Wang, Hong-Bo; Lin, Yue-Hao; Xu, Jing; Wang, Jun; Wang, Kai; Liu, Wan-Li

    2015-01-01

    Preoperative serum lactate dehydrogenase (LDH) has been used as a prognostic indicator for patients with hepatocellular carcinoma (HCC) treated with sorafenib or undergoing transcatheter arterial chemoembolization, but its significance in predicting survival of HCC patients who received curative resection remains undefined. A total of 683 patients with histopathologically confirmed HCC were enrolled in this study. The prognostic significance of preoperative serum LDH was determined by Kaplan-Meier analysis and a Cox proportional hazards regression model. The association between the preoperative serum LDH and clinicopathological parameters was evaluated by the χ2 test or linear regression analysis when appropriate. Higher preoperative serum LDH level was associated with worse prognosis. In a multivariate Cox proportional hazards analysis, the preoperative serum LDH level could predict overall survival and recurrence independently. Higher preoperative serum LDH level is associated with the elevated serum alpha-fetoprotein, the presence of hepatitis B surface antigen, larger tumor size, the presence of macrovascular invasion, the advanced tumor–lymph node–metastasis stage, worse tumor differentiation, and Child-Pugh B. Preoperative serum LDH level was an inexpensive, simple, convenient, and routinely measured biomarker exhibiting a potential to select patients at high risk with poor clinical outcome for appropriate treatment strategies. PMID:26692531

  1. A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure.

    PubMed

    Clarke, A R; Wigley, D B; Barstow, D A; Chia, W N; Atkinson, T; Holbrook, J J

    1987-05-27

    We have engineered a variant of the lactate dehydrogenase enzyme from Bacillus stearothermophilus in which arginine-173 at the proposed regulatory site has been replaced by glutamine. Like the wild-type enzyme, this mutant undergoes a reversible, protein-concentration-dependent subunit assembly, from dimer to tetramer. However, the mutant tetramer is much more stable (by a factor of 400) than the wild type and is destabilized rather than stabilized by binding the allosteric regulator, fructose 1,6-biphosphate (Fru-1,6-P2). The mutation has not significantly changed the catalytic properties of the dimer (Kd NADH, Km pyruvate, Ki oxamate and kcat), but has weakened the binding of Fru-1,6-P2 to both the dimeric and tetrameric forms of the enzyme and has almost abolished any stimulatory effect. We conclude that the Arg-173 residue in the wild-type enzyme is directly involved in the binding of Fru-1,6-P2, is important for allosteric communication with the active site, and, in part, regulates the state of quaternary structure through a charge-repulsion mechanism. PMID:3580377

  2. [Expression, purification and characterization of a thermostable lactate dehydrogenase from Thermotoga maritima].

    PubMed

    Qian, Guojun; Chen, Caiping; Zhai, Ruying; Shao, Weilan; Mei, Yanzhen

    2014-04-01

    The gene encoding thermostable lactate dehydrogenase (Tm-LDH) was cloned into the plasmid pHsh from Thermotoga maritima, and expressed in Escherichia coli JM 109. The recombinant protein was purified to homogeneity by a simple step, heat treatment. The recombinant enzyme had a molecular mass of 33 kDa. The optimal temperature and pH of Tm-LDH were observed 95 degrees C and 7.0. The purified enzyme had a half-life of 2 h at 90 degrees C, and exhibited better stability over a pH range from 5.5 to 8.0. The K(m) and V(max) values were 1.7 mmol/L, 3.8 x 10(4) U/mg of protein for pyruvate, and 7.2 mmol/L and 1.1 x 10(5) U/mg for NADH, respectively. The expression of Tm-LDH in T7 system could not obtain high efficiency, but it has been soluble over-expression in pHsh system and reached 340 mg/L. The superior stability and productivity of Tm-LDH will lay the foundation of its industrial-scale fermentation and application in the NAD regeneration. PMID:25195245

  3. Immunomagnetic capture and colorimetric detection of malarial biomarker Plasmodium falciparum lactate dehydrogenase.

    PubMed

    Markwalter, Christine F; Davis, Keersten M; Wright, David W

    2016-01-15

    We report a sensitive, magnetic bead-based colorimetric assay for Plasmodium falciparum lactate dehydrogenase (PfLDH) in which the biomarker is extracted from parasitized whole blood and purified based on antigen binding to antibody-functionalized magnetic particles. Antigen-bound particles are washed, and PfLDH activity is measured on-bead using an optimized colorimetric enzyme reaction (limit of detection [LOD] = 21.1 ± 0.4 parasites/μl). Enhanced analytical sensitivity is achieved by removal of PfLDH from the sample matrix before detection and elimination of nonspecific reductases and species that interfere with the optimal detection wavelength for measuring assay development. The optimized assay represents a simple and effective diagnostic strategy for P. falciparum malaria with time-to-result of 45 min and detection limits similar to those of commercial enzyme-linked immunosorbent assay (ELISA) kits, which can take 4-6 h. This method could be expanded to detect all species of malaria by switching the capture antibody on the magnetic particles to a pan-specific Plasmodium LDH antibody. PMID:26475567

  4. Elevation of serum l-lactate dehydrogenase B correlated with the clinical stage of lung cancer.

    PubMed

    Chen, Yue; Zhang, Hao; Xu, Anjian; Li, Na; Liu, Jifu; Liu, Chuanjun; Lv, Dongxia; Wu, Shanshan; Huang, Lingyun; Yang, Shuanying; He, Dacheng; Xiao, Xueyuan

    2006-10-01

    To identify potential biomarkers related with lung cancer metastasis, conditional media (CM) proteins collected from a primary non-small cell lung cancer (NSCLC) cell line NCI-H226 and its brain metastatic subline H226Br were analyzed by one-dimensional electrophoresis (1-D PAGE) and matrix-assisted laser desorption/time of flight mass spectrometry (MALDI-TOF-MS). Twelve biomarkers were identified, of which l-lactate dehydrogenase B (LDHB) chain was significantly up-regulated in the CM of H226Br cell and was further validated in 105 lung cancer, 93 non-lung cancer, 41 benign lung disease, as well as 65 healthy individuals sera using enzyme-linked immunosorbent assay (ELISA). It was found that the levels of LDHB were specifically elevated in NSCLC sera compared with other groups and were progressively increased with the clinical stage. At the cutoff point 0.260 (OD value) on the receiver operating characteristic (ROC) curve, LDHB could comparatively discriminate lung cancer from benign lung disease and healthy control groups with sensitivity 81%, specificity 70% and total accuracy 76%. These findings demonstrated that secretome could open up a possibility to find, identify, and characterize novel biomarkers related with invasion and metastasis. PMID:16890323

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

    PubMed

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

    2009-11-01

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

  6. Active-Loop Dynamics within the Michaelis Complex of Lactate Dehydrogenase from Bacillus stearothermophilus.

    PubMed

    Nie, Beining; Lodewyks, Kara; Deng, Hua; Desamero, Ruel Z B; Callender, Robert

    2016-07-12

    Laser-induced temperature-jump relaxation spectroscopy was used to study the active site mobile-loop dynamics found in the binding of the NADH nucleotide cofactor and oxamate substrate mimic to lactate dehydrogenase in Bacillus stearothermophilus thermophilic bacteria (bsLDH). The kinetic data can be best described by a model in which NADH can bind only to the open-loop apoenzyme, oxamate can bind only to the bsLDH·NADH binary complex in the open-loop conformation, and oxamate binding is followed by closing of the active site loop preventing oxamate unbinding. The open and closed states of the loop are in dynamic equilibrium and interconvert on the submillisecond time scale. This interconversion strongly accelerates with an increase in temperature because of significant enthalpy barriers. Binding of NADH to bsLDH results in minor changes of the loop dynamics and does not shift the open-closed equilibrium, but binding of the oxamate substrate mimic shifts this equilibrium to the closed state. At high excess oxamate concentrations where all active sites are nearly saturated with the substrate mimic, all active site mobile loops are mainly closed. The observed active-loop dynamics for bsLDH is very similar to that previously observed for pig heart LDH. PMID:27319381

  7. Detection of an intermediate late in the unfolding pathway of bacillus stearothermophilus lactate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Sleigh, Roger N.; Halsall, David J.; Clarke, Anthony R.; Behan-Martin, Moira; Holbrook, J. J.

    1994-08-01

    In vivo proteins fold to form one active structure in minutes or seconds, ruling out the possibility that a polypeptide samples all possible conformational space during folding. We have used site directed mutagenesis to produce 15 single tryptophan containing mutants of Bacillus stearothermophilus lactate dehydrogenase (BS LDH) thus enabling the equilibria of unfolding to be seen from 15 defined positions. These mutant versions of BS LDH have the same X-ray structure as the wild type protein8. Previously Smith et al.11 had detected and assigned structures to 4 folding states. The first intermediate, a monomer with secondary and super secondary structure largely intact, is formed after the dimer dissociates at 0.55 M guanidinium hydrochloride (GuHCl). The second intermediate on the unfolding pathway is stable at 2.2 M GuHCl. It had been assumed previously that the transition from this molten-globule structure to the fully denatured form occurred as a single process. We have now identified a core folding motif. In this, helix (alpha) -1F forms a helix-sheet interaction with (beta) -K and (beta) -K has interactions with both (alpha) -2G and (alpha) -3G. This super secondary interaction forms the most stable folding motif in BS LDH and is lost at 2.8 M GuHCl leaving helix (alpha) -1F, (alpha) -2G, and (alpha) -3G which are stable until 3 M GuHCl.

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

    PubMed Central

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

    2011-01-01

    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

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

    NASA Astrophysics Data System (ADS)

    Masterson, Jean E.; Schwartz, Steven D.

    2014-10-01

    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.

  10. Estrogen-related receptor alpha modulates lactate dehydrogenase activity in thyroid tumors.

    PubMed

    Mirebeau-Prunier, Delphine; Le Pennec, Soazig; Jacques, Caroline; Fontaine, Jean-Fred; Gueguen, Naig; Boutet-Bouzamondo, Nathalie; Donnart, Audrey; Malthièry, Yves; Savagner, Frédérique

    2013-01-01

    Metabolic modifications of tumor cells are hallmarks of cancer. They exhibit an altered metabolism that allows them to sustain higher proliferation rates in hostile environment outside the cell. In thyroid tumors, the expression of the estrogen-related receptor α (ERRα), a major factor of metabolic adaptation, is closely related to the oxidative metabolism and the proliferative status of the cells. To elucidate the role played by ERRα in the glycolytic adaptation of tumor cells, we focused on the regulation of lactate dehydrogenases A and B (LDHA, LDHB) and the LDHA/LDHB ratio. Our study included tissue samples from 10 classical and 10 oncocytic variants of follicular thyroid tumors and 10 normal thyroid tissues, as well as samples from three human thyroid tumor cell lines: FTC-133, XTC.UC1 and RO82W-1. We identified multiple cis-acting promoter elements for ERRα, in both the LDHA and LDHB genes. The interaction between ERRα and LDH promoters was confirmed by chromatin immunoprecipitation assays and in vitro analysis for LDHB. Using knock-in and knock-out cellular models, we found an inverse correlation between ERRα expression and LDH activity. This suggests that thyroid tumor cells may reprogram their metabolic pathways through the up-regulation of ERRα by a process distinct from that proposed by the recently revisited Warburg hypothesis. PMID:23516535

  11. Understanding interactions of functionalized nanoparticles with proteins: a case study on lactate dehydrogenase.

    PubMed

    Stueker, Oliver; Ortega, Van A; Goss, Greg G; Stepanova, Maria

    2014-05-28

    Nanomaterials in biological solutions are known to interact with proteins and have been documented to affect protein function, such as enzyme activity. Understanding the interactions of nanoparticles with biological components at the molecular level will allow for rational designs of nanomaterials for use in medical technologies. Here we present the first detailed molecular mechanics model of functionalized gold nanoparticle (NP) interacting with an enzyme (L-lactate dehydrogenase (LDH) enzyme). Molecular dynamics (MD) simulations of the response of LDH to the NP binding demonstrate that although atomic motions (dynamics) of the main chain exhibit only a minor response to the binding, the dynamics of side chains are significantly constrained in all four active sites that predict alteration in kinetic properties of the enzyme. It is also demonstrated that the 5 nm gold NPs cause a decrease in the maximal velocity of the enzyme reaction (V(max)) and a trend towards a reduced affinity (increased K(m)) for the β-NAD binding site, while pyruvate enzyme kinetics (K(m) and V(max)) are not significantly altered in the presence of the gold NPs. These results demonstrate that modeling of NP:protein interactions can be used to understand alterations in protein function. PMID:24591162

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

    PubMed

    Koenig, Samuel; Solé, Montserrat

    2014-03-01

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

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

    SciTech Connect

    Datta, T.; Doermer, P.

    1987-12-01

    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.

  14. Gene Expression Variation in Duplicate Lactate dehydrogenase Genes: Do Ecological Species Show Distinct Responses?

    PubMed Central

    Cristescu, Melania E.; Demiri, Bora; Altshuler, Ianina; Crease, Teresa J.

    2014-01-01

    Lactate dehydrogenase (LDH) has been shown to play an important role in adaptation of several aquatic species to different habitats. The genomes of Daphnia pulex, a pond species, and Daphnia pulicaria, a lake inhabitant, encode two L-LDH enzymes, LDHA and LDHB. We estimated relative levels of Ldh gene expression in these two closely related species and their hybrids in four environmental settings, each characterized by one of two temperatures (10°C or 20°C), and one of two concentrations of dissolved oxygen (DO; 6.5–7 mg/l or 2–3 mg/l). We found that levels of LdhA expression were 4 to 48 times higher than LdhB expression (p<0.005) in all three groups (the two parental species and hybrids). Moreover, levels of LdhB expression differed significantly (p<0.05) between D. pulex and D. pulicaria, but neither species differed from the hybrid. Consistently higher expression of LdhA relative to LdhB in both species and the hybrid suggests that the two isozymes could be performing different functions. No significant differences in levels of gene expression were observed among the four combinations of temperature and dissolved oxygen (p>0.1). Given that Daphnia dwell in environments characterized by fluctuating conditions with long periods of low dissolved oxygen concentration, we suggest that these species could employ regulated metabolic depression to survive in such environments. PMID:25080082

  15. Gene expression variation in duplicate lactate dehydrogenase genes: do ecological species show distinct responses?

    PubMed

    Cristescu, Melania E; Demiri, Bora; Altshuler, Ianina; Crease, Teresa J

    2014-01-01

    Lactate dehydrogenase (LDH) has been shown to play an important role in adaptation of several aquatic species to different habitats. The genomes of Daphnia pulex, a pond species, and Daphnia pulicaria, a lake inhabitant, encode two L-LDH enzymes, LDHA and LDHB. We estimated relative levels of Ldh gene expression in these two closely related species and their hybrids in four environmental settings, each characterized by one of two temperatures (10°C or 20°C), and one of two concentrations of dissolved oxygen (DO; 6.5-7 mg/l or 2-3 mg/l). We found that levels of LdhA expression were 4 to 48 times higher than LdhB expression (p<0.005) in all three groups (the two parental species and hybrids). Moreover, levels of LdhB expression differed significantly (p<0.05) between D. pulex and D. pulicaria, but neither species differed from the hybrid. Consistently higher expression of LdhA relative to LdhB in both species and the hybrid suggests that the two isozymes could be performing different functions. No significant differences in levels of gene expression were observed among the four combinations of temperature and dissolved oxygen (p>0.1). Given that Daphnia dwell in environments characterized by fluctuating conditions with long periods of low dissolved oxygen concentration, we suggest that these species could employ regulated metabolic depression to survive in such environments. PMID:25080082

  16. Evaluation on the inhibition of pyrrol-2-yl ethanone derivatives to lactate dehydrogenase and anticancer activities.

    PubMed

    Lu, Na-Na; Weng, Zhao-Yue; Chen, Qiu-Yun; Boison, Daniel; Xiao, Xin-Xin; Gao, Jing

    2016-08-01

    Lactate dehydrogenase A (LDH-A) is a potentially important metabolic target for the inhibition of the highly activated glycolysis pathway in cancer cells. In order to develop bifunctional compounds as inhibitor of LDH-A and anticancer agents, two pyrrol-2-yl methanone (or ethanone) derivatives (PM1 and PM2) were synthesized and evaluated as inhibitors of LDH-A based on the enzyme assay and cell assay by spectroscopy analysis. Fluorescence and CD spectra results demonstrated that both the change of second structure of LDH-A and the affinity interaction for compounds to LDH-A gave great effect on the activity of LDH-A. In particular, low concentration of compounds (1μμ-25μμ) could change the level of pyruvate in cancer cells. Moreover, the in vitro assay results demonstrated that pyrrol-2-yl ethanone derivatives can inhibit the proliferation of cancer cells. Therefore, pyrrol-2-yl ethanone derivatives (PM2) can be both LDH-A inhibitor and anticancer agents. PMID:27104676

  17. Regulation of cell growth and apoptosis through lactate dehydrogenase C over-expression in Chinese hamster ovary cells.

    PubMed

    Fu, Tuo; Zhang, Cunchao; Jing, Yu; Jiang, Cheng; Li, Zhenhua; Wang, Shengyu; Ma, Kai; Zhang, Dapeng; Hou, Sheng; Dai, Jianxin; Kou, Geng; Wang, Hao

    2016-06-01

    Lactate has long been credited as a by-product, which jeopardizes cell growth and productivity when accumulated over a certain concentration during the manufacturing process of therapeutic recombinant proteins by Chinese hamster ovary (CHO) cells. A number of efforts to decrease the lactate concentration have been developed; however, the accumulation of lactate is still a critical issue by the late stage of fed-batch culture. Therefore, a lactate-tolerant cell line was developed through over-expression of lactate dehydrogenase C (LDH-C). In fed-batch culture, sodium lactate or sodium pyruvate was supplemented into the culture medium to simulate the environment of lactate accumulation, and LDH-C over-expression increased the highest viable cell density by over 30 and 50 %, respectively, on day 5, meanwhile the viability was also improved significantly since day 5 compared with that of the control. The percentages of cells suffering early and late apoptosis decreased by 3.2 to 12.5 and 2.0 to 4.3 %, respectively, from day 6 onwards in the fed-batch culture when 40 mM sodium pyruvate was added compared to the control. The results were confirmed by mitochondrial membrane potential assay. In addition, the expression of cleaved caspases 3 and 7 decreased in cells over-expressing LDH-C, suggesting the mitochondrial pathway was involved in the LDH-C regulated anti-apoptosis. In conclusion, a novel cell line with higher lactate tolerance, lowered lactate production, and alleviated apoptosis response was developed by over-expression of LDH-C, which may potentially represent an efficient and labor-saving approach in generating recombinant proteins. PMID:26841889

  18. A double mutant of highly purified Geobacillus stearothermophilus lactate dehydrogenase recognises l-mandelic acid as a substrate.

    PubMed

    Binay, Barış; Sessions, Richard B; Karagüler, Nevin Gül

    2013-05-10

    Lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) (bsLDH) has a crucial role in producing chirally pure hydroxyl compounds. α-Hydroxy acids are used in many industrial situations, ranging from pharmaceutical to cosmetic dermatology products. One drawback of this enzyme is its limited substrate specificity. For instance, l-lactate dehydrogenase exhibits no detectable activity towards the large side chain of 2-hydroxy acid l-mandelic acid, an α-hydroxy acid with anti-bacterial activity. Despite many attempts to engineer bsLDH to accept α-hydroxy acid substrates, there have been no attempts to introduce the industrially important l-mandelic acid to bsLDH. Herein, we describe attempts to change the reactivity of bsLDH towards l-mandelic acid. Using the Insight II molecular modelling programme (except 'program' in computers) and protein engineering techniques, we have successfully introduced substantial mandelate dehydrogenase activity to the enzyme. Energy minimisation modelling studies suggested that two mutations, T246G and I240A, would allow the enzyme to utilise l-mandelic acid as a substrate. Genes encoding for the wild-type and mutant enzymes were constructed, and the resulting bsLDH proteins were overexpressed in Escherichia coli and purified using the TAGZyme system. Enzyme assays showed that insertion of this double mutation into highly purified bsLDH switched the substrate specificity from lactate to l-mandelic acid. PMID:23608509

  19. Amperometric lactate biosensor for flow injection analysis based on a screen-printed carbon electrode containing Meldola's Blue-Reinecke salt, coated with lactate dehydrogenase and NAD+.

    PubMed

    Piano, M; Serban, S; Pittson, R; Drago, G A; Hart, J P

    2010-06-30

    A biosensor for the measurement of lactate in serum has been developed, which is based on a screen-printed carbon electrode, modified with Meldola's Blue-Reinecke Salt (MBRS-SPCE), coated with the enzyme lactate dehydrogenase NAD(+) dependent (from Porcine heart), and NAD(+). A cellulose acetate layer was deposited on the top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V vs. Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer pH 10 containing 0.1 M potassium chloride solution; a flow rate of 0.8 ml min(-1) was used throughout the investigation. The biosensor response was linear over the range 0.55-10 mM lactate; the former represents the detection limit. The precision of the system was determined by carrying out 10 repeat injections of 10 mM l(+)lactic acid standard; the calculated coefficient of variation was 4.28%. It was demonstrated that this biosensor system could be applied to the direct measurement of lactate in serum without pre-treatment; therefore, this would allow high throughput-analysis, at low cost, for this clinically important analyte. PMID:20685431

  20. Detection of L-lactate in polyethylene glycol solutions confirms the identity of the active-site ligand in a proline dehydrogenase structure.

    PubMed

    Zhang, Min; Tanner, John J

    2004-05-01

    Polyethylene glycol (PEG) is often used in protein crystallography as a low-ionic-strength precipitant for crystallization and as a cryoprotectant for low-temperature data collection. Prompted by the discovery of an apparent L-lactate molecule bound in the active site of the Escherichia coli PutA proline dehydrogenase domain crystal structure, the L-lactate concentration of several PEG solutions was measured. 50%(w/v) solutions of PEGs with molecular weight 3000, 4000 and 8000 contain millimolar levels of L-lactate. In contrast, L-lactate was not detected in solutions of PEG monomethyl ethers or PEG 3350. These results help to explain why L-lactate was present in the proline dehydrogenase domain crystal structure. This work also has implications for the crystallization of enzymes that bind L-lactate. PMID:15103160

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

    PubMed

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

    2014-09-01

    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

  2. Cariogenicity of a lactate dehydrogenase-deficient mutant of Streptococcus mutans serotype c in gnotobiotic rats.

    PubMed Central

    Fitzgerald, R J; Adams, B O; Sandham, H J; Abhyankar, S

    1989-01-01

    A lactate dehydrogenase-deficient (Ldh-) mutant of a human isolate of Streptococcus mutans serotype c was tested in a gnotobiotic rat caries model. Compared with the wild-type Ldh-positive (Ldh+) strains, it was significantly (alpha less than or equal to 0.005) less cariogenic in experiments with two different sublines of Sprague-Dawley rats. The Ldh- mutant strain 044 colonized the oral cavity of the test animals to the same extent as its parent strain 041, although its initial implantation was slightly but not significantly (P greater than or equal to 0.2) less. Multiple oral or fecal samples plated on 2,3,5-triphenyltetrazolium indicator medium revealed no evidence of back mutation from Ldh- to Ldh+ in vivo. Both Ldh+ strain 041 and Ldh- strain 044 demonstrated bacteriocinlike activity in vitro against a number of human strains of mutans streptococci representing serotype a (S. cricetus) and serotypes c and e (S. mutans). Serotypes b (S. rattus) and f (S. mutans) and strains of S. mitior, S. sanguis, and S. salivarius were not inhibited. Thus, Ldh mutant strain 044 possesses a number of desirable traits that suggest it should be investigated further as a possible effector strain for replacement therapy of dental caries. These traits include its stability and low cariogenicity in the sensitive gnotobiotic rat caries model, its bacteriocinlike activity against certain other cariogenic S. mutans (but not against more inocuous indigenous oral streptococci), and the fact that it is a member of the most prevalent human serotype of cariogenic streptococci. PMID:2917788

  3. Regulation of the Activity of Lactate Dehydrogenases from Four Lactic Acid Bacteria*

    PubMed Central

    Feldman-Salit, Anna; Hering, Silvio; Messiha, Hanan L.; Veith, Nadine; Cojocaru, Vlad; Sieg, Antje; Westerhoff, Hans V.; Kreikemeyer, Bernd; Wade, Rebecca C.; Fiedler, Tomas

    2013-01-01

    Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (Pi), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (Kact) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of Pi are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of Pi inhibits E. faecalis LDH2, whereas in the absence of FBP, Pi is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of Pi to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of Pi, FBP, and pH that results in different regulatory effects on the LDHs of different LABs. PMID:23720742

  4. Evaluation of D-dimer and lactate dehydrogenase plasma levels in patients with relapsed acute leukemia

    PubMed Central

    HU, WANGQIANG; WANG, XIAOXIA; YANG, RONGRONG

    2016-01-01

    Despite the outstanding advances made over the past decade regarding our knowledge of acute leukemia (AL), relapsed AL remains to be associated with a dismal prognosis. A better understanding of AL relapse and monitoring of the D-dimer and lactate dehydrogenase (LDH) plasma levels following chemotherapy may aid clinicians in determining whether relapse may occur in the subsequent phases of the disease. The present study evaluated D-dimer and LDH levels in 204 patients with relapsed AL. Data were collected at the initial onset of AL, at complete remission (CR) and in patients with relapsed AL. D-dimer plasma levels were significantly increased in patients with initial AL and in patients with relapsed AL (P=0.005 and P=0.007, respectively) but not in those with CR. LDH levels were significantly increased in AL patients at the initial onset of disease and at relapse compared with patients achieving CR, irrespective of cell type. Plasma prothrombin time, activated partial thromboplastin time and fibrinogen levels were not significantly different across patients (with the exception of acute promyelocytic leukemia patients) at the initial onset, relapsed AL or CR. Routine hematological parameters (white blood cell count, hemoglobin, platelet count) were significantly different at the initial onset of AL (P=0.002, P<0.001 and P=0.001, respectively) and during relapsed AL (P=0.009, P=0.003 and P<0.001, respectively) compared with patients achieving CR, suggesting an association between D-dimer, LDH and relapsed AL. These results also indicate that determination of D-dimer and LDH levels may be useful for predicting the probability of relapse during chemotherapy, but should also be combined with routine hematological parameters. PMID:27347185

  5. An optimized lactate dehydrogenase release assay for screening of drug candidates in neuroscience

    PubMed Central

    Kaja, Simon; Payne, Andrew J.; Singh, Tulsi; Ghuman, Jasleen K.; Sieck, Erin G.; Koulen, Peter

    2015-01-01

    Background Quantification of lactate dehydrogenase (LDH) release is a widely accepted assay for the quantitative determination of cell viability and late-stage apoptosis. Major disadvantages of commercially available LDH assay kits include proprietary formulations, limited options for optimization and high cost, all resulting in limited reproducibility in research applications. New Method Here, we describe a novel, custom LDH assay suitable in the context of plate reader-based screening of drug candidates for glioprotection, but with wide applicability to other cell types and experimental paradigms. Results We developed a novel and highly reproducible LDH release assay that is more cost-effective than commercially available assays with comparable performance. The assay was validated by assessing 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid antioxidant protection against tert-butylhydroperoxide-induced oxidative stress in C6 astroglioma cells. Assay performance was validated by direct comparison and compatible with other methods of measuring cellular viability, namely 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 6-carboxy-2′, 7′ dichlorodihydrofluorescein diacetate assays. Comparison with Existing Method(s) There was no statistically significant difference between results obtained with the novel custom assay and a commercially available assay CytoTox96® (Promega, Madison, WI). Conclusions The novel custom LDH release assay allows the reproducible quantification of cell viability and is highly cost-effective when compared to commercially available assays (approximately 25 times cheaper). In addition and in contrast to commercially available assays, the identification and detailed description of all assay components and procedures provide greater control over experimental conditions and design. We provide a detailed standard operating procedure permitting our novel assay to be readily adapted depending on experimental requirements

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

    PubMed

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

    2014-08-01

    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

  7. Estimating the number of viable animal cells in multi-well cultures based on their lactate dehydrogenase activities.

    PubMed

    Haslam, G; Wyatt, D; Kitos, P A

    2000-01-01

    A method is described for estimating the numbers ofanimal cells in multi-well culture by simultaneouslymeasuring the lactate dehydrogenase activity of thetotal culture and the medium. The difference betweenthe two reflects the dehydrogenase content of thecells and correlates with cell number. This LDH/INTmethod was tested using several lines of normal andtransformed suspension and adherent cells. Thelactate dehydrogenase activities of duplicate cultureswere determined colourimetrically using reactioncocktails containing lactate, NAD(+), diaphorase,and p-iodonitrotetrazolium violet, with and withoutTriton X-100. The difference in absorbance at 490 nm(DeltaA(490) = A(490, test) - A(490, control)) was used to calculate the lactatedehydrogenase activity of the total culture (+ Triton)and the medium (- Triton). The cellular lactatedehydrogenase activity (difference between totaland medium dehydrogenaseactivities) was proportional to viable cell number. The effects on cell growth of four metabolicinhibitors, sodium azide, actinomycin D,cycloheximide, and taxol, were determined using theLDH/INT assay and direct cell counting. The inhibitorconcentrations that caused decreases in the LDHactivity and cell number by 50% were similar. TheLDH/INT assay is quick and sensitive, works equallywell for adherent and suspension cells, and providesinformation about LDH activities of both the mediumand cells. It is particularly useful for screeningpotential cell-growth inhibitors. PMID:19002967

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

    PubMed

    Wieser, W; Wright, E

    1979-04-01

    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

  9. Activity of phosphoglycerate mutase and its isoenzymes in serum after acute myocardial infarction

    PubMed Central

    Durany, N; Carballo, E; Joseph, J; Bedini, J L; Bartrons, R; Ballesta, A M; Carreras, J

    1996-01-01

    Aims/background—In humans there are three phosphoglycerate mutase (PGM, EC 5.4.12.1) isoenzymes (MM, MB and BB) which have similar distribution and developmental pathways to creatine kinase (CK, EC 2.7.3.2) isoenzymes. Total serum PGM activity increases in acute myocardial infarction with the same time course as creatine kinase activity. The present study was undertaken to determine changes in the activity of PGM and its isoenzymes after acute myocardial infarction. Methods—PGM activity was measured spectrophotometrically, by coupling the formation of 2-phosphoglycerate from 3-phosphoglycerate with enolase, pyruvate kinase and lactate dehydrogenase catalysed reactions. Inter- and intra-assay reproducibility was assessed. PGM isoenzyme activities were measured using cellulose acetate electrophoresis. Results—Total PGM activity in serum was increased in patients with a confirmed diagnosis of acute myocardial infarction. PGM activity peaked 12 to 24 hours after the onset of symptoms and returned to normal values within 48 hours. Electrophoretic analysis of serum from healthy subjects showed a band corresponding to BB-PGM and two other artefactual bands that did not correspond to adenylate kinase. After myocardial infarction, BB-PGM activity increased and MB-PGM and MM-PGM could be detected. On immunoblot analysis, normal serum contained an inactive form of MM-PGM with a smaller molecular weight than that of PGM tissue isoenzymes. Conclusions—Total serum PGM activity increased in patients with acute myocardial infarction, following the same temporal course as creatine kinase activity. The increase in MM-PGM and MB-PGM activities in these patients was not as high as expected. It is suggested that PGM isoenzymes, after release into the blood, undergo postsynthetic, probably proteolytic, transformation. Images PMID:16696092

  10. Conformational Heterogeneity in the Michaelis Complex of Lactate Dehydrogenase: An Analysis of Vibrational Spectroscopy Using Markov and Hidden Markov Models.

    PubMed

    Pan, Xiaoliang; Schwartz, Steven D

    2016-07-14

    Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate. Recent isotope-edited IR spectroscopy suggests that conformational heterogeneity exists within the Michaelis complex of LDH, and this heterogeneity affects the propensity toward the on-enzyme chemical step for each Michaelis substate. By combining molecular dynamics simulations with Markov and hidden Markov models, we obtained a detailed kinetic network of the substates of the Michaelis complex of LDH. The ensemble-average electric fields exerted onto the vibrational probe were calculated to provide a direct comparison with the vibrational spectroscopy. Structural features of the Michaelis substates were also analyzed on atomistic scales. Our work not only clearly demonstrates the conformational heterogeneity in the Michaelis complex of LDH and its coupling to the reactivities of the substates, but it also suggests a methodology to simultaneously resolve kinetics and structures on atomistic scales, which can be directly compared with the vibrational spectroscopy. PMID:27347759

  11. The Contribution of Electrostatic and van der Waals Interactions to the Stereospecificity of the Reaction Catalyzed by Lactate Dehydrogenase

    PubMed Central

    van Beek, Jeroen; Callender, Robert; Gunner, M. R.

    1997-01-01

    Continuum electrostatic calculations in conjunction with molecular dynamics simulations have been used to investigate the source of the stereospecificity in the hydride transfer reaction catalyzed by lactate dehydrogenase (LDH). These studies show that favorable electrostatic interactions between the carboxamide group of the reduced nicotinamide adenine dinucleotide coenzyme and protein residues of the active site of LDH can account for much if not all of the stereospecificity of the LDH-catalyzed reaction, with A-side hydride transfer more than 107 times greater than B-side transfer. Unfavorable steric interactions within the binding complex for B-side transfer are not found. ImagesFIGURE 2 PMID:9017191

  12. Pre-treatment serum lactate dehydrogenase and alkaline phosphatase as predictors of metastases in extremity osteosarcoma

    PubMed Central

    Marais, Leonard C.; Bertie, Julia; Rodseth, Reitze; Sartorius, Benn; Ferreira, Nando

    2015-01-01

    Background The prognosis of patients with metastatic osteosarcoma remains poor. However, the chance of survival can be improved by surgical resection of all metastases. In this study we investigate the value of serum alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in predicting the presence of metastatic disease at time of diagnosis. Methods Sixty-one patients with histologically confirmed conventional osteosarcoma of the extremity were included in the study. Only 19.7% of cases presented without evidence of systemic spread of the disease. Pre-treatment serum ALP and LDH were analysed in patients with and without skeletal or pulmonary metastases. Results Serum LDH and ALP levels were not significantly different in patients with or without pulmonary metastases (p=0.88 and p=0.47, respectively). The serum LDH and ALP levels did however differ significantly in patients with or without skeletal metastases (p<0.001 and p=0.02, respectively). The optimal breakpoint for serum LDH as a marker of skeletal metastases was 849 IU/L (AUC 0.839; Sensitivity=0.88; Specificity=0.73). LDH >454 IU/L equated to 100% sensitivity for detected bone metastases (positive diagnostic likelihood ratio (DLR)=1.32). With a cut-off of 76 IU/L a sensitivity of 100% was reached for serum ALP predicting the presence of skeletal metastases (positive DLR=1.1). In a multivariate analysis both LDH ≥850 IU/L (odds ratio [OR]=9; 95% confidence interval (CI) 1.8–44.3) and ALP ≥280 IU/L (OR=10.3; 95% CI 2.1–50.5) were predictive of skeletal metastases. LDH however lost its significance in a multivariate model which included pre-treatment tumour volume. Conclusion In cases of osteosarcoma with LDH >850 IU/L and/or ALP >280 IU/L it may be prudent to consider more sensitive staging investigations for detection of skeletal metastases. Further research is required to determine the value and the most sensitive cut-off points of serum ALP and LDH in the prediction of skeletal metastases. PMID

  13. Lactate dehydrogenase activity of rat epididymis and spermatozoa: effect of constant light.

    PubMed

    Ponc, R H; Carriazo, C S; Vermouth, N T

    2001-01-01

    During its passage through the epididymis, the gamete undergoes a process of "maturation" leading to the acquisition of its fertilizing ability. The epididymis displays regional variations in the morphology and metabolic properties of its epithelium which are relevant for the progressive development of mature sperm characteristics. The epididymis has spontaneous peristaltic contractions and receives sympathetic innervation that is modulated by melatonin, a hormone synthesized and released by the pineal gland. Constant lighting disrupts melatonin synthesis and secretion. We have studied the effect of constant light on lactate dehydrogenase (LDH; EC 1.1.1.27) and its isozyme C4 activities and protein content in whole epididymis, epididymal tissue and in spermatozoa from caput and cauda segments. Animals were exposed from birth to an illumination schedule of 14 h light:10 h dark (group L:D). At 60 days of age one group of animals was submitted to constant light over 50 days (group L:L). In order to test the fertilizing ability, the rats of each group were mated with soliciting estrous females. The percentage of pregnancies in females mated with males maintained in L:L was remarkably lower than those in females mated with males maintained in the L:D photoperiod (44% and 88% respectively). Constant light increased protein concentration and LDH activity in caput as well as in cauda of total epididymis. On the contrary, in epididymal tissue, the protein content decreased in both epididymal sections compared with controls. When enzymatic activity was expressed in Units per spermatozoa, constant light induced a significant reduction of total LDH and LDHC4 in caput and cauda spermatozoa while LDH activity of epididymal tissue was not affected. In spite of the decrease in LDH per sperm cell when rats were exposed to constant light, in total epididymis (epididymis tissue plus sperm cells content) and in spermatozoa, values of enzyme activities expressed per weight unit were

  14. Assessment of lactate dehydrogenase, alkaline phosphatase and aspartate aminotransferase activities in cow's milk as an indicator of subclinical mastitis.

    PubMed

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

    2007-05-01

    This study examined the activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in the milk of lactating Holstein cows in association with subclinical mastitis (SCM). A total of 94 milk samples were collected from 58 lactating dairy cows representing stages of lactation from the second to the tenth week after calving. Those which were classified as positive by California mastitis test (CMT) were deemed to have subclinical mastitis. All the milk samples were skimmed by centrifugation at 10 000g at 0 degrees C and were used for enzyme activities estimations. The mean activities of LDH and ALP were higher in the milk from udders with SCM than in the milk from healthy udders (p < 0.05). There were no significant differences in AST values. The maximum agreement rates between the CMT results and LDH and ALP values were seen at thresholds of > 180 IU/L and > 40 IU/L respectively (kappa values 0.65 and 0.79, respectively). However, the sensitivity of the tests for identifying SCM at these thresholds was higher for ALP (96.4%) than for LDH (68.5%). In this study, LDH and ALP tests were standardized for cow's milk and results showed that only the ALP test was reliable in the early diagnosis of subclinical mastitis. PMID:17268916

  15. Aromatic Surfactant as Aggregating Agent for Aptamer-Gold Nanoparticle-Based Detection of Plasmodium Lactate Dehydrogenase.

    PubMed

    Jain, Priyamvada; Chakma, Babina; Singh, Naveen Kumar; Patra, Sanjukta; Goswami, Pranab

    2016-07-01

    A novel ssDNA aptamer (P38), with a 40 mer random region flanked by primer-binding sites on both sides, targeting Plasmodium falciparum lactate dehydrogenase (PfLDH) has been developed through systematic evolution of ligands by exponential enrichment (SELEX), including counter SELEX against human lactate dehydrogenase A and B (hLDH A and B). The 2D structure of P38 shows the presence of three stem loops with a δG of -9.18 kcal/mol. EMSA studies on P38 complexes with the increasing concentration of PfLDH revealed a dissociation constant of 0.35 µM. P38 has been exploited for the quantitative detection of PfLDH using cationic surfactant-mediated aggregation of gold nanoparticles (16-nm diameter) as an optical probe. Among the three different cationic surfactants, characterized by different hydrocarbon tail groups, benzalkonium chloride (BCK) was found to be most efficient with a limit of detection of 281 ± 11 pM. This BCK-based approach with the novel highly selective aptamer provides simple and sensitive detection of PfLDH in the clinically relevant range. PMID:27189484

  16. The management of metastatic germ cell tumours and the clinical utility of lactate dehydrogenase estimations.

    PubMed

    Gill, P G; Abbott, R; Jones, A M; Thomas, D W

    1985-04-01

    Forty-five patients with metastatic germ cell tumour were treated with chemotherapy. Complete remission was achieved in 63% of all cases and in 65% of patients whose primary tumour arose in the testis or ovary. Surgical resection of abdominal masses persisting after chemotherapy was performed in seven patients, two of whom were found to have persistent tumours. Twenty-seven of the 33 patients with teratoma originating in the gonads remain in complete remission. Total serum LDH activity was elevated in 28 of the patients with measurable disease. The increased LDH was not accompanied by significant alteration in other hepatic enzymes nor were hepatic metastases demonstrable in these patients. Fractionation of the LDH demonstrated that the increased LDH in these patients was located in either iso-enzymes 1 or fractions 1 + 2. Alteration of the serum LDH activity correlated with the response to therapy and warrants further study. PMID:2412541

  17. The Conformation of NAD+ Bound to Lactate Dehydrogenase Determined by Nuclear Magnetic Resonance with Suppression of Spin Diffusion

    NASA Astrophysics Data System (ADS)

    Vincent, Sebastien J. F.; Zwahlen, Catherine; Post, Carol Beth; Burgner, John W.; Bodenhausen, Geoffrey

    1997-04-01

    We have reinvestigated the conformation of NAD+ bound to dogfish lactate dehydrogenase (LDH) by using an NMR experiment that allows one to exploit nuclear Overhauser effects to determine internuclear distances between pairs of protons, without perturbation of spin-diffusion effects from other protons belonging either to the cofactor or to the binding pocket of the enzyme. The analysis indicates that the structure of bound NAD+ is in accord with the conformation determined in the solid state by x-ray diffraction for the adenosine moiety, but deviates significantly from that of the nicotinamide. The NMR data indicate conformational averaging about the glycosidic bond of the nicotinamide nucleotide. In view of the strict stereospecificity of catalysis by LDH and the conformational averaging of bound NAD+ that we infer from solution-state NMR, we suggest that LDH binds the cofactor in both syn and anti conformations, but that binding interactions in the syn conformation are not catalytically productive.

  18. Isolation and characterization of two cDNA clones of anaerobically induced lactate dehydrogenase from barley roots

    SciTech Connect

    Hondred, D.; Hanson, A.D. )

    1990-05-01

    In barley roots during hypoxia, five lactate dehydrogenase (LDH) isozymes accumulate with a concomitant increase in enzyme activity ({approximately}20-fold). These isozymes are thought to be tetramers resulting from the random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH have been isolated from a {lambda}gt11 library using antiserum raised against barley LDH purified {approximately}3,000-fold and using nucleic acid probes synthesized by the polymerase chain reaction. Two cDNA clones were obtained (1,305 and 1,166 bp). The deduced amino acid sequences of the two barley LDHs are 96% identical to each other, and 50% and 40% identical to vertebrate and bacterial LDHs, respectively. Northern blots identified a single mRNA band ({approximately}1.5 kb) whose level rose 8-fold during hypoxia.

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

    PubMed

    Dragovich, Peter S; Fauber, Benjamin P; Corson, Laura B; Ding, Charles Z; Eigenbrot, Charles; Ge, HongXiu; Giannetti, Anthony M; Hunsaker, Thomas; Labadie, Sharada; Liu, Yichin; Malek, Shiva; Pan, Borlan; Peterson, David; Pitts, Keith; Purkey, Hans E; Sideris, Steve; Ultsch, Mark; VanderPorten, Erica; Wei, BinQing; Xu, Qing; Yen, Ivana; Yue, Qin; Zhang, Huihui; Zhang, Xuying

    2013-06-01

    A novel 2-thio-6-oxo-1,6-dihydropyrimidine-containing inhibitor of human lactate dehydrogenase (LDH) was identified by high-throughput screening (IC50=8.1 μM). Biochemical, surface plasmon resonance, and saturation transfer difference NMR experiments indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of the screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.48 μM). A crystal structure of an optimized compound bound to human LDHA was obtained and explained many of the observed structure-activity relationships. PMID:23628333

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

    SciTech Connect

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

    2012-01-01

    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

  1. Targeted Disruption of the kstD Gene Encoding a 3-Ketosteroid Δ1-Dehydrogenase Isoenzyme of Rhodococcus erythropolis Strain SQ1

    PubMed Central

    van der Geize, R.; Hessels, G. I.; van Gerwen, R.; Vrijbloed, J. W.; van der Meijden, P.; Dijkhuizen, L.

    2000-01-01

    Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Δ1-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Δ1-dehydrogenase genes. Heterologous expression of kstD yielded 3-ketosteroid Δ1-dehydrogenase (KSTD) activity under the control of the lac promoter in Escherichia coli. Targeted disruption of the kstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9α-hydroxy-4-androstene-3,17-dione was not abolished by the kstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of Δ1-dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation of kstD is not sufficient for inactivation of the Δ1-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that R. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain. PMID:10788377

  2. Efficient production of enantiomerically pure D-phenyllactate from phenylpyruvate by structure-guided design of an engineered D-lactate dehydrogenase.

    PubMed

    Wang, Min; Zhu, Lingfeng; Xu, Xiaoling; Wang, Limin; Yin, Ruochun; Yu, Bo

    2016-09-01

    3-Phenyllactic acid (PLA) is an antimicrobial compound with broad-spectrum activity against bacteria and fungi that could be widely used in the food industry and livestock feeds. Notably, D-PLA exhibits higher antibacterial activity, which gains more attention than L-PLA. In this report, the D-lactate dehydrogenase DLDH744 from Sporolactobacillus inulinus CASD was engineered to increase the enzymatic activities toward phenylpyruvate by protein structure-guided modeling analysis. The phenylpyruvate molecule was first docked in the active center of DLDH744. The residues that might tightly pack around the benzene ring of phenylpyruvate were all selected for mutation. The single site mutant M307L showed the highest increased activity toward bulkier substrate phenylpyruvate than the wild type. By using the engineered D-lactate dehydrogenase M307L expressed in Escherichia coli strains, without coexpression of the cofactor regeneration system, 21.43 g/L D-PLA was produced from phenylpyruvate with a productivity of 1.58 g/L/h in the fed-batch biotransformation process, which ranked in the list as the highest production titer of D-PLA by D-lactate dehydrogenase. The enantiomeric excess value of produced D-PLA in the broth was higher than 99.7 %. Additionally, the structure-guided design of this enzyme will also provide referential information for further engineering other 2-hydroxyacid dehydrogenases, which are useful for a wide range of fine chemical synthesis. PMID:27020295

  3. Protein Conformational Landscapes and Catalysis. Influence of Active Site Conformations in the Reaction Catalyzed by L-Lactate Dehydrogenase

    PubMed Central

    Świderek, Katarzyna; Tuñón, Iñaki; Martí, Sergio; Moliner, Vicent

    2015-01-01

    In the last decade L-Lactate Dehydrogenase (LDH) has become an extremely useful marker in both clinical diagnosis and in monitoring the course of many human diseases. It has been assumed from the 80s that the full catalytic process of LDH starts with the binding of the cofactor and the substrate followed by the enclosure of the active site by a mobile loop of the protein before the reaction to take place. In this paper we show that the chemical step of the LDH catalyzed reaction can proceed within the open loop conformation, and the different reactivity of the different protein conformations would be in agreement with the broad range of rate constants measured in single molecule spectrometry studies. Starting from a recently solved X-ray diffraction structure that presented an open loop conformation in two of the four chains of the tetramer, QM/MM free energy surfaces have been obtained at different levels of theory. Depending on the level of theory used to describe the electronic structure, the free energy barrier for the transformation of pyruvate into lactate with the open conformation of the protein varies between 12.9 and 16.3 kcal/mol, after quantizing the vibrations and adding the contributions of recrossing and tunneling effects. These values are very close to the experimentally deduced one (14.2 kcal·mol−1) and ~2 kcal·mol−1 smaller than the ones obtained with the closed loop conformer. Calculation of primary KIEs and IR spectra in both protein conformations are also consistent with our hypothesis and in agreement with experimental data. Our calculations suggest that the closure of the active site is mainly required for the inverse process; the oxidation of lactate to pyruvate. According to this hypothesis H4 type LDH enzyme molecules, where it has been propose that lactate is transformed into pyruvate, should have a better ability to close the mobile loop than the M4 type LDH molecules. PMID:25705562

  4. Lactation

    PubMed Central

    1989-01-01

    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

  5. Loss of NADH Oxidase Activity in Streptococcus mutans Leads to Rex-Mediated Overcompensation in NAD+ Regeneration by Lactate Dehydrogenase

    PubMed Central

    Baker, J. L.; Derr, A. M.; Faustoferri, R. C.

    2015-01-01

    ABSTRACT Previous studies of the oral pathogen Streptococcus mutans have determined that this Gram-positive facultative anaerobe mounts robust responses to both acid and oxidative stresses. The water-forming NADH oxidase (Nox; encoded by nox) is thought to be critical for the regeneration of NAD+, for use in glycolysis, and for the reduction of oxygen, thereby preventing the formation of damaging reactive oxygen species. In this study, the free NAD+/NADH ratio in a nox deletion strain (Δnox) was discovered to be remarkably higher than that in the parent strain, UA159, when the strains were grown in continuous culture. This unanticipated result was explained by significantly elevated lactate dehydrogenase (Ldh; encoded by ldh) activity and ldh transcription in the Δnox strain, which was mediated in part by the redox-sensing regulator Rex. cDNA microarray analysis of S. mutans cultures exposed to simultaneous acid stress (growth at a low pH) and oxidative stress (generated through the deletion of nox or the addition of exogenous oxygen) revealed a stress response synergistically heightened over that with either stress alone. In the Δnox strain, this elevated stress response included increased glucose phosphoenolpyruvate phosphotransferase system (PTS) activity, which appeared to be due to elevated manL transcription, mediated in part, like elevated ldh transcription, by Rex. While the Δnox strain does possess a membrane composition different from that of the parent strain, it did not appear to have defects in either membrane permeability or ATPase activity. However, the altered transcriptome and metabolome of the Δnox strain were sufficient to impair its ability to compete with commensal peroxigenic oral streptococci during growth under aerobic conditions. IMPORTANCE Streptococcus mutans is an oral pathogen whose ability to outcompete commensal oral streptococci is strongly linked to the formation of dental caries. Previous work has demonstrated that the S

  6. Additive effects of clofibric acid and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high-saturated fat diet

    PubMed Central

    Hwang, Byounghoon; Wu, Pengfei; Harris, Robert A.

    2012-01-01

    SUMMARY Although improving glucose metabolism by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) might prove beneficial in the treatment of type 2 diabetes or diet-induced obesity, it might induce detrimental effects by inhibiting fatty acid oxidation. PPARα agonists are often used to treat dyslipidemia in patients, especially in type 2 diabetes. Combinational treatment with a PDK4 inhibitor and PPARα agonists may prove beneficial. However, PPARα agonists may be less effective in the presence of a PDK4 inhibitor because PPARα agonists induce PDK4 expression. In the present study, the effects of clofibric acid, a PPARα agonist, on blood and liver lipids were determined in wild type and PDK4 knockout mice fed a high fat diet. As expected, treatment of wild type mice with clofibric acid resulted in less body weight gain, smaller epididymal fat pads, greater insulin sensitivity, and lower levels of serum and liver triacylglycerol. Surprisingly, rather than decreasing the effectiveness of clofibric acid, PDK4 deficiency enhanced the beneficial effects of clofibric acid on hepatic steatosis, lowered blood glucose levels, and did not prevent the positive effects of clofibric acid on serum triacylglycerols and free fatty acids. The metabolic effects of clofibric acid are therefore independent of the induction of PDK4 expression. The additive beneficial effects on hepatic steatosis may be due to induction of increased capacity for fatty acid oxidation and partial uncoupling of oxidative phosphorylation by clofibric acid and a reduction in the capacity for fatty acid synthesis by PDK4 deficiency. PMID:22429297

  7. Lactate dehydrogenase A as a highly abundant eye lens protein in platypus (Ornithorhynchus anatinus): upsilon (upsilon)-crystallin.

    PubMed

    van Rheede, Teun; Amons, Reinout; Stewart, Niall; de Jong, Wilfried W

    2003-06-01

    Vertebrate eye lenses mostly contain two abundant types of proteins, the alpha-crystallins and the beta/gamma-crystallins. In addition, certain housekeeping enzymes are highly expressed as crystallins in various taxa. We now observed an unusual approximately 41-kd protein that makes up 16% to 18% of the total protein in the platypus eye lens. Its cDNA sequence was determined, which identified the protein as muscle-type lactate dehydrogenase A (LDH-A). It is the first observation of LDH-A as a crystallin, and we designate it upsilon (upsilon)-crystallin. Interestingly, the related heart-type LDH-B occurs as an abundant lens protein, known as epsilon-crystallin, in many birds and crocodiles. Thus, two members of the ldh gene family have independently been recruited as crystallins in different higher vertebrate lineages, suggesting that they are particularly suited for this purpose in terms of gene regulatory or protein structural properties. To establish whether platypus LDH-A/upsilon-crystallin has been under different selective constraints as compared with other vertebrate LDH-A sequences, we reconstructed the vertebrate ldh-a gene phylogeny. No conspicuous rate deviations or amino acid replacements were observed. PMID:12716980

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

    PubMed

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

    2014-12-01

    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

  9. Cryptosporidium Lactate Dehydrogenase Is Associated with the Parasitophorous Vacuole Membrane and Is a Potential Target for Developing Therapeutics.

    PubMed

    Zhang, Haili; Guo, Fengguang; Zhu, Guan

    2015-01-01

    The apicomplexan, Cryptosporidium parvum, possesses a bacterial-type lactate dehydrogenase (CpLDH). This is considered to be an essential enzyme, as this parasite lacks the Krebs cycle and cytochrome-based respiration, and mainly-if not solely, relies on glycolysis to produce ATP. Here, we provide evidence that in extracellular parasites (e.g., sporozoites and merozoites), CpLDH is localized in the cytosol. However, it becomes associated with the parasitophorous vacuole membrane (PVM) during the intracellular developmental stages, suggesting involvement of the PVM in parasite energy metabolism. We characterized the biochemical features of CpLDH and observed that, at lower micromolar levels, the LDH inhibitors gossypol and FX11 could inhibit both CpLDH activity (Ki = 14.8 μM and 55.6 μM, respectively), as well as parasite growth in vitro (IC50 = 11.8 μM and 39.5 μM, respectively). These observations not only reveal a new function for the poorly understood PVM structure in hosting the intracellular development of C. parvum, but also suggest LDH as a potential target for developing therapeutics against this opportunistic pathogen, for which fully effective treatments are not yet available. PMID:26562790

  10. Prognostic implications of dynamic serum lactate dehydrogenase assessments in nasopharyngeal carcinoma patients treated with intensity-modulated radiotherapy

    PubMed Central

    Zhou, Guan-Qun; Ren, Xian-Yue; Mao, Yan-Ping; Chen, Lei; Sun, Ying; Liu, Li-Zhi; Li, Li; Lin, Ai-Hua; Mai, Hai-Qiang; Ma, Jun

    2016-01-01

    The prognostic value of dynamic serum lactate dehydrogenase (LDH) levels in patients with nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiotherapy (IMRT) hasn’t been explored. We retrospectively analyzed 1,428 cases of NPC treated with IMRT with or without chemotherapy. Elevated pre- and/or post-treatment LDH levels were found to be associated with unfavorable overall survival (OS), disease-free survival (DFS) and distant metastasis-free survival (DMFS), but not with local relapse-free survival (LRFS). The dynamic variations in LDH levels were prognostic factors for OS, DFS and DMFS, but not for LRFS. Multivariate analysis revealed that the N category, T category, post-treatment serum LDH level and age were independent prognostic factors for OS. Our results demonstrated that dynamic variations in LDH levels were associated with risk of distant failure and death, which may shed light on the dynamics of the disease and the response to therapy. We consider that LDH measurements will be of great clinical importance in the management of NPC, especially, when considering “decision points” in treatment algorithms. Therefore, we strongly recommend that LDH levels should be determined before and after treatment in NPC patients and the results integrated into decisions regarding treatment strategies. PMID:26928265

  11. The value of lactate dehydrogenase serum levels as a prognostic and predictive factor for advanced pancreatic cancer patients receiving sorafenib

    PubMed Central

    Faloppi, Luca; Bianconi, Maristella; Giampieri, Riccardo; Sobrero, Alberto; Labianca, Roberto; Ferrari, Daris; Barni, Sandro; Aitini, Enrico; Zaniboni, Alberto; Boni, Corrado; Caprioni, Francesco; Mosconi, Stefania; Fanello, Silvia; Berardi, Rossana; Bittoni, Alessandro; Andrikou, Kalliopi; Cinquini, Michela; Torri, Valter; Scartozzi, Mario; Cascinu, Stefano

    2015-01-01

    Although lactate dehydrogenase (LDH) serum levels, indirect markers of angiogenesis, are associated with a worse outcome in several tumours, their prognostic value is not defined in pancreatic cancer. Moreover, high levels are associated even with a lack of efficacy of tyrosine kinase inhibitors, contributing to explain negative results in clinical trials. We assessed the role of LDH in advanced pancreatic cancer receiving sorafenib. Seventy-one of 114 patients included in the randomised phase II trial MAPS (chemotherapy plus or not sorafenib) and with available serum LDH levels, were included in this analysis. Patients were categorized according to serum LDH levels (LDH ≤vs.> upper normal rate). A significant difference was found in progression free survival (PFS) and in overall survival (OS) between patients with LDH values under or above the cut-off (PFS: 5.2 vs. 2.7 months, p = 0.0287; OS: 10.7 vs. 5.9 months, p = 0.0021). After stratification according to LDH serum levels and sorafenib treatment, patients with low LDH serum levels treated with sorafenib showed an advantage in PFS (p = 0.05) and OS (p = 0.0012). LDH appears to be a reliable parameter to assess the prognosis of advanced pancreatic cancer patients, and it may be a predictive parameter to select patients candidate to receive sorafenib. PMID:26397228

  12. Effects and Mechanism of Atmospheric-Pressure Dielectric Barrier Discharge Cold Plasma on Lactate Dehydrogenase (LDH) Enzyme

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Xu, Zimu; Shen, Jie; Li, Xu; Ding, Lili; Ma, Jie; Lan, Yan; Xia, Weidong; Cheng, Cheng; Sun, Qiang; Zhang, Zelong; Chu, Paul K.

    2015-05-01

    Proteins are carriers of biological functions and the effects of atmospheric-pressure non-thermal plasmas on proteins are important to applications such as sterilization and plasma-induced apoptosis of cancer cells. Herein, we report our detailed investigation of the effects of helium-oxygen non-thermal dielectric barrier discharge (DBD) plasmas on the inactivation of lactate dehydrogenase (LDH) enzyme solutions. Circular dichroism (CD) and dynamic light scattering (DLS) indicate that the loss of activity stems from plasma-induced modification of the secondary molecular structure as well as polymerization of the peptide chains. Raising the treatment intensity leads to a reduced alpha-helix content, increase in the percentage of the beta-sheet regions and random sequence, as well as gradually decreasing LDH activity. However, the structure of the LDH plasma-treated for 300 seconds exhibits a recovery trend after storage for 24 h and its activity also increases slightly. By comparing direct and indirect plasma treatments, plasma-induced LDH inactivation can be attributed to reactive species (RS) in the plasma, especially ones with a long lifetime including hydrogen peroxide, ozone, and nitrate ion which play the major role in the alteration of the macromolecular structure and molecular diameter in lieu of heat, UV radiation, and charged particles.

  13. The Arabidopsis KS-type dehydrin recovers lactate dehydrogenase activity inhibited by copper with the contribution of His residues.

    PubMed

    Hara, Masakazu; Monna, Shuhei; Murata, Takae; Nakano, Taiyo; Amano, Shono; Nachbar, Markus; Wätzig, Hermann

    2016-04-01

    Dehydrin, which is one of the late embryogenesis abundant (LEA) proteins, is involved in the ability of plants to tolerate the lack of water. Although many reports have indicated that dehydrins bind heavy metals, the physiological role of this metal binding has not been well understood. Here, we report that the Arabidopsis KS-type dehydrin (AtHIRD11) recovered the lactate dehydrogenase (LDH) activity denatured by Cu(2+). The LDH activity was partially inhibited by 0.93 μM Cu(2+) but totally inactivated by 9.3 μM Cu(2+). AtHIRD11 recovered the activity of LDH treated with 9.3 μM Cu(2+) in a dose-dependent manner. The recovery activity of AtHIRD11 was significantly higher than those of serum albumin and lysozyme. The conversion of His residues to Ala in AtHIRD11 resulted in the loss of the Cu(2+) binding of the protein as well as the disappearance of the conformational change induced by Cu(2+) that is observed by circular dichroism spectroscopy. The mutant protein showed lower recovery activity than the original AtHIRD11. These results indicate that AtHIRD11 can reactivate LDH inhibited by Cu(2+) via the His residues. This function may prevent physiological damage to plants due to heavy-metal stress. PMID:26940498

  14. Effects and Mechanism of Atmospheric-Pressure Dielectric Barrier Discharge Cold Plasma on Lactate Dehydrogenase (LDH) Enzyme

    PubMed Central

    Zhang, Hao; Xu, Zimu; Shen, Jie; Li, Xu; Ding, Lili; Ma, Jie; Lan, Yan; Xia, Weidong; Cheng, Cheng; Sun, Qiang; Zhang, Zelong; Chu, Paul K.

    2015-01-01

    Proteins are carriers of biological functions and the effects of atmospheric-pressure non-thermal plasmas on proteins are important to applications such as sterilization and plasma-induced apoptosis of cancer cells. Herein, we report our detailed investigation of the effects of helium-oxygen non-thermal dielectric barrier discharge (DBD) plasmas on the inactivation of lactate dehydrogenase (LDH) enzyme solutions. Circular dichroism (CD) and dynamic light scattering (DLS) indicate that the loss of activity stems from plasma-induced modification of the secondary molecular structure as well as polymerization of the peptide chains. Raising the treatment intensity leads to a reduced alpha-helix content, increase in the percentage of the beta-sheet regions and random sequence, as well as gradually decreasing LDH activity. However, the structure of the LDH plasma-treated for 300 seconds exhibits a recovery trend after storage for 24 h and its activity also increases slightly. By comparing direct and indirect plasma treatments, plasma-induced LDH inactivation can be attributed to reactive species (RS) in the plasma, especially ones with a long lifetime including hydrogen peroxide, ozone, and nitrate ion which play the major role in the alteration of the macromolecular structure and molecular diameter in lieu of heat, UV radiation, and charged particles. PMID:25992482

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

    PubMed Central

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

    2014-01-01

    The thermostable d-lactate dehydrogenase from Lactobacillus jensenii (Lj d-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 Lj d-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 (V M) is 2.58 Å3 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

  16. Semi-Rational Design of Geobacillus stearothermophilus L-Lactate Dehydrogenase to Access Various Chiral α-Hydroxy Acids.

    PubMed

    Aslan, Aşkın Sevinç; Birmingham, William R; Karagüler, Nevin Gül; Turner, Nicholas J; Binay, Barış

    2016-06-01

    Chiral α-hydroxy acids (AHAs) are rapidly becoming important synthetic building blocks, in particular for the production of pharmaceuticals and other fine chemicals. Chiral compounds of a variety of functionalities are now often derived using enzymes, and L-lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (bsLDH) has the potential to be employed for the industrial synthesis of chiral α-hydroxy acids. Despite the thorough characterization of this enzyme, generation of variants with high activity on non-natural substrates has remained difficult and therefore limits the use of bsLDH in industry. Here, we present the engineering of bsLDH using semi-rational design as a method of focusing screening in a small and smart library for novel biocatalysts. In this study, six mutant libraries were designed in an effort to expand the substrate range of bsLDH. The eight variants identified as having enhanced activity toward the selected α-keto acids belonged to the same library, which targeted two positions simultaneously. These new variants now may be useful biocatalysts for chiral synthesis of α-hydroxy acids. PMID:26852025

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

    PubMed

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

    2013-03-19

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

  18. The value of lactate dehydrogenase serum levels as a prognostic and predictive factor for advanced pancreatic cancer patients receiving sorafenib.

    PubMed

    Faloppi, Luca; Bianconi, Maristella; Giampieri, Riccardo; Sobrero, Alberto; Labianca, Roberto; Ferrari, Daris; Barni, Sandro; Aitini, Enrico; Zaniboni, Alberto; Boni, Corrado; Caprioni, Francesco; Mosconi, Stefania; Fanello, Silvia; Berardi, Rossana; Bittoni, Alessandro; Andrikou, Kalliopi; Cinquini, Michela; Torri, Valter; Scartozzi, Mario; Cascinu, Stefano

    2015-10-27

    Although lactate dehydrogenase (LDH) serum levels, indirect markers of angiogenesis, are associated with a worse outcome in several tumours, their prognostic value is not defined in pancreatic cancer. Moreover, high levels are associated even with a lack of efficacy of tyrosine kinase inhibitors, contributing to explain negative results in clinical trials. We assessed the role of LDH in advanced pancreatic cancer receiving sorafenib. Seventy-one of 114 patients included in the randomised phase II trial MAPS (chemotherapy plus or not sorafenib) and with available serum LDH levels, were included in this analysis. Patients were categorized according to serum LDH levels (LDH ≤ vs.> upper normal rate). A significant difference was found in progression free survival (PFS) and in overall survival (OS) between patients with LDH values under or above the cut-off (PFS: 5.2 vs. 2.7 months, p = 0.0287; OS: 10.7 vs. 5.9 months, p = 0.0021). After stratification according to LDH serum levels and sorafenib treatment, patients with low LDH serum levels treated with sorafenib showed an advantage in PFS (p = 0.05) and OS (p = 0.0012). LDH appears to be a reliable parameter to assess the prognosis of advanced pancreatic cancer patients, and it may be a predictive parameter to select patients candidate to receive sorafenib. PMID:26397228

  19. Pressure-adaptive differences in lactate dehydrogenases of three hagfishes: Eptatretus burgeri, Paramyxine atami and Eptatretus okinoseanus.

    PubMed

    Nishiguchi, Yoshikazu; Miwa, Tetsuya; Abe, Fumiyoshi

    2008-05-01

    The tolerance of abyssal pressures likely depends on adaptive modifications of fish proteins. However, structural modifications of proteins which allow functioning at high pressure remain unclear. We compared the activities of lactate dehydrogenase (LDH), an important enzyme in glycolytic reaction, in three hagfishes inhabiting different depths under increased pressure. LDH in Eptatretus okinoseanus, found at a depth of 1,000 m, was highly active at high pressure of 100 MPa maintaining the activity at 70% of that at 0.1 MPa. In contrast, LDH activity in Paramyxine atami, found at 250-400 m, decreased to 55% at 15 MPa, and that in Eptatretus burgeri, found at 45-60 m, was completely absent at 5 MPa. The result suggests that subunit interaction of the LDH-tetramer is more stable in E. okinoseanus than that in P. atami and E. burgeri under high-pressure conditions. We found six amino acid substitutions between the three LDH primary structures. Accordingly, these amino acid residues are likely to contribute to the stability of the E. okinoseanus LDH under high-pressure conditions. PMID:18299796

  20. Synbiotics suppress the release of lactate dehydrogenase, promote non-specific immunity and integrity of jejunum mucosa in piglets.

    PubMed

    Andrejčáková, Zuzana; Sopková, Drahomíra; Vlčková, Radoslava; Kulichová, Lucia; Gancarčíková, Soňa; Almášiová, Viera; Holovská, Katarína; Petrilla, Vladimír; Krešáková, Lenka

    2016-09-01

    The aim of our experiment was to study how synbiotics are able to deal with the problems of post-weaning piglets. Lactobacillus plantarum - Biocenol(TM) LP96 (CCM 7512), Lactobacillus fermentum - Biocenol(TM) LF99 (CCM 7514) and flaxseed (rich in n-3 polyunsaturated fatty acids) were administered to 36 conventional piglets from a problematic breed with confirmed presence of enterotoxigenic Escherichia coli and Coronavirus. The experimental piglets were supplied with probiotic cheeses and crushed flax-seed in the period starting 10 days before weaning and lasting up to 14 days post-weaning. Piglets in the control group were supplied only control cheese. The impact of such additives on the release of lactate dehydrogenase (LDH; spectroscopic and electrophoretic assay), alteration of immunity (index of metabolic activity), jejunum histology (light microscopy), and health of conventional piglets from a problematic breed (monitoring of hematology, consistency and moisture of feces and body temperature) were examined. We found significant decrease in LDH leakage in the blood serum and tissue extracts, indicating better cell membrane integrity in the individual organs of animals. Probiotics and flaxseed applied together seem to be a good source of nutrients to improve the immune status and the integrity of jejunum mucosa during infection. © 2015 Japanese Society of Animal Science. PMID:27581561

  1. Cryptosporidium Lactate Dehydrogenase Is Associated with the Parasitophorous Vacuole Membrane and Is a Potential Target for Developing Therapeutics

    PubMed Central

    Zhu, Guan

    2015-01-01

    Abstract The apicomplexan, Cryptosporidium parvum, possesses a bacterial-type lactate dehydrogenase (CpLDH). This is considered to be an essential enzyme, as this parasite lacks the Krebs cycle and cytochrome-based respiration, and mainly–if not solely, relies on glycolysis to produce ATP. Here, we provide evidence that in extracellular parasites (e.g., sporozoites and merozoites), CpLDH is localized in the cytosol. However, it becomes associated with the parasitophorous vacuole membrane (PVM) during the intracellular developmental stages, suggesting involvement of the PVM in parasite energy metabolism. We characterized the biochemical features of CpLDH and observed that, at lower micromolar levels, the LDH inhibitors gossypol and FX11 could inhibit both CpLDH activity (Ki = 14.8 μM and 55.6 μM, respectively), as well as parasite growth in vitro (IC50 = 11.8 μM and 39.5 μM, respectively). These observations not only reveal a new function for the poorly understood PVM structure in hosting the intracellular development of C. parvum, but also suggest LDH as a potential target for developing therapeutics against this opportunistic pathogen, for which fully effective treatments are not yet available. PMID:26562790

  2. Ontogenetic changes in citrate synthase and lactate dehydrogenase activity in the jumping muscle of the American locust (Schistocerca americana).

    PubMed

    Kirkton, Scott D; Nyberg, Elizabeth T; Fox, Kristin M

    2011-10-01

    Intraspecific studies have repeatedly shown that muscle-specific oxidative enzyme activities scale negatively with body mass while muscle-specific glycolytic enzyme activities scale positively. However, most of these studies have not included juveniles. In this study, we examined how citrate synthase (CS, EC 2.3.3.1) and lactate dehydrogenase (LDH; EC 1.1.1.27) activity in the jumping muscle of Schistocerca americana grasshoppers varied with ontogeny across a 40-fold increase in body size. In contrast to the pattern observed when adult conspecifics are compared, we show that jumping muscle CS activity increased more than 2-fold from 2nd instars to adults, while jumping muscle LDH activity increased more than 5-fold. The increased LDH activity in older grasshoppers supports previous data that older grasshoppers have a reduced jumping endurance. The increased CS activity with age may help older grasshoppers efficiently produce aerobic ATP to bend cuticular springs for energy storage before a jump or alternatively recover from anaerobic metabolism after jumping. Metabolic changes in S. americana jumping muscle are similar to other developing taxa and highlight the importance of including juveniles within intraspecific studies. When compared to adults, juvenile locomotion may have increased selection pressure because of both greater energetic demands during growth and higher predation rates. PMID:21807111

  3. The effect of 3-ketosteroid-Δ(1)-dehydrogenase isoenzymes on the transformation of AD to 9α-OH-AD by Rhodococcus rhodochrous DSM43269.

    PubMed

    Liu, Yang; Shen, Yanbing; Qiao, Yuqian; Su, Liqiu; Li, Can; Wang, Min

    2016-09-01

    Rhodococcus rhodochrous DSM43269 is well known for its 3-ketosteroid-9α-hydroxylases. However, the function of its 3-ketosteroid-Δ(1)-dehydrogenases (KSDD) remains unknown. This study compared the involvement of ksdds in the strain's androst-4-ene-3,17-dione (AD) transformation via gene deletion. The conversion was performed using AD as substrate or directly with 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD). The single deletion of ksdd1 or ksdd3 did not appear to result in the accumulation of 9α-OH-AD, whereas the single mutant △ksdd2 could preserve this compound to some extent. To further compare the role of ksdds in this strain, double mutants were constructed. All ksdd2 mutants combined with ksdd1 and/or ksdd3 resulted in the accumulation of 9α-OH-AD, among which the double mutant △ksdd2,3 behaved similarly to the single mutant △ksdd2 in this process. The mutant that lacked both ksdd1 and ksdd3 was still displayed, with no effect on the degradation of 9α-OH-AD. The triple mutant △ksdd1,2,3 was then constructed and exhibited the same capability as △ksdd1,2, accumulating more 9α-OH-AD than △ksdd2,3 and △ksdd2. The transcription of KSDD1 and KSDD2 increased, whereas that of KSDD3 seemed to exhibit no change, despite the use of the inducer AD or 9α-OH-AD. Thus, only ksdd1 and ksdd2 were involved in the transformation of AD to 9α-OH-AD. ksdd2 had the main role, ksdd1 had a minor effect on 9α-OH-AD degradation, and ksdd3 did not exhibit any action in this course. PMID:27377798

  4. Biochemical and in silico Characterization of Recombinant L-Lactate Dehydrogenase of Theileria annulata.

    PubMed

    Nural, Belma; Erdemir, Aysegul; Mutlu, Ozal; Yakarsonmez, Sinem; Danis, Ozkan; Topuzogullari, Murat; Turgut-Balik, Dilek

    2016-04-01

    Theileria annulata is a parasite that causes theileriosis in cattle. Reports about drug resistance made essential to develop new drug. LDH of Theileria schizonts is the vital enzyme for its anaerobic metabolism. TaLDH gene was first cloned into pGEM-T cloning vector with two introns in our previous study. Here we report cloning of TaLDH without introns into pLATE 31 vector in E. coli BL21(DE3). Protein was in an inactive form. Two mutations were fixed to express the active protein. Protein was purified by affinity chromatography and evaluated by SDS-PAGE and size exclusion chromatography. Optimum pH of enzyme was performed in pH 7.5, and enzyme was stabilized at 20-40 °C. Enzyme kinetics of recombinant TaLDH were found to be in the direction of pyruvate to lactate K m 0.1324 and K i 4.295 mM, k cat, 44.55/s and k cat /K m, 3.3693 × 10(5)/M/s. 3D structure of TaLDH was predicted, and possible drug binding sites were determined by homology modelling. PMID:26921192

  5. Distribution of metabolic fluxes towards glycerol phosphate and L-lactate from fructose 1,6-biphosphate in vitro: effect of glycerol phosphate dehydrogenase.

    PubMed

    Riol-Cimas, J M; Meléndez-Hevia, E

    1986-01-01

    A metabolic system in vitro, which converts fructose 1,6-biphosphate into the two alternative products, lactate and glycerol phosphate, was designed to study the distribution of metabolic fluxes and, specifically, the control of glycerol phosphate production rate in rat muscle extract. Experiments were carried out at several protein concentrations by dilution of rat muscle extract, showing non-linear behaviours of flux versus protein concentration. These were hyperbolic for glycerol phosphate and logarithmic for L-lactate. The influence of the flux towards any alternate product on the rate giving the other was studied by stimulation of each. Results obtained show that in this system, flux towards glycerol phosphate is not affected by lactate production and the same occurs for the contrary case. Glycerol phosphate dehydrogenase seems to be the only enzyme in this system whose activity controls the flux towards glycerol phosphate. PMID:3758466

  6. ALP isoenzyme test

    MedlinePlus

    Alkaline phosphatase isoenzyme test ... anything for 10 to 12 hours before the test, unless your health care provider tells you to do so. Many medicines can interfere with blood test results. Your health care provider will tell you ...

  7. Impact of Pre-Treatment Lactate Dehydrogenase Levels on Prognosis and Bevacizumab Efficacy in Patients with Metastatic Colorectal Cancer

    PubMed Central

    Passardi, Alessandro; Scarpi, Emanuela; Tamberi, Stefano; Cavanna, Luigi; Tassinari, Davide; Fontana, Annalisa; Pini, Sara; Bernardini, Ilaria; Accettura, Caterina; Ulivi, Paola; Frassineti, Giovanni Luca; Amadori, Dino

    2015-01-01

    Background To investigate the impact of pre-treatment lactate dehydrogenase (LDH) levels on the outcome of patients with metastatic colorectal cancer treated with first-line chemotherapy with or without the anti-VEGF monoclonal antibody, bevacizumab, in a phase III prospective multicentre randomized ITACa (Italian Trial in Advanced Colorectal Cancer) trial. Methods Three hundred and seventy patients enrolled onto the ITACa first-line trial were considered for this study, 176 receiving chemotherapy (either FOLFIRI or FOLFOX) plus bevacizumab and 194 receiving chemotherapy only. Pre-treatment LDH levels were evaluated to identify a potential correlation with progression-free survival (PFS), overall survival (OS) and objective response rate. Results Information on pre-treatment LDH levels was available for 344 patients. High LDH levels were predictive of a lower median PFS (8.1 months vs. 9.2 months, p< 0.0001) and median OS (16.1 months vs. 25.2 months, p< 0.0001) in the overall population. In the chemotherapy plus bevacizumab group, median PFS was 9.1 and 9.8 months in patients with high LDH and low LDH, respectively (p= 0.073), whereas in the chemotherapy-only arm it was 6.9 and 9.1 months, respectively (p < 0.0001). In patients with high LDH, the addition of bevacizumab to chemotherapy led to a reduction in the rate of progressive disease (16.4 vs. 30.5%, p= 0.081) and to a prolonged PFS (p= 0.028). Conclusion A high LDH value was confirmed as a marker of poor prognosis. Bevacizumab reduced the progressive disease rate and improved PFS in the high-LDH subgroup, making serum LDH a potentially effective an easily available and marker to select patients who benefit from bevacizumab. Trial Registration NCT01878422 ClinicalTrials.gov PMID:26244985

  8. Prognostic value of combined preoperative lactate dehydrogenase and alkaline phosphatase levels in patients with resectable pancreatic ductal adenocarcinoma.

    PubMed

    Ji, Fei; Fu, Shun-Jun; Guo, Zhi-Yong; Pang, Hui; Ju, Wei-Qiang; Wang, Dong-Ping; Hua, Yun-Peng; He, Xiao-Shun

    2016-07-01

    Serum enzymes, including lactate dehydrogenase (LDH) and alkaline phosphatase (ALP), have recently been reported to play important roles in tumor growth. Increases in LDH and ALP have been confirmed to predict poor prognosis in patients with various cancers. However, their prognostic value in pancreatic cancer has not been well studied. Therefore, we reviewed the preoperative data on LDH and ALP in 185 pancreatic ductal adenocarcinoma (PDAC) patients who underwent surgery between July 2005 and December 2010 to explore the prognostic value of these markers. The cutoff points were determined based on the upper limit of their normal values. The Chi-square test was used to analyze the relationships between LDH/ALP and clinical characteristics. Univariate and multivariate analyses were performed to identify the predictive value of the above factors for disease-free survival (DFS) and overall survival (OS). We found that elevation of LDH was related to carbohydrate antigen 19-9 (CA19-9), lymph node involvement, tumor size, TNM, distant metastasis, and recurrence. Additionally, ALP was correlated to perineural invasion. After multivariate analysis, LDH and ALP were identified as independent prognostic factors for DFS and OS, and elevation of LDH/ALP was correlated with poor DFS and OS. Notably, there was a positive correlation between LDH and ALP. The predictive power of LDH combined with ALP was more sensitive than that of either one alone. Therefore, we conclude that the preoperative LDH and ALP values are prognostic factors for PADC, and the prognostic accuracy of testing can be enhanced by the combination of LDH and ALP. PMID:27399091

  9. Effect of Follicular Fluid and Platelet-Activating Factor on Lactate Dehydrogenase C Expression in Human Asthenozoospermic Samples

    PubMed Central

    Esmaeilpour, Tahereh; Zarei, Mohmmad-Reza; Bahmanpour, Soghra; Aliabadi, Elham; Hosseini, Ahmad; Jaberipour, Mansooreh

    2014-01-01

    Background: Application of follicular fluid (FF) and platelet-activating factor (PAF) in artificial insemination improves sperm motility. Lactate dehydrogenase C (LDH-C) is a key enzyme for sperm motility. In this study, the effects of FF and PAF on the sperm motility index and LDH-C expression were investigated. Moreover, LDH-C expression was compared between asthenozoospermic and normozoospermic samples. Methods: The expression of LDH-C was examined by quantitative real-time polymerase chain reaction (q-RT PCR) and western blotting after it was treated with optimized concentrations of FF and PAF in twenty asthenozoospermic samples. Also, LDH-C expression was evaluated in five normozoospermic samples. Results: Samples with 75% FF and 100 nM of PAF had an increase in their percentages of progressive and slowly motile sperms and a decrease in their percentages of non-progressive and non-motile sperms. Moreover, LDH-C mRNA transcripts were not changed following PAF and FF treatment, and LDH-C protein was detected in highly progressive motile specimens treated with FF in the asthenozoospermic samples. Furthermore, LDH-C expression was more detectable in the normal sperms. Conclusion: Our results indicated that PAF had more beneficial effects than FF on sperm motility in the asthenozoospermic samples (P=0.0001), although the LDH-C expressions of the sperms were not changed significantly in both groups. We found no association between LDH-C expression and sperm motility after FF and PAF actions. This finding, however, requires further investigation. The fact that LDH-C protein was detected in the normozoospermic, but not asthenozoospermic, samples could be cited as a reason for the infertility in these patients. PMID:24453390

  10. Lactate dehydrogenase release as an indicator of dithranol-induced membrane injury in cultured human keratinocytes. A time profile study.

    PubMed

    Bonnekoh, B; Farkas, B; Geisel, J; Mahrle, G

    1990-01-01

    HaCaT cells, a rapidly multiplying human keratinocyte line, were tested for their sensitivity to antipsoriatic dithranol with regard to classical proliferation parameters and for the drug's action on the plasma membrane integrity by the dose- and time-dependent release of cytosolic lactate dehydrogenase (LDH). In the case of 3H thymidine as well as 14C amino acid incorporation the 50% inhibition concentration (IC50) was 0.2 microM dithranol 24 h after initial exposure to the drug. For protein content of attached cells the IC50 proved to be greater than 3.0 microM. Using 0.3, 1.0 and 3.0 microM dithranol, significant (p less than 0.05) dose dependent LDH release of 0.866 +/- 0.387, 1.842 +/- 1.127 and 2.938 +/- 1.635 mU per hour and cm2 confluent culture area was measured between the 5th and the 24th hour, compared to an acetone control of 0.504 +/- 0.299 mU/h x cm2. Between the 2nd and the 4th hour as well as from the 25th to the 48th hour and the 49th to the 72nd hour the LDH release after dithranol treatment did not exceed the control value. In accordance with these findings dose-dependent morphological signs of cell injury were detected by phase contrast microscopy beyond the 4th hour. The data reveal that: HaCaT cells are a very sensitive target for the antiproliferative action of dithranol; the drug causes considerable plasma membrane damage even at concentrations as low as 0.3 microM; and this membrane damage becomes evident after a latency of at least 4 h and for a limited period of up to 24 h. PMID:2221984

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

    SciTech Connect

    Liao, Ya-Tang; Chen, Chien-Jen; Li, Wan-Fen; Hsu, Ling-I; Tsai, Li-Yu; Huang, Yeou-Lih; Sun, Chien-Wen; Chen, Wei J.; Wang, Shu-Li

    2012-08-01

    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.

  12. Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome

    PubMed Central

    Mohammad, Goran Hamid; Olde Damink, S. W. M.; Malago, Massimo; Dhar, Dipok Kumar; Pereira, Stephen P.

    2016-01-01

    Pancreatic cancer has a 5-year survival rate of less than 4%. Despite advances in diagnostic technology, pancreatic cancer continues to be diagnosed at a late and incurable stage. Accurate biomarkers for early diagnosis and to predict treatment response are urgently needed. Since alteration of glucose metabolism is one of the hallmarks of cancer cells, we proposed that pyruvate kinase type M2 (M2PK) and lactate dehydrogenase A (LDHA) enzymes could represent novel diagnostic markers and potential therapeutic targets in pancreatic cancer. In 266 tissue sections from normal pancreas, pancreatic cystic neoplasms, pancreatic intraepithelial neoplasia (PanIN) and cancer, we evaluated the expression of PKM2, LDHA, Ki-67 and CD8+ by immunohistochemistry and correlated these markers with clinicopathological characteristics and patient survival. PKM2 and LDHA expression was also assessed by Western blot in 10 human pancreatic cancer cell lines. PKM2 expression increased progressively from cyst through PanIN to cancer, whereas LDHA was overexpressed throughout the carcinogenic process. All but one cell line showed high expression of both proteins. Patients with strong PKM2 and LDHA expression had significantly worse survival than those with weak PKM2 and/or LDHA expression (7.0 months vs. 27.9 months, respectively, p = 0.003, log rank test). The expression of both PKM2 and LDHA correlated directly with Ki-67 expression, and inversely with intratumoral CD8+ cell count. PKM2 was significantly overexpressed in poorly differentiated tumours and both PKM2 and LDHA were overexpressed in larger tumours. Multivariable analysis showed that combined expression of PKM2 and LDHA was an independent poor prognostic marker for survival. In conclusion, our results demonstrate a high expression pattern of two major glycolytic enzymes during pancreatic carcinogenesis, with increased expression in aggressive tumours and a significant adverse effect on survival. PMID:26989901

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

    PubMed Central

    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

    2014-01-01

    Summary The lactate dehydrogenase-A (LDH-A) enzyme catalyzes the inter-conversion of pyruvate and lactate, is upregulated in human cancers and is associated with aggressive tumor outcomes. Here we use a novel 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 re-activation 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

  14. Contribution of a buried aspartate residue towards the catalytic efficiency and structural stability of Bacillus stearothermophilus lactate dehydrogenase.

    PubMed Central

    Nobbs, T J; Cortés, A; Gelpi, J L; Holbrook, J J; Atkinson, T; Scawen, M D; Nicholls, D J

    1994-01-01

    The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195. This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein. A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior. In this respect the side-chain carboxylate group of Asp-143 appears to be important for maintaining tertiary structure by providing a common linkage point between three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H. The contribution of the Asp-143 side-chain to the structure and function of Bacillus stearothermophilus LDH was assessed by creating a mutant enzyme containing Asn-143. The decreased thermal stability of both unactivated and fructose-1,6-diphosphate (Fru-1,6-P2)-activated forms of the mutant enzyme support a structural role for Asp-143. Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chloride suggested that the carboxylate group of Asp-143 contributes at least 22 kJ/mol to the conformational stability of the wild-type enzyme. However, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation caused a structural weakness rather than a gross conformational change. Comparison of the wild-type and mutant enzyme steady-state parameters for various 2-keto acid substrates showed the mutation to have a general effect on catalysis, with an average difference in binding energy of 11 kJ/mol for the transition-state complexes. The different effects of pH and Fru-1,6-P2 on the wild-type and

  15. Cloning of the Staphylococcus aureus ddh gene encoding NAD+-dependent D-lactate dehydrogenase and insertional inactivation in a glycopeptide-resistant isolate.

    PubMed Central

    Boyle-Vavra, S; de Jonge, B L; Ebert, C C; Daum, R S

    1997-01-01

    The mechanism of low-level glycopeptide resistance among staphylococci is not known. A cytoplasmic protein, provisionally called Ddh (W. M. Milewski, S. Boyle-Vavra, B. Moreira, C. C. Ebert, and R. S. Daum, Antimicrob. Agents Chemother. 40:166-172, 1996), and the RNA transcript that contains the ddh gene, which encodes Ddh, are present in increased amounts in a vancomycin-resistant isolate, 523k, compared with the susceptible parent isolate, 523. Sequence analysis had previously revealed that Ddh is related to NAD+-dependent D-lactate dehydrogenase (D-nLDH) and VanH. This latter protein is essential for high-level glycopeptide resistance in Enterococcus faecium and Enterococcus faecalis by synthesizing the D-lactate needed for biosynthesis of D-lactate-terminating peptidoglycan precursors with low affinity for vancomycin. We now provide the direct evidence that the ddh gene product is Staphylococcus aureus D-nLDH and hereafter refer to the protein as D-nLDH. However, overproduction of this protein in isolate 523k did not result in production of D-lactate-containing peptidoglycan precursors, and susceptibility testing of ddh mutants of 523k demonstrated that S. aureus D-nLDH is not necessary for glycopeptide resistance in this isolate. We conclude that the mechanism of glycopeptide resistance in this isolate is distinct from that in enterococci. PMID:9352927

  16. Screening and isolation of potential lactate dehydrogenase inhibitors from five Chinese medicinal herbs: Soybean, Radix pueraria, Flos pueraria, Rhizoma belamcandae, and Radix astragali.

    PubMed

    Tang, Ying; Li, Senlin; Li, Sainan; Yang, Xiaojing; Qin, Yao; Zhang, Yuchi; Liu, Chunming

    2016-06-01

    Stroke is among the leading causes of death and severe disability worldwide. Flavonoids have been extensively used in the treatment of ischemic stroke by reducing lactate dehydrogenase levels and thereby enhancing blood perfusion to the ischemic region. Here, we used ultrafiltration high-performance liquid chromatography coupled with diode array detection and mass spectrometry for the rapid screening and identification of flavonoids from five Chinese medicinal herbs: soybean, Radix pueraria, Flos pueraria, Rhizoma belamcandae, and Radix astragali. Using PC12 cells as a suitable in vitro model of toxicity, cell viability was quantitated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results showed that the extracts of soybean and the six major components, namely, acetyldaidzin, malonylgenistin, daidiain, glycitin, genistin, and acetylcitin; the extract of R. pueraria and its main component daidzein; the extract of F. pueraria and its three major components, tectorigenin, tectoridin, and tectorigenin-7-O-xylosylglucosid; and the extract of R. belamcandae and its main component, tectoridin, were strong lactate dehydrogenase inhibitors. Also, the components of R. astragali showed no bioactivity. These findings indicate that the ultrafltration high-performance liquid chromatography coupled with diode array detection and mass spectrometry method could be utilized in rapid screening and separation of bioactive compounds from a complex matrix. PMID:27059876

  17. Changes in milk yield, lactate dehydrogenase, milking frequency, and interquarter yield ratio persist for up to 8 weeks after antibiotic treatment of mastitis.

    PubMed

    Fogsgaard, K K; Løvendahl, P; Bennedsgaard, T W; Østergaard, S

    2015-11-01

    Within the dairy industry, the appearance of milk and withdrawal time due to antibiotic residuals in the milk are used to determine recovery status after cases of treated mastitis. However, both milk production and dairy cow behavior have been shown to be affected after the normalization of milk appearance, indicating that animals may not have fully recovered. The aim of the present study was to describe the changes in milk yield, lactate dehydrogenase activity, milking frequency, and interquarter yield ratio (defined as the coefficient of variation between the active quarters) after cases of naturally occurring mastitis with special focus on the recovery period after antibiotic treatment. A second aim was to examine whether these changes were affected by the pathogens present at the time of mastitis diagnosis. This retrospective study was based on a cohort data set including 1,032 lactations from 795 dairy cows kept on 2 Danish farms and milked by an automatic milking system. A total of 174 treated mastitis cases were compared with nontreated control cows from 5 wk before treatment and until 8 wk after. Treated mastitis resulted in reduced milk yield, elevated lactate dehydrogenase activity, lower milking frequency, and elevated interquarter yield ratio. Within these measures, deviations from baseline levels and from the control cows were found as early as 1 to 3 wk before the antibiotic treatment and peaked around the days of treatment. In some cases, the mastitic cows returned to premastitis levels, whereas in others they remained affected throughout the rest of the observation period. To correctly estimate the effects of treated mastitis and the recovery status of cows, it is important to take the individual cow into account and not only compare with herd levels, as this might mask the true degree of the changes. The effects on each outcome variable depended on the involved pathogen and differences were found between primiparous cows and older animals. However

  18. Evaluation of three parasite lactate dehydrogenase-based rapid diagnostic tests for the diagnosis of falciparum and vivax malaria

    PubMed Central

    Ashley, Elizabeth A; Touabi, Malek; Ahrer, Margareta; Hutagalung, Robert; Htun, Khayae; Luchavez, Jennifer; Dureza, Christine; Proux, Stephane; Leimanis, Mara; Lwin, Myo Min; Koscalova, Alena; Comte, Eric; Hamade, Prudence; Page, Anne-Laure; Nosten, François; Guerin, Philippe J

    2009-01-01

    Background In areas where non-falciparum malaria is common rapid diagnostic tests (RDTs) capable of distinguishing malaria species reliably are needed. Such tests are often based on the detection of parasite lactate dehydrogenase (pLDH). Methods In Dawei, southern Myanmar, three pLDH based RDTs (CareStart™ Malaria pLDH (Pan), CareStart™ Malaria pLDH (Pan, Pf) and OptiMAL-IT®)were evaluated in patients presenting with clinically suspected malaria. Each RDT was read independently by two readers. A subset of patients with microscopically confirmed malaria had their RDTs repeated on days 2, 7 and then weekly until negative. At the end of the study, samples of study batches were sent for heat stability testing. Results Between August and November 2007, 1004 patients aged between 1 and 93 years were enrolled in the study. Slide microscopy (the reference standard) diagnosed 213 Plasmodium vivax (Pv) monoinfections, 98 Plasmodium falciparum (Pf) mono-infections and no malaria in 650 cases. The sensitivities (sens) and specificities (spec), of the RDTs for the detection of malaria were- CareStart Malaria™ pLDH (Pan) test: sens 89.1% [CI95 84.2-92.6], spec 97.6% [CI95 96.5-98.4] OptiMal-IT®: Pf+/- other species detection: sens 95.2% [CI95 87.5-98.2], spec 94.7% [CI95 93.3-95.8]; non-Pf detection alone: sens 89.6% [CI95 83.6-93.6], spec 96.5% [CI95 94.8-97.7] CareStart Malaria™ pLDH (Pan, Pf): Pf+/- other species: sens 93.5% [CI9585.4-97.3], spec 97.4% [95.9-98.3]; non-Pf: sens 78.5% [CI9571.1-84.4], spec 97.8% [CI95 96.3-98.7] Inter-observer agreement was excellent for all tests (kappa > 0.9). The median time for the RDTs to become negative was two days for the CareStart™ Malaria tests and seven days for OptiMAL-IT®. Tests were heat stable up to 90 days except for OptiMAL-IT® (Pf specific pLDH stable to day 20 at 35°C). Conclusion None of the pLDH-based RDTs evaluated was able to detect non-falciparum malaria with high sensitivity, particularly at low

  19. d-Lactate Dehydrogenase Gene (ldhD) Inactivation and Resulting Metabolic Effects in the Lactobacillus johnsonii Strains La1 and N312

    PubMed Central

    Lapierre, Luciane; Germond, Jacques-Edouard; Ott, Andreas; Delley, Michele; Mollet, Beat

    1999-01-01

    Lactobacillus johnsonii La1, a probiotic bacterium with demonstrated health effects, grows in milk, where it ferments lactose to d- and l-lactate in a 60:40% ratio. The d-lactate dehydrogenase (D-LDH) gene (ldhD) of this strain was isolated, and an in vitro-truncated copy of that gene was used to inactivate the genomic copy in two strains, La1 and N312, by gene replacement. For that, an 8-bp deletion was generated within the cloned ldhD gene to inactivate its function. The plasmid containing the altered ldhD was transferred to L. johnsonii via conjugative comobilization with Lactococcus lactis carrying pAMβ1. Crossover integrations of the plasmid at the genomic ldhD site were selected, and appropriate resolution of the cointegrate structures resulted in mutants that had lost the plasmid and in which the original ldhD was replaced by the truncated copy. These mutants completely lacked D-LDH activity. Nevertheless, the lower remaining L-LDH activity of the cells was sufficient to reroute most of the accumulating pyruvate to l-lactate. Only a marginal increase in production of the secondary end products acetaldehyde, diacetyl, and acetoin was observed. It can be concluded that in L. johnsonii D- and L-LDH are present in substantial excess for their role to eliminate pyruvate and regenerate NAD+ and that accumulated pyruvate is therefore not easily redirected in high amounts to secondary metabolic routes. PMID:10473408

  20. Carbon Flux Trapping: Highly Efficient Production of Polymer-Grade d-Lactic Acid with a Thermophilic d-Lactate Dehydrogenase.

    PubMed

    Li, Chao; Tao, Fei; Xu, Ping

    2016-08-17

    High production of polymer-grade d-lactic acid is urgently required, particularly for the synthesis of polylactic acid. High-temperature fermentation has multiple advantages, such as lower equipment requirement and energy consumption, which are essential for lowering operating costs. We identified and introduced a unique d-lactate dehydrogenase into a thermotolerant butane-2,3-diol-producing strain. Carbon flux "trapping" was achieved by a "trapping point" created by combination of the introduced enzyme and the host efflux pump, which afforded irreversible transport of d-lactic acid. The overall carbon flux of the engineered strain was significantly enhanced and was redistributed predominantly to d-lactic acid. Under optimized conditions at 50 °C, d-lactic acid reached the highest titer (226.6 g L(-1) ) reported to date. This discovery allows us to extend the carbon flux trapping strategy to engineering complex metabolic networks. PMID:27237045

  1. Immobilized metal ion affinity chromatography on Co2+-carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle.

    PubMed

    Chaga, G; Hopp, J; Nelson, P

    1999-02-01

    A rapid method for the purification of lactate dehydrogenase from whole chicken muscle extract in one chromatographic step is reported. The purification procedure can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent is used that can be utilized at linear flow rates higher than 5 cm/min. The final preparation of the enzyme was with purity higher than 95% as ascertained by SDS-PAGE. Three immobilized metal ions (Ni2+, Zn2+ and Co2+) were compared for their binding properties towards the purified enzyme. The binding site of the enzyme for immobilized intermediate metal ions was determined after cleavage with CNBr and binding studies of the derivative peptides on immobilized Co2+. A peptide located on the N-terminus of the enzyme, implicated in the binding, has great potential as a purification tag in fusion proteins. PMID:9889081

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

    PubMed

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

    2006-03-01

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

  3. Major Role of NAD-Dependent Lactate Dehydrogenases in the Production of l-Lactic Acid with High Optical Purity by the Thermophile Bacillus coagulans.

    PubMed

    Wang, Limin; Cai, Yumeng; Zhu, Lingfeng; Guo, Honglian; Yu, Bo

    2014-12-01

    Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production-NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)-were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6. PMID:25217009

  4. Major Role of NAD-Dependent Lactate Dehydrogenases in the Production of l-Lactic Acid with High Optical Purity by the Thermophile Bacillus coagulans

    PubMed Central

    Wang, Limin; Cai, Yumeng; Zhu, Lingfeng; Guo, Honglian

    2014-01-01

    Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production—NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)—were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6. PMID:25217009

  5. Elevated pretreatment serum levels of soluble vascular cell adhesion molecule 1 and lactate dehydrogenase as predictors of survival in cutaneous metastatic malignant melanoma.

    PubMed Central

    Franzke, A.; Probst-Kepper, M.; Buer, J.; Duensing, S.; Hoffmann, R.; Wittke, F.; Volkenandt, M.; Ganser, A.; Atzpodien, J.

    1998-01-01

    Very rapid progression of disease with a median survival of 6-9 months is a common feature of metastatic cutaneous malignant melanoma. Nevertheless, substantial variability of survival suggests that metastatic cutaneous malignant melanoma can be divided into several biological subgroups. Pretreatment serum levels of soluble adhesion molecules and various clinical parameters in cutaneous metastatic malignant melanoma were evaluated to determine their prognostic value. In this study pretreatment serum levels of soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble intercellular cell adhesion molecule 1 (sICAM-1), soluble endothelial leukocyte adhesion molecule 1 (sE-selectin) and multiple clinical factors were assessed in relation to overall survival of 97 consecutive patients with metastatic cutaneous malignant melanoma seen at our institution between May 1990 and April 1996. For statistical analysis, both univariate and multivariate Cox proportional-hazards models were used. Elevated pretreatment serum levels of sVCAM-1 (P < 0.005) and of lactate dehydrogenase (P < 0.002) were rendered statistically independent and were significantly associated with unfavourable outcome. Patients were assigned to one of three risk categories (low, intermediate and high) according to a cumulative risk score defined as the function of the sum of these two variables. There were significant differences in overall survival (P < 0.0001) between low- (n = 53, 5-year survival probability of 23.3%), intermediate- (n = 29, 5-year survival probability of 9.9%) and high-risk (n = 15) patients. Elevated pretreatment serum levels of sVCAM-1 and of lactate dehydrogenase correlate with poor outcome in metastatic cutaneous malignant melanoma. These data support risk stratification for future therapeutic trials and identify factors that need to be validated in prospective studies and may potentially influence decision-making in palliative management of patients with disseminated cutaneous

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

    PubMed Central

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

    2013-01-01

    An NAD-dependent d-lactate dehydrogenase (d-nLDH) of Lactobacillus bulgaricus ATCC 11842 was rationally re-designed for asymmetric reduction of a homologous series of α-keto carboxylic acids such as phenylpyruvic acid (PPA), α-ketobutyric acid, α-ketovaleric acid, β-hydroxypyruvate. Compared with wild-type d-nLDH, the Y52L mutant d-nLDH showed elevated activities toward unnatural substrates especially with large substitutes at C-3. By the biocatalysis combined with a formate dehydrogenase for in situ generation of NADH, the corresponding (R)-α-hydroxy carboxylic acids could be produced at high yields and highly optical purities. Taking the production of chiral (R)-phenyllactic acid (PLA) from PPA for example, 50 mM PPA was completely reduced to (R)-PLA in 90 min with a high yield of 99.0% and a highly optical purity (>99.9% e.e.) by the coupling system. The results presented in this work suggest a promising alternative for the production of chiral α-hydroxy carboxylic acids. PMID:24292439

  7. A chemically modified carbon paste electrode with d-lactate dehydrogenase and alanine aminotranferase enzyme sequences for d-lactic acid analysis.

    PubMed

    Shu, H C; Wu, N P

    2001-04-12

    An amperometric biosensor was constructed for the analysis of d-lactic acid based on immobilizing d-lactate dehydrogenase(d-LDH), alanine aminotransferase (ALT), NAD(+), a redox polymer and polyethylenimine in carbon paste. The effect of addition of ALT in the paste, using enzyme sequences of ALT/d-LDH, was insignificant for d-lactic acid analysis. The responses of d-lactic acid in ALT/d-LDH paste electrode are the same as those in d-LDH paste electrode. However, the interference effect of pyruvate in the sample can be substantially reduced if sodium glutamate was applied in the carrier solution. When ALT immobilized in control porous glass as an immobilized enzyme reactor (IMER) was mounted in flow injection analysis system with the d-LDH paste electrode as detector for d-lactate analysis, the interference of the pyruvate can be significantly eliminated. The adverse effect of pyruvate in the samples for d-lactic acid analysis was reduced more effectively in ALT IMER with d-LDH electrode than in ALT/d-LDH electrode. PMID:18968259

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

    SciTech Connect

    Zhang, Yanfeng; Gao, Xiaoli

    2012-08-31

    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.

  9. Stable Suppression of Lactate Dehydrogenase Activity during Anoxia in the Foot Muscle of Littorina littorea and the Potential Role of Acetylation as a Novel Posttranslational Regulatory Mechanism.

    PubMed

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

    2013-01-01

    The intertidal marine snail, Littorina littorea, has evolved to withstand extended bouts of oxygen deprivation brought about by changing tides or other potentially harmful environmental conditions. Survival is dependent on a strong suppression of its metabolic rate and a drastic reorganization of its cellular biochemistry in order to maintain energy balance under fixed fuel reserves. Lactate dehydrogenase (LDH) is a crucial enzyme of anaerobic metabolism as it is typically responsible for the regeneration of NAD(+), which allows for the continued functioning of glycolysis in the absence of oxygen. This study compared the kinetic and structural characteristics of the D-lactate specific LDH (E.C. 1.1.1.28) from foot muscle of aerobic control versus 24 h anoxia-exposed L. littorea. Anoxic LDH displayed a near 50% decrease in V max (pyruvate-reducing direction) as compared to control LDH. These kinetic differences suggest that there may be a stable modification and regulation of LDH during anoxia, and indeed, subsequent dot-blot analyses identified anoxic LDH as being significantly less acetylated than the corresponding control enzyme. Therefore, acetylation may be the regulatory mechanism that is responsible for the suppression of LDH activity during anoxia, which could allow for the production of alternative glycolytic end products that in turn would increase the ATP yield under fixed fuel reserves. PMID:24233354

  10. Stable Suppression of Lactate Dehydrogenase Activity during Anoxia in the Foot Muscle of Littorina littorea and the Potential Role of Acetylation as a Novel Posttranslational Regulatory Mechanism

    PubMed Central

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

    2013-01-01

    The intertidal marine snail, Littorina littorea, has evolved to withstand extended bouts of oxygen deprivation brought about by changing tides or other potentially harmful environmental conditions. Survival is dependent on a strong suppression of its metabolic rate and a drastic reorganization of its cellular biochemistry in order to maintain energy balance under fixed fuel reserves. Lactate dehydrogenase (LDH) is a crucial enzyme of anaerobic metabolism as it is typically responsible for the regeneration of NAD+, which allows for the continued functioning of glycolysis in the absence of oxygen. This study compared the kinetic and structural characteristics of the D-lactate specific LDH (E.C. 1.1.1.28) from foot muscle of aerobic control versus 24 h anoxia-exposed L. littorea. Anoxic LDH displayed a near 50% decrease in Vmax (pyruvate-reducing direction) as compared to control LDH. These kinetic differences suggest that there may be a stable modification and regulation of LDH during anoxia, and indeed, subsequent dot-blot analyses identified anoxic LDH as being significantly less acetylated than the corresponding control enzyme. Therefore, acetylation may be the regulatory mechanism that is responsible for the suppression of LDH activity during anoxia, which could allow for the production of alternative glycolytic end products that in turn would increase the ATP yield under fixed fuel reserves. PMID:24233354

  11. The lactate dehydrogenase--reduced nicotinamide--adenine dinucleotide--pyruvate complex. Kinetics of pyruvate binding and quenching of coeznyme fluorescence.

    PubMed

    Südi, J

    1974-04-01

    The stopped-flow kinetic studies described in this and the following paper (Südi, 1974) demonstrate that a Haldane-type description of the reversible lactate dehydrogenase reaction presents an experimentally feasible task. Combined results of these two papers yield numerical values for the six rate constants defined by the following equilibrium scheme, where E represents lactate dehydrogenase: [Formula: see text] The experiments were carried out at pH8.4 at a relatively low temperature (6.3 degrees C) with the pig heart enzyme. Identification of the above two intermediates and determination of the corresponding rate constants actually involve four series of independent observations in these studies, since (a) the reaction can be followed in both directions, and (b) both the u.v. absorption and the fluorescence of the coenzymes are altered in the reaction, and it is shown that these two spectral changes do not occur simultaneously. Kinetic observations made in the reverse direction are reported in this paper. It is demonstrated that the fluorescence of NADH can no longer be observed in the ternary complex E(NADH) (Pyr). Even though the oxidation-reduction reaction rapidly follows the formation of this complex, the numerical values of k(-4) (8.33x10(5)m(-1).s(-1)) and k(+4) (222s(-1)) are easily obtained from a directly observed second-order reaction step in which fluorescent but not u.v.-absorbing material is disappearing. U.v.-absorption measurements do not clearly resolve the subsequent oxidation-reduction step from the dissociation of lactate. It is shown that this must be due partly to the instrumental dead time, and partly to a low transient concentration of E(NAD+) (Lac) in the two-step sequential reaction in which the detectable disappearance of u.v.-absorbing material takes place. It is estimated that about one-tenth of the total change in u.v. absorption is due to a ;burst reaction' in which E(NAD+) (Lac) is produced, and this estimation yields, from k

  12. Differentiating inflamed and normal lungs by the apparent reaction rate constants of lactate dehydrogenase probed by hyperpolarized 13C labeled pyruvate

    PubMed Central

    Xu, He N.; Kadlececk, Stephen; Shaghaghi, Hoora; Zhao, Huaqing; Profka, Harilla; Pourfathi, Mehrdad; Rizi, Rahim

    2016-01-01

    Background Clinically translatable hyperpolarized (HP) 13C-NMR can probe in vivo enzymatic reactions, e.g., lactate dehydrogenase (LDH)-catalyzed reaction by injecting HP 13C-pyruvate into the subject, which is converted to 13C labeled lactate by the enzyme. Parameters such as 13C-lactate signals and lactate-to-pyruvate signal ratio are commonly used for analyzing the HP 13C-NMR data. However, the biochemical/biological meaning of these parameters remains either unclear or dependent on experimental settings. It is preferable to quantify the reaction rate constants with a clearer physical meaning. Here we report the extraction of the kinetic parameters of the LDH reaction from HP 13C-NMR data and investigate if they can be potential predictors of lung inflammation. Methods Male Sprague-Dawley rats (12 controls, 14 treated) were used. One dose of bleomycin (2.5 U/kg) was administered intratracheally to the treatment group. The lungs were removed, perfused, and observed by the HP-NMR technique, where a HyperSense dynamic nuclear polarization system was used to generate the HP 13C-pyruvate for injecting into the lungs. A 20 mm 1H/13C dual-tuned coil in a 9.4-T Varian vertical bore NMR spectrometer was employed to acquire the 13C spectral data every 1 s over a time period of 300 s using a non-selective, 15-degree radiofrequency pulse. The apparent rate constants of the LDH reaction and their ratio were quantified by applying ratiometric fitting analysis to the time series data of 13C labeled pyruvate and lactate. Results The apparent forward rate constant kp=(3.67±3.31)×10−4 s−1, reverse rate constant kl=(4.95±2.90)×10−2 s−1, rate constant ratio kp/kl=(7.53±5.75)×10−3 for the control lungs; kp=(11.71±4.35)×10−4 s−1, kl=(9.89±3.89)×10−2 s−1, and kp/kl=(12.39±4.18)×10−3 for the inflamed lungs at the 7th day post treatment. Wilcoxon rank-sum test showed that the medians of these kinetic parameters of the 7-day cohort were significantly

  13. Beta-2 microglobulin and lactate dehydrogenase levels are useful prognostic markers in early stage primary gastric lymphoma.

    PubMed

    Avilés, A; Narváez, B R

    1998-10-01

    The optimal management of primary gastric lymphoma (PGL) remains undecided because a definitive classification for therapeutic decision is not available. The International Index Project has proved to be useful in patients with nodal disease, but in extranodal presentation it has not been tested. We reviewed 297 patients with early stage PGL. They were initially classified according to the prognostic features of the International Index Project. No influence on duration of time to treatment failure (TTF) or overall survival was observed. For this reason we developed a logistical model to identify prognostic factors in patients with early stage PGL. Levels of beta-2 microglobulin and lactic dehydrogenase were observed to have prognostic significance in both univariate and multivariate analysis. With these parameters we constructed a logistical model to identify patients at low risk (TTF = 76%; at 7 years overall survival was 96%), statistically different to patients at high risk (TTF = 34% and overall survival = 22%). The number of patients at intermediate risk were too small to compare with the other groups. Because pathological or other clinical or laboratory prognostic features cannot help in the identification of a prognostic model, we propose that the use beta-2 microglobulin and lactic dehydrogenase can define different groups at risk and develop a prognostic system to define the best therapeutic approach in this patients. PMID:9807677

  14. Engineering a d-lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors

    PubMed Central

    Meng, Hengkai; Liu, Pi; Sun, Hongbing; Cai, Zhen; Zhou, Jie; Lin, Jianping; Li, Yin

    2016-01-01

    Engineering the cofactor specificity of a natural enzyme often results in a significant decrease in its activity on original cofactor. Here we report that a NADH-dependent dehydrogenase (d-LDH) from Lactobacillus delbrueckii 11842 can be rationally engineered to efficiently use both NADH and NADPH as cofactors. Point mutations on three amino acids (D176S, I177R, F178T) predicted by computational analysis resulted in a modified enzyme designated as d-LDH*. The Kcat/Km of the purified d-LDH* on NADPH increased approximately 184-fold while the Kcat/Km on NADH also significantly increased, showing for the first time that a rationally engineered d-LDH could exhibit comparable activity on both NADPH and NADH. Further kinetic analysis revealed that the enhanced affinity with NADH or NADPH and the significant increased Kcat of d-LDH* resulted in the significant increase of d-LDH* activity on both NADPH and NADH. This study thus demonstrated that the cofactor specificity of dehydrogenase can be broadened by using targeted engineering approach, and the engineered enzyme can efficiently function in NADH-rich, or NADPH-rich, or NADH and NADPH-rich environment. PMID:27109778

  15. Purification and partial kinetic and physical characterization of two NADP-specific glutamate dehydrogenase isoenzymes and their protein precursors, and measurement of the patterns of accumulation and rates of degradation of their nonidentical subunits in synchronized cells of Chlorella cultured in different concentrations of ammonia

    SciTech Connect

    Bascomb, N.F.

    1986-01-01

    Two ammonium-inducible, chloroplast-localized, NADP-specific glutamate dehydrogenases were purified from Chlorella sorokiniana. They were homopolymers of either alpha or beta subunits with molecular weights of 55,500 and 53,000, respectively. These isoenzymes were separated by their differential binding to the substrate affinity column. Peptide mapping of purified alpha and beta subunits showed them to have a high degree of sequence homology. By use of SDS slab-gel electrophoresis and a Western blot/immunodetection procedure, patterns of accumulation of alpha and beta subunits (in their holoenzyme) were measured in cells cultured in media, containing different concentrations of ammonia. Pulse-chase experiments with (/sup 35/S)sulfate were performed to measured the rates of degradation of the two isoenzymes. When the culture medium contained 2 mM ammonia or lower, cells accumulated only the alpha holoenzyme. Above 2 mM ammonia, cells contained both enzymes; however, their patterns of accumulation and rates of degradation were very different. The physiological role of alpha and beta holoenzymes appears to be ammonia assimilation at low and high external ammonia concentrations, respectively. From in vitro-translation studies with total cellular poly(A)/sup +/RNA, isolated from cells engaged in synthesis of alpha or beta holoenzymes or both, it was concluded that alpha and beta subunits have protein precursor(s) or identical molecular weight (M/sub r/ = 58,500). When the putative protein-precursor(s) were incubated in vitro, with cell-free extracts from Chlorella cells, they were processed to proteins the size of alpha and beta subunits.

  16. The intrinsically disordered protein LEA7 from Arabidopsis thaliana protects the isolated enzyme lactate dehydrogenase and enzymes in a soluble leaf proteome during freezing and drying.

    PubMed

    Popova, Antoaneta V; Rausch, Saskia; Hundertmark, Michaela; Gibon, Yves; Hincha, Dirk K

    2015-10-01

    The accumulation of Late Embryogenesis Abundant (LEA) proteins in plants is associated with tolerance against stresses such as freezing and desiccation. Two main functions have been attributed to LEA proteins: membrane stabilization and enzyme protection. We have hypothesized previously that LEA7 from Arabidopsis thaliana may stabilize membranes because it interacts with liposomes in the dry state. Here we show that LEA7, contrary to this expectation, did not stabilize liposomes during drying and rehydration. Instead, it partially preserved the activity of the enzyme lactate dehydrogenase (LDH) during drying and freezing. Fourier-transform infrared (FTIR) spectroscopy showed no evidence of aggregation of LDH in the dry or rehydrated state under conditions that lead to complete loss of activity. To approximate the complex influence of intracellular conditions on the protective effects of a LEA protein in a convenient in-vitro assay, we measured the activity of two Arabidopsis enzymes (glucose-6-P dehydrogenase and ADP-glucose pyrophosphorylase) in total soluble leaf protein extract (Arabidopsis soluble proteome, ASP) after drying and rehydration or freezing and thawing. LEA7 partially preserved the activity of both enzymes under these conditions, suggesting its role as an enzyme protectant in vivo. Further FTIR analyses indicated the partial reversibility of protein aggregation in the dry ASP during rehydration. Similarly, aggregation in the dry ASP was strongly reduced by LEA7. In addition, mixtures of LEA7 with sucrose or verbascose reduced aggregation more than the single additives, presumably through the effects of the protein on the H-bonding network of the sugar glasses. PMID:25988244

  17. Insulin, CCAAT/Enhancer-Binding Proteins and Lactate Regulate the Human 11β-Hydroxysteroid Dehydrogenase Type 2 Gene Expression in Colon Cancer Cell Lines

    PubMed Central

    Alikhani-Koupaei, Rasoul; Ignatova, Irena D.; Guettinger, Andreas; Frey, Felix J.; Frey, Brigitte M.

    2014-01-01

    11β-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon. PMID:25133511

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

    PubMed Central

    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

    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

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

    PubMed Central

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

    1993-01-01

    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

  20. Inhibition of Growth by Combined Treatment with Inhibitors of Lactate Dehydrogenase and either Phenformin or Inhibitors of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3.

    PubMed

    Lea, Michael A; Guzman, Yolanda; Desbordes, Charles

    2016-04-01

    Enhanced glycolysis in cancer cells presents a target for chemotherapy. Previous studies have indicated that proliferation of cancer cells can be inhibited by treatment with phenformin and with an inhibitor of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB) namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). In the present work, the action of two inhibitors that are effective at lower concentrations than 3PO, namely 1-(3-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PQP) and 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) were investigated. The inhibitors of lactate dehydrogenase (LDHA) studied in order of half-maximal inhibitory concentrations were methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (NHI-2) < isosafrole < oxamate. In colonic and bladder cancer cells, additive growth inhibitory effects were seen with the LDHA inhibitors, of which NHI-2 was effective at the lowest concentrations. Growth inhibition was generally greater with PFK15 than with PQP. The increased acidification of the culture medium and glucose uptake caused by phenformin was blocked by combined treatment with PFKFB3 or LDHA inhibitors. The results suggest that combined treatment with phenformin and inhibitors of glycolysis can cause additive inhibition of cell proliferation and may mitigate lactic acidosis caused by phenformin when used as a single agent. PMID:27069123

  1. Reactive oxygen species (ROS) production triggered by prostaglandin D2 (PGD2) regulates lactate dehydrogenase (LDH) expression/activity in TM4 Sertoli cells.

    PubMed

    Rossi, Soledad P; Windschüttl, Stefanie; Matzkin, María E; Rey-Ares, Verónica; Terradas, Claudio; Ponzio, Roberto; Puigdomenech, Elisa; Levalle, Oscar; Calandra, Ricardo S; Mayerhofer, Artur; Frungieri, Mónica B

    2016-10-15

    Reactive oxygen species (ROS) regulate testicular function in health and disease. We previously described a prostaglandin D2 (PGD2) system in Sertoli cells. Now, we found that PGD2 increases ROS and hydrogen peroxide (H2O2) generation in murine TM4 Sertoli cells, and also induces antioxidant enzymes expression suggesting that defense systems are triggered as an adaptive stress mechanism that guarantees cell survival. ROS and specially H2O2 may act as second messengers regulating signal transduction pathways and gene expression. We describe a stimulatory effect of PGD2 on lactate dehydrogenase (LDH) expression via DP1/DP2 receptors, which is prevented by the antioxidant N-acetyl-L-cysteine and the PI3K/Akt pathway inhibitor LY 294002. PGD2 also enhances Akt and CREB/ATF-1 phosphorylation. Our results provide evidence for a role of PGD2 in the regulation of the oxidant/antioxidant status in Sertoli cells and, more importantly, in the modulation of LDH expression which takes place through ROS generation and the Akt-CREB/ATF-1 pathway. PMID:27329155

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

    PubMed Central

    2012-01-01

    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

  3. Oligosaccharide-based Surfactant/Citric Acid Buffer System Stabilizes Lactate Dehydrogenase during Freeze-drying and Storage without the Addition of Natural Sugar.

    PubMed

    Ogawa, Shigesaburo; Kawai, Ryuichiro; Koga, Maito; Asakura, Kouichi; Takahashi, Isao; Osanai, Shuichi

    2016-06-01

    Experiments were conducted to assess the maintenance effects of oligosaccharide-based surfactants on the enzymatic activity of a model protein, lactate dehydrogenase (LDH), during freeze-drying and room temperature storage using the citric acid buffer system. Oligosaccharide-based surfactants, which exhibit a high glass transition temperature (Tg), promoted the eminent retention of enzymatic activity during these protocols, whereas monosaccharide-based surfactants with a low Tg displayed poor performance at high concentration, albeit much better than that of Tween 80 at middle concentration. The increase in the alkyl chain length did not exert positive effects as observed for the maintenance effect during freeze-thawing, but an amphiphilic nature and a glass forming ability were crucial for the effective stabilization at a low excipient concentration during freeze-drying. Even a low oligosaccharide-based surfactant content (0.1 mg mL(-1)) could maintain LDH activity during freeze-drying, but a high surfactant content (1.0 mg mL(-1)) was required to prevent buffer precipitation and retain high LDH activity on storage. Regarding storage, glass formation restricted molecular mobility in the lyophilized matrix, and LDH activity was effectively retained. The present results describe a strategy based on the glass-forming ability of surfactant-type excipients that affords a natural sugar-free formulation or an alternative use for polysorbate-type surfactants. PMID:27181251

  4. A Streamlined, Automated Protocol for the Production of Milligram Quantities of Untagged Recombinant Rat Lactate Dehydrogenase A Using ÄKTAxpressTM

    PubMed Central

    Nowicki, Matthew W.; Blackburn, Elizabeth A.; McNae, Iain W.; Wear, Martin A.

    2015-01-01

    We developed an efficient, automated 2-step purification protocol for the production of milligram quantities of untagged recombinant rat lactate dehydrogenase A (rLDHA) from E. coli, using the ÄKTAxpress™ chromatography system. Cation exchange followed by size exclusion results in average final purity in excess of 93% and yields ~ 14 milligrams per 50 ml of original cell culture in EnPresso B media, in under 8 hrs, including all primary sample processing and column equilibration steps. The protein is highly active and coherent biophysically and a viable alternative to the more problematic human homolog for structural and ligand-binding studies; an apo structure of untagged rLDHA was solved to a resolution 2.29 Å (PDB ID 5ES3). Our automated methodology uses generic commercially available pre-packed columns and simple buffers, and represents a robust standard method for the production of milligram amounts of untagged rLDHA, facilitating a novel fragment screening approach for new inhibitors. PMID:26717415

  5. 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

    Sassaman, Clay

    1978-01-01

    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

  6. 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

    Guha, Anirban; Gera, Sandeep; Sharma, Anshu

    2012-01-01

    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

  7. 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

    Zhou Guanqun; Tang Linglong; Mao Yanping; Chen Lei; Li Wenfei; Sun Ying; Liu Lizhi; Li Li; Lin Aihua; Ma Jun

    2012-03-01

    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.

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

    SciTech Connect

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

    2008-02-01

    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.

  9. Insufficient filling of vacuum tubes as a cause of microhemolysis and elevated serum lactate dehydrogenase levels. Use of a data-mining technique in evaluation of questionable laboratory test results.

    PubMed

    Tamechika, Yoshie; Iwatani, Yoshinori; Tohyama, Kaoru; Ichihara, Kiyoshi

    2006-01-01

    Experienced physicians noted unexpectedly elevated concentrations of lactate dehydrogenase in some patient samples, but quality control specimens showed no bias. To evaluate this problem, we used a "latent reference individual extraction method", designed to obtain reference intervals from a laboratory database by excluding individuals who have abnormal results for basic analytes other than the analyte in question, in this case lactate dehydrogenase. The reference interval derived for the suspected year was 264-530 U/L, while that of the previous year was 248-495 U/L. The only change we found was the introduction of an order entry system, which requests precise sampling volumes rather than complete filling of vacuum tubes. The effect of vacuum persistence was tested using ten freshly drawn blood samples. Compared with complete filling, 1/5 filling resulted in average elevations of lactate dehydrogenase, aspartic aminotransferase, and potassium levels of 8.0%, 3.8%, and 3.4%, respectively (all p<0.01). Microhemolysis was confirmed using a urine stick method. The length of time before centrifugation determined the degree of hemolysis, while vacuum during centrifugation did not affect it. Microhemolysis is the probable cause of the suspected pseudo-elevation noted by the physicians. Data-mining methodology represents a valuable tool for monitoring long-term bias in laboratory results. PMID:16681441

  10. Pretreatment serum lactate dehydrogenase is an independent prognostic factor for patients receiving neoadjuvant chemotherapy for locally advanced cervical cancer.

    PubMed

    Li, Jing; Wu, Miao-Fang; Lu, Huai-Wu; Chen, Qing; Lin, Zhong-Qiu; Wang, Li-Juan

    2016-08-01

    For locally advanced cervical cancer (LACC), hypoxia is a characteristic property. This study aimed to investigate whether baseline lactic dehydrogenase (LDH) level, which is a marker of hypoxia, had clinical value in determining neoadjuvant chemotherapy (NACT) response and prognosis for LACC patients. The study cohort included 418 patients with a median follow-up of 37.5 months. Cox proportional hazards models were used to assess the prognostic value of baseline LDH levels. Multivariate logistic regression analysis was performed to identify independent predictors of complete response after NACT. Backward stepwise selection with the Akaike information criterion was used to identify factors that could be entered into the multivariate regression model. Compared with patients with LDH levels <252.0 μ/L, patients with LDH levels ≥252.0 μ/L were more likely to have an elevated level of squamous cell carcinoma antigen, lymphatic vascular space involvement, lymph node metastasis, and positive parametrium and achieved lower complete remission rates. Baseline LDH levels ≥252.0 μ/L was an independent prognosticator for recurrence-free survival (adjusted hazard ratio [HR], 3.56; 95% confidence interval [CI] 2.22-5.69; P < 0.0001) and cancer-specific survival (adjusted HR, 3.08; 95% CI, 1.89-5.01; P < 0.0001). The predictive value of baseline LDH value remained significant in the subgroup analysis. LDH level ≥252.0 μ/L was identified as an independent predictor of complete remission after NACT (adjusted odds ratio [OR], 0.29; 95% CI, 0.15-0.58; P < 0.0001). Baseline LDH ≥252.0 μ/L is an independent prognostic predictor for patients receiving neoadjuvant chemotherapy for LACC. It helps distinguish patients with different prognosis and select patients who are more likely to benefit from NACT. PMID:27350066

  11. A reagentless amperometric electrode based on carbon paste, chemically modified with D-lactate dehydrogenase, NAD(+), and mediator containing polymer for D-lactic acid analysis. I. Construction, composition, and characterization.

    PubMed

    Shu, H C; Mattiasson, B; Persson, B; Nagy, G; Gorton, L; Sahni, S; Geng, L; Boguslavsky, L; Skotheim, T

    1995-05-01

    A reagentless carbon paste electrode was designed for D-lactic acid analysis in a flow injection system for the monitoring of the production of D-lactate in a batch fermentation. D-Lactate dehydrogenase, nicotinamide adenine dinucleotide (NAD(+)), a synthetic redox polymer containing covalently attached toluidine blue O as mediator, graphite powder, and paraffin oil were used for the construction of the modified carbon paste electrode. D-Lactate selectivity was indicated by insignificant responses from a variety of possible interfernces including L-lactate. The electrodes gave a linear response in the range between 0.05 and 5 mM D-lactate, with a detecting limit of 30 muM, allowing a sample throughput of 20 h(-1). Preliminary investigations were made by covering the electrode surface with electropolymerized membranes. Satisfactory stability was observed, indicated by a reproducibility of 3.3% relative standard deviation (RSD, n = 31), with a non-membrane-covered electrode for the analysis of D-lactate in fermentation broth. A long-term stability (230 broth samples) was proven, suggesting the electrodes to have a good potential for use in on-line monitoring of fermentation processes. (c) 1995 John Wiley & Sons, Inc. PMID:18623311

  12. Testis-Specific Lactate Dehydrogenase (LDH-C4) in Skeletal Muscle Enhances a Pika's Sprint-Running Capacity in Hypoxic Environment.

    PubMed

    Wang, Yang; Wei, Lian; Wei, Dengbang; Li, Xiao; Xu, Lina; Wei, Linna

    2015-08-01

    LDH-C4 is a lactate dehydrogenase that catalyzes the conversion of pyruvate to lactate. In mammals, ldh-c was originally thought to be expressed only in testis and spermatozoa. Plateau pika (Ochotona curzoniae), which belongs to the genus Ochotona of the Ochotonidea family, is a hypoxia tolerant mammal living 3000-5000 m above sea level on the Qinghai-Tibet Plateau, an environment which is strongly hypoxic. Ldh-c is expressed not only in testis and sperm but also in somatic tissues of plateau pika. In this study, the effects of N-propyl oxamate and N-isopropyl oxamate on LDH isozyme kinetics were compared to screens for a selective inhibitor of LDH-C4. To reveal the role and physiological mechanism of LDH-C4 in skeletal muscle of plateau pika, we investigated the effect of N-isopropyl oxamate on the pika exercise tolerance as well as the physiological mechanism. Our results show that Ki of N-propyl oxamate and N-isopropyl oxamate for LDH-A4, LDH-B4, and LDH-C4 were 0.094 mmol/L and 0.462 mmol/L, 0.119 mmol/L and 0.248 mmol/L, and 0.015 mmol/L and 0.013 mmol/L, respectively. N-isopropyl oxamate is a powerful selective inhibitor of plateau pika LDH-C4. In our exercise tolerance experiment, groups treated with inhibitors had significantly lower swimming times than the uninhibited control group. The inhibition rates of LDH, LD, and ATP were 37.12%, 66.27%, and 32.42%, respectively. Our results suggested that ldh-c is expressed in the skeletal muscle of plateau pika, and at least 32.42% of ATP in the skeletal muscle is catalyzed by LDH-C4 by anaerobic glycolysis. This suggests that pika has reduced dependence on oxygen and enhanced adaptation to hypoxic environment due to increased anaerobic glycolysis by LDH-C4 in skeletal muscle. LDH-C4 in plateau pika plays the crucial role in anaerobic glycolysis and generates ATP rapidly since this is the role of LDH-A4 in most species on plain land, which provide evidence that the native humans and animals in Qinghai-Tibet plateau

  13. Molecular characterization of CcpA and involvement of this protein in transcriptional regulation of lactate dehydrogenase and pyruvate formate-lyase in the ruminal bacterium Streptococcus bovis.

    PubMed

    Asanuma, Narito; Yoshii, Takahiro; Hino, Tsuneo

    2004-09-01

    A ccpA gene that encodes global catabolite control protein A (CcpA) in Streptococcus bovis was identified and characterized, and the involvement of CcpA in transcriptional control of a gene (ldh) encoding lactate dehydrogenase (LDH) and a gene (pfl) encoding pyruvate formate-lyase (PFL) was examined. The ccpA gene was shown to be transcribed as a monocistronic operon. A catabolite-responsive element (cre) was found in the promoter region of ccpA, suggesting that ccpA transcription in S. bovis is autogenously regulated. CcpA required HPr that was phosphorylated at the serine residue at position 46 (HPr-[Ser-P]) for binding to the cre site, but glucose 6-phosphate, fructose 1,6-bisphosphate, and NADP had no effect on binding. Diauxic growth was observed when S. bovis was grown in a medium containing glucose and lactose, but it disappeared when ccpA was disrupted, which indicates that CcpA is involved in catabolite repression in S. bovis. The level of ccpA mRNA was higher when cells were grown on glucose than when they were grown on lactose, which was in line with the level of ldh mRNA. When cells were grown on glucose, the ldh mRNA level was lower but the pfl mRNA level was higher in a ccpA-disrupted mutant than in the parent strain, which suggests that ldh transcription is enhanced and pfl transcription is suppressed by CcpA. The ccpA-disrupted mutant produced less lactate and more formate than the parent, probably because the mutant had reduced LDH activity and elevated PFL activity. In the upper region of both ldh and pfl, a cre-like sequence was found, suggesting that the complex consisting of CcpA and HPr-[Ser-P] binds to the possible cre sites. Thus, CcpA appears to be involved in the global regulation of sugar utilization in S. bovis. PMID:15345406

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

    PubMed Central

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

    2013-01-01

    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-1α and subsequent accelerated HIF-1α proteasome degradation. In vivo study also demonstrated that epigallocatechin could significantly inhibit breast cancer growth, HIF-1α/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

  15. 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

    Partl, Richard; Richtig, Erika; Avian, Alexander; Berghold, Andrea; Kapp, Karin S.

    2013-03-01

    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.

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

    PubMed Central

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

    1998-01-01

    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

  17. Serum lactate dehydrogenase levels at presentation in stage IV non-small cell lung cancer: predictive value of metastases and relation to survival outcomes.

    PubMed

    Lee, Dong Soo; Park, Kyung Ran; Kim, Seung Joon; Chung, Mi Joo; Lee, Yun Hee; Chang, Ji Hyun; Kang, Jin Hyoung; Hong, Sook Hee; Kim, Myung Sin; Kim, Yeon Sil

    2016-01-01

    This study aimed to evaluate the clinical correlations between serum lactate dehydrogenase (LDH) levels and tumor characteristics and to investigate the prognostic impact of serum LDH levels in advanced non-small cell lung cancer (NSCLC). A total of 394 patients were included in the present study between June 2007 and January 2013. All eligible patients had serum LDH levels available before treatment, and whole-body metastatic extent was measured using whole-body metastatic scores, as determined by 18(F)-fludeoxyglucose positron emission tomography scans from 1 to 7 as the sum of each metastatic region. The diagnostic cutoff value for an abnormal serum LDH level was 450 IU/L. The median serum LDH level was 477 IU/L (range, 113-2850), and 224 (56.9 %) patients had abnormal serum LDH levels. The serum LDH levels showed no significant associations with age, gender, histology, tumor differentiation, and smoking history. However, the proportion of patients with abnormal serum LDH levels was statistically significantly higher in the high total metastatic score group (scores 3-7) than in the low total metastatic score group (scores 1-2) (65.3 vs 50.4 %, p = 0.001). In a multivariate survival analysis, age (p = 0.001), gender (p = 0.001), histology (p = 0.003), tumor differentiation (p = 0.001), Eastern Cooperative Oncology Group (ECOG) performance status (p = 0.001), LDH levels (p = 0.046), and treatment factors (p = 0.001) proved to be independent prognostic factors for survival outcomes. The results of this study suggest that the serum LDH levels at presentation may be significantly correlated with whole-body tumor extent and might independently but modestly prognosticate OS in stage IV NSCLC. PMID:26240025

  18. Effects of L-carnitine and Pentoxifylline on the Activity of Lactate Dehydrogenase C4 isozyme and Motility of Testicular Spermatozoa in Mice

    PubMed Central

    Aliabadi, Elham; Karimi, Fatemeh; Rasti, Mozhgan; Akmali, Masoumeh; Esmaeilpour, Tahereh

    2013-01-01

    Background Extracted sperm from the testis have poor motility. Moreover, their motility changes during their journey through epidydimis. Meanwhile, they face high concentration of L-carnitin. In addition, lactate dehydrogenase C4 (LDH-C4) gene disorders has been shown to cause impaired sperm motility, leading to infertility in male mice. The aim of this study was to evaluate sperm motility and LDH-C4 enzyme activity upon L-carnitine (LC) and Pentoxifylline (PTX) administrations in mice. Methods We extracted testicular sperm of 48 mice and divided them into three equal parts. One part was incubated with Ham's F10 medium (control), the other parts were treated with Ham's F10 containing LC and PTX with a final concentration of 1.76 mM, for 30 min at room temperature. Sperm motility was assessed according to the World Health Organization (WHO) criteria. Sperm LDH-C4 enzyme activity was measured by spectrophotometery method. Statistical analyses were performed using ANOVA and Fisher's LSD test, and a p-value less than 0.05 was considered as a statistically significant difference. Results Sperm motility increased after 30 min of incubation in LC- and PTX-treated group (p<0.001). LC and PTX administrations showed a significant increase in the LDHC4 enzyme activity of sperm compared to that of the controls after 30 min (P=0.04 and 0.01, respectively). Conclusion The effects of LC and PTX on motility of sperm can be explained by an increase in LDH-C4 enzyme activity that may influence male fertility status. We suggest that LC as a non-toxic antioxidant is more suitable for use in assisted reproductive technique protocols than PTX. PMID:23926565

  19. The prognostic role of lactate dehydrogenase serum levels in patients with hepatocellular carcinoma who are treated with sorafenib: the influence of liver fibrosis

    PubMed Central

    Yada, Masayoshi; Miyazaki, Masayuki; Motomura, Kenta; Masumoto, Akihide; Nakamuta, Makoto; Kohjima, Motoyuki; Sugimoto, Rie; Aratake, Yoshifusa; Higashi, Nobuhiko; Morizono, Shusuke; Takao, Shinichiro; Yamashita, Naoki; Satoh, Takeaki; Yamashita, Shinsaku; Kuniyoshi, Masami

    2016-01-01

    Background Serum lactate dehydrogenase (LDH) levels could be a prognostic factor for sorafenib-treated patients with several types of solid tumor because it reflects hypoxic circumstances in aggressive tumors. For hepatocellular carcinoma (HCC), however, the prognostic role of LDH has been controversial. Liver fibrosis can potentially cause hypoxia in the liver, which has not been previously studied in the patients with advanced HCC. Thus, we aimed to analyze the prognostic role of LDH based on the degree of fibrosis. Methods Eighty-nine consecutive patients with HCC (Child-Pugh class A) who were treated using sorafenib were enrolled into this study. Pretreatment characteristics and changes in hepatic functional tests based on early response to sorafenib and serum LDH levels were analyzed. The degree of fibrosis was estimated using the aspartate aminotransferase (AST) to platelet ratio index (APRI), and the tumor response was evaluated after 3 months of sorafenib treatment. Results Overall, five patients discontinued sorafenib within 4 weeks. For the other 84 patients, those with progressive disease (PD) had significantly high pretreatment LDH levels, which correlated with the APRI score but not with the tumor stage. Multivariate logistic analysis revealed that older age and lower pretreatment LDH levels were independent prognostic factors for a better response to sorafenib. In patients who discontinued sorafenib early, three experienced acute liver failure accompanied with an increase in serum LDH. Conclusions We demonstrated that baseline serum LDH levels in HCC patients were affected by liver fibrosis but not by the tumor stage, and these LDH levels could be a marker for early response to sorafenib. A marked increase in serum LDH levels during sorafenib administration might also indicate subsequent acute liver failure. Close observation of serum LDH levels before and during sorafenib treatment could be useful in managing treatment of patients receiving this

  20. 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

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

    2014-06-01

    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

  1. 200th anniversary of lactate research in muscle.

    PubMed

    Gladden, L Bruce

    2008-07-01

    This year, 2008, marks the bicentennial of research into lactate metabolism in muscle. Berzelius linked lactate accumulation to exercise in 1807/1808 when he noted the presence of lactate in the muscles of "hunted stags." Today, the exact mechanism of intramuscular lactate oxidation and the relationship of lactate dehydrogenase to mitochondria remain unresolved as animated debate surrounds the intracellular lactate shuttle. PMID:18580290

  2. Discovery of 2-((3-cyanopyridin-2-yl)thio)acetamides as human lactate dehydrogenase A inhibitors to reduce the growth of MG-63 osteosarcoma cells: Virtual screening and biological validation.

    PubMed

    Cui, Wei; Lv, Wei; Qu, Ying; Ma, Rui; Wang, Yi-Wei; Xu, Yong-Jun; Wu, Di; Chen, Xuanhuang

    2016-08-15

    Lactate dehydrogenase A (LDHA) has emerged as an attractive target in the oncology field. In this paper, we present the identification of 2-((3-cyanopyridin-2-yl)thio)acetamide-containing compounds as LDHA inhibitors. The in vitro enzymatic assay suggested that inhibitor 9 had good inhibitory potency against LDHA with IC50 value as 1.24μM. Cytotoxicity assay showed that inhibitor 9 strongly inhibited the proliferation of cancer cell MG-63 (EC50=0.98μM). These findings indicated that inhibitor 9 could be employed as a lead for developing more potent LDHA inhibitor with anti-proliferative potency. PMID:27406795

  3. Chitosan promotes immune responses, ameliorates glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, but enhances lactate dehydrogenase levels in normal mice in vivo

    PubMed Central

    YEH, MING-YANG; SHIH, YUNG-LUEN; CHUNG, HSUEH-YU; CHOU, JASON; LU, HSU-FENG; LIU, CHIA-HUI; LIU, JIA-YOU; HUANG, WEN-WEN; PENG, SHU-FEN; WU, LUNG-YUAN; CHUNG, JING-GUNG

    2016-01-01

    Chitosan, a naturally derived polymer, has been shown to possess antimicrobial and anti-inflammatory properties; however, little is known about the effect of chitosan on the immune responses and glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) activities in normal mice. The aim of the present study was to investigate whether chitosan has an effect on the immune responses and GOT, GPT and LDH activities in mice in vivo. BALB/c mice were divided into four groups. The negative control group was treated with a normal diet; the positive control group was treated with a normal diet plus orally administered acetic acid and two treatment groups were treated with a normal diet plus orally administered chitosan in acetic acid at doses of 5 and 20 mg/kg, respectively, every other day for 24 days. Mice were weighed during the treatment, and following the treatment, blood was collected, and liver and spleen samples were isolated and weighted. The blood samples were used for measurement of white blood cell markers, and the spleen samples were used for analysis of phagocytosis, natural killer (NK) cell activity and cell proliferation using flow cytometry. The results indicated that chitosan did not markedly affect the body, liver and spleen weights at either dose. Chitosan increased the percentages of CD3 (T-cell marker), CD19 (B-cell marker), CD11b (monocytes) and Mac-3 (macrophages) when compared with the control group. However, chitosan did not affect the phagocytic activity of macrophages in peripheral blood mononuclear cells, although it decreased it in the peritoneal cavity. Treatment with 20 mg/kg chitosan led to a reduction in the cytotoxic activity of NK cells at an effector to target ratio of 25:1. Chitosan did not significantly promote B-cell proliferation in lipopolysaccharide-pretreated cells, but significantly decreased T-cell proliferation in concanavalin A-pretreated cells, and decreased the activity of

  4. Salivary carbonic anhydrase isoenzyme VI

    PubMed Central

    Kivelä, Jyrki; Parkkila, Seppo; Parkkila, Anna-Kaisa; Leinonen, Jukka; Rajaniemi, Hannu

    1999-01-01

    The carbonic anhydrases (CAs) participate in the maintenance of pH homeostasis in various tissues and biological fluids of the human body by catalysing the reversible reaction CO2+ H2O ⇌ HCO3−+ H+ (Davenport & Fisher, 1938; Davenport, 1939; Maren, 1967). Carbonic anhydrase isoenzyme VI (CA VI) is the only secretory isoenzyme of the mammalian CA gene family. It is exclusively expressed in the serous acinar cells of the parotid and submandibular glands, from where it is secreted into the saliva. In this review, we will discuss recent advances in research focused on the physiological role of salivary CA VI in the oral cavity and upper alimentary canal. PMID:10523402

  5. Pathogenic mechanisms underlying X-linked Charcot-Marie-Tooth neuropathy (CMTX6) in patients with a pyruvate dehydrogenase kinase 3 mutation.

    PubMed

    Perez-Siles, Gonzalo; Ly, Carolyn; Grant, Adrienne; Drew, Alexander P; Yiu, Eppie M; Ryan, Monique M; Chuang, David T; Tso, Shih-Chia; Nicholson, Garth A; Kennerson, Marina L

    2016-10-01

    Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. An X-linked form of CMT (CMTX6) is caused by a missense mutation (R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. PDK3 is one of 4 isoenzymes that negatively regulate the activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation of its first catalytic component pyruvate dehydrogenase (designated as E1). Mitochondrial PDC catalyses the oxidative decarboxylation of pyruvate to acetyl CoA and links glycolysis to the energy-producing Krebs cycle. We have previously shown the R158H mutation confers PDK3 enzyme hyperactivity. In this study we demonstrate that the increased PDK3 activity in patient fibroblasts (PDK3(R158H)) leads to the attenuation of PDC through hyper-phosphorylation of E1 at selected serine residues. This hyper-phosphorylation can be reversed by treating the PDK3(R158H) fibroblasts with the PDK inhibitor dichloroacetate (DCA). In the patient cells, down-regulation of PDC leads to increased lactate, decreased ATP and alteration of the mitochondrial network. Our findings highlight the potential to develop specific drug targeting of the mutant PDK3 as a therapeutic approach to treating CMTX6. PMID:27388934

  6. Isoenzyme characterization of Leishmania isolated from human cases with localized cutaneous leishmaniasis from the State of Campeche, Yucatan Peninsula, Mexico.

    PubMed

    Canto-Lara, S B; Cardenas-Maruffo, M F; Vargas-Gonzalez, A; Andrade-Narvaez, F

    1998-04-01

    Seventy-five isolates from the State of Campeche, Mexico, an area endemic for localized cutaneous leishmaniasis (LCL), were characterized by isoenzyme markers (glucose phosphate isomerase, mannose phospate isomerase, nucleoside hydrolase, phosphoglucomutase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase). Seventy (93.3%) were identified as Leishmania (Leishmania) mexicana and 5 (6.7%) as L. (Viannia) braziliensis. This is the first report of authochthonus human LCL caused by L. (V.) braziliensis in the State of Campeche, Yucatan Peninsula, Mexico. PMID:9574789

  7. Inflammation increases pyruvate dehydrogenase kinase 4 (PDK4) expression via the Jun N-Terminal Kinase (JNK) pathway in C2C12 cells.

    PubMed

    Park, Hana; Jeoung, Nam Ho

    2016-01-22

    Chronic inflammation augments the deleterious effects of several diseases, particularly diabetes, cancer, and sepsis. It is also involved in the process of metabolic shift from glucose oxidation to lactate production. Although several studies suggest that the change in activity of the pyruvate dehydrogenase complex (PDC) is a major factor causing this metabolic change, the exact mechanism of the inflammatory state remains unclear. In this study, we investigated the effect of lipopolysaccharide (LPS) on the expression of pyruvate dehydrogenase kinase 4 (PDK4), which is strongly associated with inactivation of the PDC in C2C12 myoblasts. In C2C12 myoblasts, LPS exposure led to increased PDK4 mRNA and protein expression levels as well as lactate production in culture medium. However, the expression levels of other PDK isoenzymes (PDK1 - 3) remained unchanged. Additionally, we observed that LPS treatment induced phosphorylation of Jun N-Terminal Kinases (JNK). To confirm the role of JNK, we inhibited the JNK pathway and observed that PDK4 expression and lactate production were decreased, but p38 and ERK were not significantly changed. Taken together, our results suggest that LPS induces PDK4 expression and alters glucose metabolism via the JNK pathway. PMID:26740179

  8. Equating salivary lactate dehydrogenase (LDH) with LDH-5 expression in patients with oral squamous cell carcinoma: An insight into metabolic reprogramming of cancer cell as a predictor of aggressive phenotype.

    PubMed

    Saluja, Tajindra Singh; Spadigam, Anita; Dhupar, Anita; Syed, Shaheen

    2016-04-01

    Oral squamous cell carcinoma (OSCC) is the sixth most common human malignancy. According to World Health Organization, oral cancer has been reported to have the highest morbidity and mortality and a survival rate of approximately 50 % at 5 years from diagnosis. This is attributed to the subjectivity in TNM staging and histological grading which may result in less than optimum treatment outcomes including tumour recurrence. One of the hallmarks of cancer is aerobic glycolysis also known as the Warburg effect. This glycolytic phenotype (hypoxic state) not only confers immortality to cancer cells, but also correlates with the belligerent behaviour of various malignancies and is reflected as an increase in the expression of lactate dehydrogenase 5 (LDH-5), the main isoform of LDH catalysing the conversion of pyruvate to lactate during glycolysis. The diagnostic role of salivary LDH in assessing the metabolic phenotype of oral cancer has not been studied. Since salivary LDH is mainly sourced from oral epithelial cells, any pathological changes in the epithelium should reflect diagnostically in saliva. Thus in our current research, we made an attempt to ascertain the biological behaviour and aggressiveness of OSCC by appraising its metabolic phenotype as indirectly reflected in salivary LDH activity. We found that salivary LDH can be used to assess the aggressiveness of different histological grades of OSCC. For the first time, an evidence of differing metabolic behaviour in similar histologic tumour grade is presented. Taken together, our study examines the inclusion of salivary LDH as potential diagnostic parameter and therapeutic index in OSCC. PMID:26577856

  9. Indoleamine 2,3‑dioxygenase downregulates T‑cell receptor complex ζ‑chain and c‑Myc, and reduces proliferation, lactate dehydrogenase levels and mitochondrial glutaminase in human T‑cells.

    PubMed

    Eleftheriadis, Theodoros; Pissas, Georgios; Antoniadi, Georgia; Tsogka, Konstantina; Sounidaki, Maria; Liakopoulos, Vassilios; Stefanidis, Ioannis

    2016-01-01

    Indoleamine 2,3‑dioxygenase (IDO), through L‑tryptophan depletion, activates general control non‑derepressible (GCN) 2 kinase and suppresses T‑cell proliferation, in addition to suppressing aerobic glycolysis and glutaminolysis, which are required for these rapidly proliferating cells. A number of, however not all of these alterations, are partially mediated through IDO‑induced p53 upregulation. In two‑way mixed lymphocyte reactions (MLRs), IDO reduced cellular proliferation. In MLR‑derived T‑cells, IDO induced the expression levels of p53 and p21, however concurrently reduced the levels of ζ‑chain, c‑Myc, lactate dehydrogenase A (LDH‑A) and glutaminase (GLS)2. However, p53 had no effect on the expression of the above proteins. These results were recapitulated in T‑cells activated with anti‑CD2, anti‑CD3 and anti‑CD28 by direct activation of the GCN2 kinase with tryptophanol. In conclusion, IDO, through GCN2 kinase activation, downregulates the levels of TCR‑complex ζ‑chain and c‑Myc, resulting in the suppression of T‑cell proliferation and a reduction in the levels of LDH‑A and GLS2, which are key enzymes involved in aerobic glycolysis and glutaminolysis, respectively. PMID:26647830

  10. Efficient Production of Optically Pure d-Lactic Acid from Raw Corn Starch by Using a Genetically Modified l-Lactate Dehydrogenase Gene-Deficient and α-Amylase-Secreting Lactobacillus plantarum Strain▿

    PubMed Central

    Okano, Kenji; Zhang, Qiao; Shinkawa, Satoru; Yoshida, Shogo; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-01-01

    In order to achieve direct and efficient fermentation of optically pure d-lactic acid from raw corn starch, we constructed l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 α-amylase (AmyA). The resulting strain produced only d-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct d-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct d-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct d-lactic acid fermentation from raw starch. PMID:19011066

  11. Efficient production of optically pure D-lactic acid from raw corn starch by using a genetically modified L-lactate dehydrogenase gene-deficient and alpha-amylase-secreting Lactobacillus plantarum strain.

    PubMed

    Okano, Kenji; Zhang, Qiao; Shinkawa, Satoru; Yoshida, Shogo; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

    2009-01-01

    In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 alpha-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch. PMID:19011066

  12. A quantitative study of the biospecific desorption of rat liver (M4) lactate dehydrogenase from 10-carboxydecylamino-Sepharose. Determination of the number of ligand-binding sites blocked on adsorption.

    PubMed

    Kyprianou, P; Yon, R J

    1982-12-01

    1. The theory of Nichol, Ogston, Winzor & Sawyer [(1974) Biochem. J. 143, 435-443] for quantitative affinity chromatography, when adapted for use with a non-specific column from which a multi-site protein can be specifically desorbed by its free ligand, permits determination of the concentration of adsorption sites on the column, their adsorptive affinity (as an association constant) and either the intrinsic (site) constant for ligand-binding to the protein or an 'occlusion coefficient' (defined as the number of ligand-binding sites blocked on adsorption), one of which must be known. 2. The theory has been applied to the NADH-specific desorption of rat liver M4 lactate dehydrogenase from 10-carboxydecylamino-Sepharose. It suggests that most of the enzyme molecules are adsorbed with at least two NADH-binding sites blocked, indicating an extensive adsorption interface in relation to the protein surface. Other chromatographic parameters were also determined for the system. 3. Among topics discussed are (a) factors affecting the experimentally determined value for the number of blocked sites, (b) the nature of the adsorption sites on the column and (c) the similarity of the analysis to that for determining Hill coefficients, and other possible applications. PMID:7165707

  13. Leukemic cell creatine kinase and its isoenzymes.

    PubMed

    Fang, S R; Yao, E G; Wei, S Z; Fan, H; Dong, Z R

    1989-06-01

    Using malachite green single agent coloration and acetate membrane electrophoresis, we studied the cellular creatine kinase (CK) activity and its isoenzymes in 7 normal controls and 26 leukemia patients. The leukemic cellular CK activity was 12.62 +/- 4.86 u/mg protein, 2.2 times higher than the normal value (5.73 +/- 2.66 u/mg protein, p less than 0.05). Only 2 of 5 normal leukocyte samples showed '+' CK isoenzyme MM. 22 leukemia patients had CK isoenzyme. CK-BB appeared mainly in acute granulocytic leukemic, and CK-MM mainly in other types. CK-MB was also found in 6 patients. The recurrence of CK-BB may indicate atavism, and the enhanced anaerobic glycolysis and the accelerated energetic turnover may be on of the metabolic characteristics of leukemic cell. PMID:2512061

  14. IL-4 and IL-13 induce protection from complement and melittin in endothelial cells despite initial loss of cytoplasmic proteins: membrane resealing impairs quantifying cytotoxicity with the lactate dehydrogenase permeability assay.

    PubMed

    Benson, Barbara A; Vercellotti, Gregory M; Dalmasso, Agustin P

    2015-01-01

    Endothelial cell activation and injury by the terminal pathway of complement is important in various pathobiological processes, including xenograft rejection. Protection against injury by human complement can be induced in porcine endothelial cells (ECs) with IL-4 and IL-13 through metabolic activation. However, despite this resistance, the complement-treated ECs were found to lose membrane permeability control assessed with the small molecule calcein. Therefore, to define the apparent discrepancy of permeability changes vis-à-vis the protection from killing, we now investigated whether IL-4 and IL-13 influence the release of the large cytoplasmic protein lactate dehydrogenase (LDH) in ECs incubated with complement or the pore-forming protein melittin. Primary cultures of ECs were pre-treated with IL-4 or IL-13 and then incubated with human serum as source of antibody and complement or melittin. Cell death was assessed using neutral red. Membrane permeability was quantitated measuring LDH release. We found that IL-4-/IL-13-induced protection of ECs from killing by complement or melittin despite loss of LDH in amounts similar to control ECs. However, the cytokine-treated ECs that were protected from killing rapidly regained effective control of membrane permeability. Moreover, the viability of the protected ECs was maintained for at least 2 days. We conclude that the protection induced by IL-4/IL-13 in ECs against lethal attack by complement or melittin is effective and durable despite severe initial impairment of membrane permeability. The metabolic changes responsible for protection allow the cells to repair the membrane injury caused by complement or melittin. PMID:26031609

  15. IL-4 and IL-13 induce protection from complement and melittin in endothelial cells despite initial loss of cytoplasmic proteins: Membrane resealing impairs quantifying cytotoxicity with the lactate dehydrogenase permeability assay

    PubMed Central

    Benson, Barbara A.; Vercellotti, Gregory M.; Dalmasso, Agustin P.

    2015-01-01

    Background Endothelial cell activation and injury by the terminal pathway of complement is important in various pathobiological processes, including xenograft rejection. Protection against injury by human complement can be induced in porcine endothelial cells (ECs) with IL-4 and IL-13 through metabolic activation. However, despite this resistance, the complement-treated ECs were found to lose membrane permeability control assessed with the small molecule calcein. Therefore to define the apparent discrepancy of permeability changes vis-à-vis the protection from killing we now investigated whether IL-4 and IL-13 influence the release of the large cytoplasmic protein lactate dehydrogenase (LDH) in ECs incubated with complement or the pore-forming protein melittin. Methods Primary cultures of ECs were pretreated with IL-4 or IL-13 and then incubated with human serum as source of antibody and complement or melittin. Cell death was assessed using neutral red. Membrane permeability was quantitated measuring LDH release. Results We found that IL-4/IL-13 induced protection of ECs from killing by complement or melittin despite loss of LDH in amounts similar to control ECs. However, the cytokine-treated ECs that were protected from killing rapidly regained effective control of membrane permeability. Moreover, the viability of the protected ECs was maintained for at least 2 days. Conclusion We conclude that the protection induced by IL-4/IL-13 in ECs against lethal attack by complement or melittin is effective and durable despite severe initial impairment of membrane permeability. The metabolic changes responsible for protection allow the cells to repair the membrane injury caused by complement or melittin. PMID:26031609

  16. Creatine kinase MB isoenzyme in dermatomyositis: a noncardiac source

    SciTech Connect

    Larca, L.J.; Coppola, J.T.; Honig, S.

    1981-03-01

    Three patients with polymyositis had elevated serum levels of creatine kinase MB isoenzyme. The presence of this isoenzyme is used extensively to diagnose myocardial infarction, but the isoenzyme is also found in sera of patients with primary muscular and neuromuscular disorders. Researchers studied cardiac function in two of our patients with electrocardiograms, technetium stannous pyrophosphate scanning, and technetium 99m-labeled erythrocyte gated blood pool imaging and in the third patient by postmortem examination. There was no evidence of myocardial involvement to account for the high serum levels of isoenzyme. Creatine kinase MB in the sera of patients with polymyositis does not necessarily indicate myocardial necrosis.

  17. The prognostic roles of ALDH1 isoenzymes in gastric cancer

    PubMed Central

    Li, Kai; Guo, Xiaoguang; Wang, Ziwei; Li, Xiaofeng; Bu, Youquan; Bai, Xuefeng; Zheng, Liansheng; Huang, Ying

    2016-01-01

    Increased aldehyde dehydrogenase 1 (ALDH1) activity has been determined to be present in the stem cells of several kinds of cancers including gastric cancer (GC). Nevertheless, which ones of ALDH1’s isoenzymes are leading to ALDH1 activity remains elusive. In this study, we examined the prognostic value and hazard ratio (HR) of individual ALDH1 isoenzymes in patients with GC using “The Kaplan–Meier plotter” database. mRNA high expression level of ALDH1A1 was not found to be significantly correlated with the overall survival (OS) of all patients with GC followed for 20 years, HR =0.86 (95% confidence interval [CI]: 0.7–1.05), P=0.13. mRNA high expression level of ALDH1A2 was also not significantly correlated with OS for all patients with GC, HR =1.13 (95% CI: 0.91–1.41), P=0.25. mRNA high expression level of ALDH1A3 was found to be significantly correlated with worsened OS in either intestinal-type patients, HR =2.24 (95% CI: 1.44–3.49), P=0.00026, or diffuse-type patients, HR =1.91 (95% CI: 1.02–3.59), P=0.04. Interestingly, mRNA high expression level of ALDH1B1 was found to be significantly correlated with better OS for all patients with GC, HR =0.66 (95% CI: 0.53–0.81), P=7.8e–05, and mRNA high expression level of ALDH1L1 was found to be significantly correlated with worsened OS for all patients with GC, HR =1.23 (95% CI: 1–1.51), P=0.048. Furthermore, our results also indicate that ALDH1A3 and ALDH1L1 are potential major contributors to the ALDH1 activity in GC, since mRNA high expression levels of ALDH1A3 and ALDH1L1 were found to be significantly correlated with worsened OS for all patients with GC. Based on our study, ALDH1A3 and ALDH1L1 are potential prognostic markers and therapeutic targets for patients with GC. PMID:27354812

  18. L-Lactate transport into rat heart mitochondria and reconstruction of the L-lactate/pyruvate shuttle.

    PubMed Central

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

    2002-01-01

    In vitro reconstruction of the L-lactate/pyruvate shuttle has been performed, which allows NADH oxidation outside rat heart mitochondria. Such a shuttle occurs due to the combined action of the novel mitochondrial L-lactate/pyruvate antiporter, which differs from the monocarboxylate carrier, and the mitochondrial L-lactate dehydrogenase. The rate of L-lactate/pyruvate antiport proved to regulate the shuttle in vitro. PMID:11988081

  19. L-Lactate transport into rat heart mitochondria and reconstruction of the L-lactate/pyruvate shuttle.

    PubMed

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

    2002-05-15

    In vitro reconstruction of the L-lactate/pyruvate shuttle has been performed, which allows NADH oxidation outside rat heart mitochondria. Such a shuttle occurs due to the combined action of the novel mitochondrial L-lactate/pyruvate antiporter, which differs from the monocarboxylate carrier, and the mitochondrial L-lactate dehydrogenase. The rate of L-lactate/pyruvate antiport proved to regulate the shuttle in vitro. PMID:11988081

  20. Analysis of the phosphofructokinase subunits and isoenzymes in human tissues.

    PubMed Central

    Dunaway, G A; Kasten, T P; Sebo, T; Trapp, R

    1988-01-01

    The 6-phosphofructo-1-kinase (PFK) subunits and isoenzymes were studied in human muscle, heart, brain, liver, platelets, fibroblasts, erythrocytes, placenta and umbilical cord. In each tissue, the subunit types in the native isoenzymes were characterized by immunological titration with subunit-specific antibodies and by column chromatography on QAE (quaternary aminoethyl)-Sephadex. Further, the subunits of the partially purified native isoenzymes were resolved by SDS/polyacrylamide-gel electrophoresis, identified by immunoblotting, and quantified by scanning gel densitometry of silver-stained gels and immunoblots. Depending on the type of tissue, one to three subunits were detected. The Mr values of the L, M and C subunits regardless of tissue were 76,700 +/- 1400, 82,500 +/- 1640 and 86,500 +/- 1620. Of the tissues studied, only the muscle PFK isoenzymes exhibited one subunit, which was the M-type subunit. Of the other tissues studied, the PFK isoenzymes contained various amounts of all three subunits. Considering the properties of the native PFK isoenzymes, it is clear that, in human tissues, they are not simply various combinations of two or three homotetrameric isoenzymes, but complex mixtures of homotetramers and heterotetramers. The kinetic/regulatory properties of the various isoenzyme pools were found to be dependent on subunit composition. Images Fig. 1. PMID:2970843

  1. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 7. Certification of four reference materials for the determination of enzymatic activity of gamma-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase accord.

    PubMed

    Siekmann, Lothar; Bonora, Roberto; Burtis, Carl A; Ceriotti, Ferruccio; Clerc-Renaud, Pascale; Férard, Georges; Ferrero, Carlo A; Forest, Jean-Claude; Franck, Paul F H; Gella, F-Javier; Hoelzel, Wieland; Jørgensen, Poul Jørgen; Kanno, Takashi; Kessner, Art; Klauke, Rainer; Kristiansen, Nina; Lessinger, Jean-Marc; Linsinger, Thomas P J; Misaki, Hideo; Mueller, Mathias M; Panteghini, Mauro; Pauwels, Jean; Schiele, Françoise; Schimmel, Heinz G; Vialle, Arlette; Weidemann, Gerhard; Schumann, Gerhard

    2002-07-01

    This paper is the seventh in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 4. Reference Procedure for the Measurement of Catalytic Concentration of Alanine Aminotransferase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of Gamma-Glutamyltransferase. A document describing the determination of preliminary reference values is also in preparation. The certification of the catalytic activity concentrations as determined by the recently elaborated IFCC primary reference methods at 37 degrees C of four enzyme preparations, namely IRMM/IFCC 452 (gamma-glutamyltransferase), IRMM/IFCC 453 (lactate dehydrogenase 1), IRMM/IFCC 454 (alanine aminotransferase) and IRMM/IFCC 455 (creatine kinase) is described. Homogeneity data were derived from previous results. Stability was assessed using recently obtained data as well as data from previous stability studies. The collaborative study for value assignment was performed under a strict quality control scheme to ensure traceability to the primary reference method. Uncertainty of the materials was assessed in compliance with the Guide to the Expression of Uncertainty in Measurement. The certified values obtained at 37 degrees C are 1.90 microkat/l +/- 0.04 microkat/l (114.1 U/l +/- 2.4 U/l), for gamma-glutamyltransferase, 8.37 microkat/l +/- 0.12 microkat/l (502 U/l +/- 7 U/l), for lactate dehydrogenase 1, 3.09 microkat/l +/- 0.07 microkat

  2. Chemical modification of aldehyde dehydrogenase by a vinyl ketone analogue of an insect pheromone.

    PubMed

    Blatter, E E; Tasayco, M L; Prestwich, G; Pietruszko, R

    1990-12-01

    A major component of the sex pheromone from the tobacco budworm moth Heliothis virescens is a C16 straight-chain aldehyde with a single unsaturation at the eleventh position. The sex pheromones are inactivated when metabolized to their corresponding acids by insect aldehyde dehydrogenase. During this investigation it was demonstrated that the C16 aldehyde is a good substrate for human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 with Km and Kcat. values at pH 7.0 of 2 microM and 0.4 mumol of NADH/min per mg and of 0.6 microM and 0.24 mumol of NADH/min per mg respectively. A vinyl ketone analogue of the pheromone inhibited insect pheromone metabolism; it also inactivated human aldehyde dehydrogenase. Total inactivation of both isoenzymes was achieved at stoichiometric (equal or less than the subunit number) concentrations of vinyl ketone, incorporating 2.1-2.6 molecules/molecule of enzyme. Substrate protection was observed in the presence of the parent aldehyde and 5'-AMP. Peptide maps of tryptic digests of the E2 isoenzyme modified with 3H-labelled vinyl ketone showed that incorporation occurred into a single peptide peak. The labelled peptide of E2 isoenzyme was further purified on h.p.l.c. and sequenced. The label was incorporated into cysteine-302 in the primary structure of E2 isoenzyme, thus indicating that cysteine-302 is located in the aldehyde substrate area of the active site of aldehyde dehydrogenase. Affinity labelling of aldehyde dehydrogenase with vinyl ketones may prove to be of general utility in biochemical studies of these enzymes. PMID:2268265

  3. Lactate metabolism in the fetal rabbit lung

    SciTech Connect

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

    1986-05-01

    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.

  4. Kinetic studies of the uptake of aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro.

    PubMed Central

    Marra, E; Passarella, S; Casamassima, E; Perlino, E; Doonan, S; Quagliariello, E

    1985-01-01

    Kinetic measurements of the uptake of native mitochondrial aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro were carried out. The uptake of both the enzymes is essentially complete in 1 min and shows saturation characteristics. The rate of uptake of aspartate aminotransferase into mitochondria is decreased by malate dehydrogenase, and vice versa. The inhibition is exerted by isoenzyme remaining outside the mitochondria rather than by isoenzyme that has been imported. The thiol compound beta-mercaptoethanol decreases the rate of uptake of the tested enzymes; inhibition is a result of interaction of beta-mercaptoethanol with the mitochondria and not with the enzymes themselves. The rate of uptake of aspartate aminotransferase is inhibited non-competitively by malate dehydrogenase, but competitively by beta-mercaptoethanol. The rate of uptake of malate dehydrogenase is inhibited non-competitively by aspartate aminotransferase and by beta-mercaptoethanol. beta-Mercaptoethanol prevents the inhibition of the rate of uptake of malate dehydrogenase by aspartate aminotransferase. These results are interpreted in terms of a model system in which the two isoenzymes have separate but interacting binding sites within a receptor in the mitochondrial membrane system. PMID:4015628

  5. Proton nuclear magnetic resonance spectroscopy of horseradish peroxidase isoenzymes: correlation of distinctive spectra with isoenzyme specific activities.

    PubMed

    Gonzalez-Vergara, E; Meyer, M; Goff, H M

    1985-11-01

    High-resolution proton NMR spectra are reported for the paramagnetic ferric native and cyano complexes of the five major horseradish root peroxidase (HRP) isoenzymes (A1, A2, A3, B, and C). Axial imidazole resonances are observed in the native and cyano-complex spectra of all the isoenzymes, thus indicating the presence of a common axial histidine ligand. Proton NMR spectra outside the usual diamagnetic region are identical for sets of A1 and A2 isoenzymes and for the B and C isoenzyme set. Variation in heme residue chemical shift positions may be controlled in part by porphyrin vinyl side chain-protein interactions. Diverse upfield spectra among the isoenzymes reflect amino acid substitutions and/or conformational differences near the prosthetic group, as signals in this region must result from amino acid residues in proximity to the heme center. Acid-base dependence studies reveal an "alkaline" transition that converts the native high-spin iron (III) porphyrin to the low-spin state. The transition occurs at pH 9.3, 9.4, 9.8, and 10.9 for respective HRP A1, A2, A3, and C isoenzymes, respectively. Significantly, this ordering also reflects specific activities for the isoenzymes in the order A1 = A2 greater than A3 greater than B = C. Identical proton NMR spectra for A1/A2 and B/C isoenzyme sets parallel equivalent specific activities for members of a particular set. Proton NMR spectra thus appear to be highly sensitive to protein modifications that affect catalytic activity. PMID:4084538

  6. Hemichannel-mediated release of lactate.

    PubMed

    Karagiannis, Anastassios; Sylantyev, Sergiy; Hadjihambi, Anna; Hosford, Patrick S; Kasparov, Sergey; Gourine, Alexander V

    2016-07-01

    In the central nervous system lactate contributes to the extracellular pool of readily available energy substrates and may also function as a signaling molecule which mediates communication between glial cells and neurons. Monocarboxylate transporters are believed to provide the main pathway for lactate transport across the membranes. Here we tested the hypothesis that lactate could also be released via opening of pannexin and/or functional connexin hemichannels. In acute slices prepared from the brainstem, hippocampus, hypothalamus and cortex of adult rats, enzymatic amperometric biosensors detected significant tonic lactate release inhibited by compounds, which block pannexin/connexin hemichannels and facilitated by lowering extracellular [Ca(2+)] or increased PCO2 Enhanced lactate release triggered by hypoxia was reduced by ∼50% by either connexin or monocarboxylate transporter blockers. Stimulation of Schaffer collateral fibers triggered lactate release in CA1 area of the hippocampus, which was facilitated in conditions of low extracellular [Ca(2+)], markedly reduced by blockade of connexin hemichannels and abolished by lactate dehydrogenase inhibitor oxamate. These results indicate that lactate transport across the membranes may occur via mechanisms other than monocarboxylate transporters. In the central nervous system, hemichannels may function as a conduit of lactate release, and this mechanism is recruited during hypoxia and periods of enhanced neuronal activity. PMID:26661210

  7. Isoenzyme clustering of Trypanosomatidae Colombian populations.

    PubMed

    Montilla, Marleny M; Guhl, Felipe; Jaramillo, Carlos; Nicholls, Santiago; Barnabe, Christian; Bosseno, Marie F; Breniere, Simone F

    2002-04-01

    Thirty-six Trypanosomatidae stocks isolated from various hosts and geographical areas in Colombia and 7 others from Bolivia, Chile, Honduras and Panama have been surveyed by Multilocus Enzyme Electrophoresis (MLEE). Part of the Colombian stocks were previously characterized by morphology and biological behavior as belonging to Trypanosoma cruzi and Trypanosoma rangeli taxa, others were unknown species. The genetic variability observed at 13 different loci was considerable, since 38 zymodemes could be distinguished and 2 upper branches were observed. The first branch corresponded to T. cruzi and was divided in the two major phylogenetic subdivision of T. cruzi (T. cruzi I, T. cruzi II). The majority of the Colombian T. cruzi stocks (92%) felt into T. cruzi I. Only two stocks, isolated from sylvatic mammals, belonged to T. cruzi II. Among T. cruzi I, we did not observed any additional phylogenetic subdivision and host-dependent genotype specificity. The second branch was genetically very heterogeneous and included all T. rangeli stocks, the stocks isolated from bats and one stock isolated from a sylvatic R. prolixus vector. The stocks belonging to T. rangeli presented only one locus instead of two for the malic enzyme system. Since, the upper level of resolution of the isoenzyme method was exceeded, the current clustering study failed to draw a clear distinction between such a diverse set of Trypanosomatidae species. PMID:12164294

  8. D-lactate metabolism in the alga, Chlamydomonas Reinhardtii

    SciTech Connect

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

    1986-05-01

    (/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.

  9. Glycolysis and the significance of lactate in traumatic brain injury

    PubMed Central

    Carpenter, Keri L. H.; Jalloh, Ibrahim; Hutchinson, Peter J.

    2015-01-01

    In traumatic brain injury (TBI) patients, elevation of the brain extracellular lactate concentration and the lactate/pyruvate ratio are well-recognized, and are associated statistically with unfavorable clinical outcome. Brain extracellular lactate was conventionally regarded as a waste product of glucose, when glucose is metabolized via glycolysis (Embden-Meyerhof-Parnas pathway) to pyruvate, followed by conversion to lactate by the action of lactate dehydrogenase, and export of lactate into the extracellular fluid. In TBI, glycolytic lactate is ascribed to hypoxia or mitochondrial dysfunction, although the precise nature of the latter is incompletely understood. Seemingly in contrast to lactate's association with unfavorable outcome is a growing body of evidence that lactate can be beneficial. The idea that the brain can utilize lactate by feeding into the tricarboxylic acid (TCA) cycle of neurons, first published two decades ago, has become known as the astrocyte-neuron lactate shuttle hypothesis. Direct evidence of brain utilization of lactate was first obtained 5 years ago in a cerebral microdialysis study in TBI patients, where administration of 13C-labeled lactate via the microdialysis catheter and simultaneous collection of the emerging microdialysates, with 13C NMR analysis, revealed 13C labeling in glutamine consistent with lactate utilization via the TCA cycle. This suggests that where neurons are too damaged to utilize the lactate produced from glucose by astrocytes, i.e., uncoupling of neuronal and glial metabolism, high extracellular levels of lactate would accumulate, explaining the association between high lactate and poor outcome. Recently, an intravenous exogenous lactate supplementation study in TBI patients revealed evidence for a beneficial effect judged by surrogate endpoints. Here we review the current state of knowledge about glycolysis and lactate in TBI, how it can be measured in patients, and whether it can be modulated to achieve better

  10. Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.

    PubMed

    Hoey, M E; Allison, N; Scott, A J; Fewson, C A

    1987-12-15

    L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus. PMID:3325042

  11. Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.

    PubMed Central

    Hoey, M E; Allison, N; Scott, A J; Fewson, C A

    1987-01-01

    L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus. PMID:3325042

  12. D-Lactate transport and metabolism in rat liver mitochondria.

    PubMed Central

    de Bari, Lidia; Atlante, Anna; Guaragnella, Nicoletta; Principato, Giovanni; Passarella, Salvatore

    2002-01-01

    In the present study we investigated whether isolated rat liver mitochondria can take up and metabolize D-lactate. We found the following: (1) externally added D-lactate causes oxygen uptake by mitochondria [P/O ratio (the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation)=2] and membrane potential (Delta(psi)) generation in processes that are rotenone-insensitive, but inhibited by antimycin A and cyanide, and proton release from coupled mitochondria inhibited by alpha-cyanocinnamate, but not by phenylsuccinate; (2) the activity of the putative flavoprotein (D-lactate dehydrogenase) was detected in inside-out submitochondrial particles, but not in mitochondria and mitoplasts, as it is localized in the matrix phase of the mitochondrial inner membrane; (3) three novel separate translocators exist to mediate D-lactate traffic across the mitochondrial inner membrane: the D-lactate/H(+) symporter, which was investigated by measuring fluorimetrically the rate of endogenous flavin reduction, the D-lactate/oxoacid antiporter (which mediates both the D-lactate/pyruvate and D-lactate/oxaloacetate exchanges) and D-lactate/malate antiporter studied by monitoring photometrically the appearance of the D-lactate counteranions outside mitochondria. The D-lactate translocators, in the light of their different inhibition profiles separate from the monocarboxylate carrier, were found to differ from each other in the V(max) values and in the inhibition and pH profiles and were shown to regulate mitochondrial D-lactate metabolism in vitro. The D-lactate translocators and the D-lactate dehydrogenase could account for the removal of the toxic methylglyoxal from cytosol, as well as for D-lactate-dependent gluconeogenesis. PMID:11955284

  13. Evaluation of NAD(P)-Dependent Dehydrogenase Activities in Neutrophilic Granulocytes by the Bioluminescent Method.

    PubMed

    Savchenko, A A

    2015-09-01

    Bioluminescent method for measurements of the neutrophilic NAD(P)-dependent dehydrogenases (lactate dehydrogenase, NAD-dependent malate dehydrogenase, NADP-dependent decarboxylating malate dehydrogenase, NAD-dependent isocitrate dehydrogenase, and glucose- 6-phosphate dehydrogenase) is developed. The sensitivity of the method allows minimization of the volume of biological material for measurements to 104 neutrophils per analysis. The method is tried in patients with diffuse purulent peritonitis. Low levels of NADPH synthesis enzymes and high levels of enzymes determining the substrate flow by the Krebs cycle found in these patients can lead to attenuation of functional activity of cells. PMID:26468025

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

    SciTech Connect

    Miller, Matthew; Suominen, Pirkko; Aristidou, Aristos; Hause, Benjamin Matthew; Van Hoek, Pim; Dundon, Catherine Asleson

    2012-03-20

    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.

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

    PubMed Central

    Crow, Vaughan L.

    1986-01-01

    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

  16. The primary pathway for lactate oxidation in Desulfovibrio vulgaris

    PubMed Central

    Vita, Nicolas; Valette, Odile; Brasseur, Gaël; Lignon, Sabrina; Denis, Yann; Ansaldi, Mireille; Dolla, Alain; Pieulle, Laetitia

    2015-01-01

    The ability to respire sulfate linked to lactate oxidation is a key metabolic signature of the Desulfovibrio genus. Lactate oxidation by these incomplete oxidizers generates reductants through lactate dehydrogenase (LDH) and pyruvate-ferredoxin oxidoreductase (PFOR), with the latter catalyzing pyruvate conversion into acetyl-CoA. Acetyl-CoA is the source of substrate-level phosphorylation through the production of ATP. Here, we show that these crucial steps are performed by enzymes encoded by a nonacistronic transcriptional unit named now as operon luo (for lactate utilization operon). Using a combination of genetic and biochemical techniques, we assigned a physiological role to the operon genes DVU3027-28 and DVU3032-33. The growth of mutant Δ26-28 was highly disrupted on D-lactate, whereas the growth of mutant Δ32-33 was slower on L-lactate, which could be related to a decrease in the activity of D-lactate or L-lactate oxidase in the corresponding mutants. The DVU3027-28 and DVU3032-33 genes thus encode functional D-LDH and L-LDH enzymes, respectively. Scanning of the genome for lactate utilization revealed several lactate permease and dehydrogenase homologs. However, transcriptional compensation was not observed in any of the mutants except for lactate permease. Although there is a high degree of redundancy for lactate oxidase, it is not functionally efficient in LDH mutants. This result could be related to the identification of several operon enzymes, including LDHs, in the PFOR activity bands, suggesting the occurrence of a lactate-oxidizing supermolecular structure that can optimize the performance of lactate utilization in Desulfovibrio species. PMID:26167158

  17. 21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alkaline phosphatase or isoenzymes test system... Test Systems § 862.1050 Alkaline phosphatase or isoenzymes test system. (a) Identification. An alkaline phosphatase or isoenzymes test system is a device intended to measure alkaline phosphatase or its...

  18. 21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alkaline phosphatase or isoenzymes test system... Test Systems § 862.1050 Alkaline phosphatase or isoenzymes test system. (a) Identification. An alkaline phosphatase or isoenzymes test system is a device intended to measure alkaline phosphatase or its...

  19. Purification and properties of the cytoplasmic glucose-6-phosphate dehydrogenase from pea leaves.

    PubMed

    Fickenscher, K; Scheibe, R

    1986-06-01

    A method involving affinity chromatography on the yellow dye Remazol Brilliant Gelb GL to highly purify the cytoplasmic isoenzyme of glucose-6-phosphate dehydrogenase from pea shoots is described. Purification is at least 6000-fold. The specific activity of the purified enzyme is 185 mumol NADP reduced/min per mg protein. The preparation was free from any contamination of chloroplastic isoenzyme. The purified enzyme retains its activity in the presence of reducing agents which, in contrast, inactivate the chloroplast enzyme. The state of activity of the cytoplasmic and the chloroplastic isoenzyme in illuminated or darkened pea leaves was investigated using specific antibodies. While upon illumination the chloroplastic isoenzyme was inactivated by 80 to 90%, we could not find any change in activity of the cytoplasmic glucose-6-phosphate dehydrogenase. ATP, ADP, NAD, NADH, and various sugar phosphates do not inhibit the enzyme activity. Only NADPH is a strong competitive inhibitor with respect to NADP, suggesting that the enzyme is regulated by feedback inhibition by one of its products. Mg2+ ions have no influence on the activity of the enzyme. The molecular weight has found to be 240,000 for the native enzyme and 60,000 for the subunit. Throughout the purification procedure the enzyme was very unstable unless NADP was present in the buffer. PMID:3717951

  20. Isoenzymes of Pyruvate Kinase in Etioplasts and Chloroplasts 1

    PubMed Central

    Ireland, Robert J.; Deluca, Vincenzo; Dennis, David T.

    1979-01-01

    Isoenzymes of pyruvate kinase from green leaves of castor bean and etiolated leaves of pea plants have been separated by ion filtration chromatography. One of the isoenzymes is localized in the plastid, whereas the other is in the cytosol. The cytosolic enzyme has a pH optimum from pH 7 to pH 9, and is able to utilize nucleotides other than ADP as the phosphoryl acceptor. The plastid enzyme has a much sharper optimum at pH 8, and is less efficient at using alternative nucleotides. The plastic pyruvate kinase, unlike the cytosolic enzyme, requires the presence of dithiothreitol or 2-mercaptoethanol during isolation and storage to stabilize the activity. PMID:16660835

  1. Plasma intestinal alkaline phosphatase isoenzymes in neonates with bowel necrosis.

    PubMed Central

    McLachlan, R; Coakley, J; Murton, L; Campbell, N

    1993-01-01

    AIM--To determine if the intestinal isoenzymes of alkaline phosphatase (ALP) are biochemical markers of bowel necrosis in neonates. METHODS--Plasma ALP isoenzymes were measured in 22 babies with bowel necrosis, histologically confirmed, and in 22 matched controls. The isoenzymes were also measured in 16 infants with signs of necrotising enterocolitis, who recovered without histological confirmation of bowel necrosis. The isoenzymes were separated by polyacrylamide gel electrophoresis. Auxiliary tests for identification included neuraminidase digestion and treatment with monoclonal and polyclonal antiplacental antibodies. RESULTS--Intestinal ALP was detected in 16 infants with bowel necrosis--13 had fetal intestinal ALP (FI-ALP) and three had adult intestinal ALP (AI-ALP). FI-ALP was detected in nine of the controls. In the babies with bowel necrosis intestinal ALP was found over all gestations, but in the controls only in those less than 34 weeks. The percentages of total ALP activity due to intestinal ALP were significantly higher in those with bowel necrosis compared with matched controls (p = 0.028). In babies of all gestations diagnostic sensitivity for the presence of intestinal ALP as a marker of bowel necrosis was 73% and diagnostic specificity 59%. In babies greater than 34 weeks' gestation, diagnostic sensitivity fell to 60% but the test became completely specific. In two babies FI-ALP increased from zero/trace to high activity coincident with the episode of bowel necrosis. In 16 babies with signs of necrotising enterocolitis but unconfirmed bowel necrosis FI-ALP was detected in four. CONCLUSION--Intestinal ALP seems to be released into the circulation in some babies with bowel necrosis, but its detection does not have the diagnostic sensitivity and specificity to be a reliable biochemical marker of the condition. Images PMID:8157755

  2. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a type of ...

  3. Advanced Stage, Increased Lactate Dehydrogenase, and Primary Site, but Not Adolescent Age (≥ 15 Years), Are Associated With an Increased Risk of Treatment Failure in Children and Adolescents With Mature B-Cell Non-Hodgkin's Lymphoma: Results of the FAB LMB 96 Study

    PubMed Central

    Cairo, Mitchell S.; Sposto, Richard; Gerrard, Mary; Auperin, Anne; Goldman, Stanton C.; Harrison, Lauren; Pinkerton, Ross; Raphael, Martine; McCarthy, Keith; Perkins, Sherrie L.; Patte, Catherine

    2012-01-01

    Purpose Adolescents (age 15 to 21 years) compared with younger children with mature B-cell non-Hodgkin's lymphoma (NHL) have been historically considered to have an inferior prognosis. We therefore analyzed the impact of age and other diagnostic factors on the risk of treatment failure in children and adolescents treated on the French-American-British Mature B-Cell Lymphoma 96 (FAB LMB 96) trial. Patients and Methods Patients were divided by risk: group A (limited), group B (intermediate), and group C (advanced), as previously described. Prognostic factors analyzed for event-free survival (EFS) included age (< 15 v ≥ 15 years), stage (I/II v III/IV), primary site, lactate dehydrogenase (LDH), bone marrow/CNS (BM/CNS) involvement, and histology (diffuse large B-cell lymphoma v mediastinal B-cell lymphoma v Burkitt lymphoma or Burkitt-like lymphoma). Results The 3-year EFS for the whole cohort was 88% ± 1%. Age was not associated as a risk factor for increased treatment failure in either univariate analysis (P = .15) or multivariate analysis (P = .58). Increased LDH (≥ 2 × upper limit of normal [ULN] v < 2 × ULN), primary site, and BM-positive/CNS-positive disease were all independent risk factors associated with a significant increase in treatment failure rate (relative risk, 2.0; P < .001, P < .012, and P < .001, respectively). Conclusion LDH level at diagnosis, mediastinal disease, and combined BM-positive/CNS-positive involvement are independent risk factors in children with mature B-cell NHL. Future studies should be developed to identify specific therapeutic strategies (immunotherapy) to overcome these risk factors and to identify the biologic basis associated with these prognostic factors in children with mature B-cell NHL. PMID:22215753

  4. 21 CFR 862.1360 - Gamma-glutamyl transpeptidase and isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1360 Gamma-glutamyl transpeptidase and isoenzymes test system....

  5. 21 CFR 862.1360 - Gamma-glutamyl transpeptidase and isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1360 Gamma-glutamyl transpeptidase and isoenzymes test system....

  6. Utilization of d-Lactate as an Energy Source Supports the Growth of Gluconobacter oxydans

    PubMed Central

    Sheng, Binbin; Xu, Jing; Zhang, Yingxin; Jiang, Tianyi; Deng, Sisi; Kong, Jian; Ma, Cuiqing; Xu, Ping

    2015-01-01

    d-Lactate was identified as one of the few available organic acids that supported the growth of Gluconobacter oxydans 621H in this study. Interestingly, the strain used d-lactate as an energy source but not as a carbon source, unlike other lactate-utilizing bacteria. The enzymatic basis for the growth of G. oxydans 621H on d-lactate was therefore investigated. Although two putative NAD-independent d-lactate dehydrogenases, GOX1253 and GOX2071, were capable of oxidizing d-lactate, GOX1253 was the only enzyme able to support the d-lactate-driven growth of the strain. GOX1253 was characterized as a membrane-bound dehydrogenase with high activity toward d-lactate, while GOX2071 was characterized as a soluble oxidase with broad substrate specificity toward d-2-hydroxy acids. The latter used molecular oxygen as a direct electron acceptor, a feature that has not been reported previously in d-lactate-oxidizing enzymes. This study not only clarifies the mechanism for the growth of G. oxydans on d-lactate, but also provides new insights for applications of the important industrial microbe and the novel d-lactate oxidase. PMID:25862219

  7. Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

    SciTech Connect

    Boernke, W. E.; Millard, C. S.; Stevens, P. W.; Kakar, S. N.; Stevens, F. J.; Donnelly, M. I.; Nebraska Wesleyan Univ.

    1995-09-10

    Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the native enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the mutant

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

    PubMed Central

    2014-01-01

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

  9. KINETICS OF BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P450 ISOENZYMES IN HUMAN LIVER MICROSOMES

    EPA Science Inventory

    Kinetics of Bromodichloromethane Metabolism by
    Cytochrome P450 Isoenzymes in Human Liver Microsomes

    Guangyu Zhao and John W. Allis

    ABSTRACT
    The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have ...

  10. The effects of some bromophenols on human carbonic anhydrase isoenzymes.

    PubMed

    Taslimi, Parham; Gülçin, İlhami; Öztaşkın, Necla; Çetinkaya, Yasin; Göksu, Süleyman; Alwasel, Saleh H; Supuran, Claudiu T

    2016-08-01

    Carbonic anhydrases (CAs, EC 4.2.1.1), which are involved in a variety of physiological and pathological processes, are ubiquitous metalloenzymes mainly catalyzing the reversible hydration of carbon dioxide (CO2) to bicarbonate ([Formula: see text]) and proton (H(+)). In this study, a dozen of bromophenol derivatives (1-12) were evaluated as metalloenzyme CA (EC 4.2.1.1) inhibitors against the human carbonic anhydrase isoenzymes I and II (hCA I and II). Cytosolic hCA I and II isoenzymes were effectively inhibited by bromophenol derivatives (1-12) with Kis in the low nanomolar range of 1.85 ± 0.58 to 5.04 ± 1.46 nM against hCA I and in the range of 2.01 ± 0.52 to 2.94 ± 1.31 nM against hCA II, respectively. PMID:26133541

  11. Changes in arginase isoenzymes pattern in human hepatocellular carcinoma

    SciTech Connect

    Chrzanowska, Alicja; Krawczyk, Marek; Baranczyk-Kuzma, Anna

    2008-12-12

    Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide affecting preferentially patients with liver cirrhosis. The studies were performed on tissues obtained during surgery from 50 patients with HCC, 40 with liver cirrhosis and 40 control livers. It was found that arginase activity in HCC was nearly 5- and 15-fold lower than in cirrhotic and normal livers, respectively. Isoenzymes AI (so-called liver-type arginase) and AII (extrahepatic arginase) were identified by Western blotting in all studied tissues, however the amount of AI, as well as the expression of AI-mRNA were lower in HCC, in comparison with normal liver, and those of AII were significantly higher. Since HCC is arginine-dependent, and arginine is essential for cells growth, the decrease of AI may preserve this amino acid within tumor cells. Concurrently, the rise of AII can increase the level of polyamines, compounds crucial for cells proliferation. Thus, both arginase isoenzymes seem to participate in liver cancerogenesis.

  12. Purification, characterization and application of lipoxygenase isoenzymes from Lasiodiplodia theobromae.

    PubMed

    Patel, Disha D; Patel, Ravi R; Thakkar, Vasudev R

    2015-01-01

    Lipoxygenase oxidizes linoleic acid into hydroperoxy octadecadienoic acid (HPOD), which is important in food and flavour industries for production of bread and flavouring compounds. As Lasiodiplodia theobromae is an unexplored, good source of lipoxygenase, it was purified from it by size-exclusion (Sephadex G100) and ion-exchange (DEAE-cellulose) chromatography and characterized. Upon purification, L. theobromae was found to contain two different lipoxygenases, one of 93 kDa (LOX1) and another of 45 kDa (LOX2). Both the isoenzymes were having optimum pH 6.0 and optimum temperatures 50 and 40 °C, respectively. The catalytic efficiency of LOX1 and LOX2 was found to be 1300 and 1.67 × 10(9), respectively. The catalytic efficiency of LOX2 is higher than the catalytic efficiency of soya bean LOX1 that is 10.9 × 10(6). Both the isoenzymes of LOX oxidized linoleic acid to produce 9-HPOD and 13-HPOD both; however, LOX1 produced more of 9-HPOD and LOX2 produced more of 13-HPOD. Both the LOXes were not inhibited by jasmonic acid. Addition of LOX1 and LOX2 altered the elasticity as well as viscosity of dough prepared from bleached wheat flour. PMID:25326184

  13. Transport and metabolism of D-lactate in Jerusalem artichoke mitochondria.

    PubMed

    Atlante, Anna; de Bari, Lidia; Valenti, Daniela; Pizzuto, Roberto; Paventi, Gianluca; Passarella, Salvatore

    2005-06-01

    We report here initial studies on D-lactate metabolism in Jerusalem artichoke. It was found that: 1) D-lactate can be synthesized by Jerusalem artichoke by virtue of the presence of glyoxalase II, the activity of which was measured photometrically in both isolated Jerusalem artichoke mitochondria and cytosolic fraction after the addition of S-D-lactoyl-glutathione. 2) Externally added D-lactate caused oxygen consumption by mitochondria, mitochondrial membrane potential increase and proton release, in processes that were insensitive to rotenone, but inhibited by both antimycin A and cyanide. 3) D-lactate was metabolized inside mitochondria by a flavoprotein, a putative D-lactate dehydrogenase, the activity of which could be measured photometrically in mitochondria treated with Triton X-100. 4) Jerusalem artichoke mitochondria can take up externally added D-lactate by means of a D-lactate/H(+) symporter investigated by measuring the rate of reduction of endogenous flavins. The action of the d-lactate translocator and of the mitochondrial D-lactate dehydrogenase could be responsible for the subsequent metabolism of d-lactate formed from methylglyoxal in the cytosol of Jerusalem artichoke. PMID:15949980

  14. Structure and Function of Plasmodium falciparum malate dehydrogenase: Role of Critical Amino Acids in C-substrate Binding Procket

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our lab have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal g...

  15. Analysis of Quaternary Structure of a [LDH-like] Malate Dehydrogenase of Plasmodium falciparum with Oligomeric Mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to L-lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archea and other prokaryotes. Initial sequence a...

  16. Lactation and reproduction*

    PubMed Central

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

    1975-01-01

    The authors review the literature on the effect of lactation on fertility in the absence of contraception and on the effects of contraceptive measures on lactation. They examine data from several countries on the intervals between births and on the return of menstruation and ovulation after childbirth, comparing lactating with nonlactating women. They conclude that lactation is an inefficient contraceptive for the individual, but that in populations sustained lactation is associated with reduced fertility. Possible physiological mechanisms causing lactation amenorrhoea are discussed. Though much of the literature on the effect of contraceptives on lactation is inadequate, there is general agreement that the estrogen component of hormonal preparations has an adverse effect on lactation, but that progestins alone do not. Many questions remain. Is this effect seen in established lactation, or only in the puerperal period? Is it a direct pharmacological effect, or are pill-users the mothers least motivated to maintain breast-feeding? Does a close relationship exist between hormones given and lactation performance? The authors comment on some of the technical deficiencies of previous studies in this field and discuss practical possibilities of, and limitations to, obtaining adequate scientific information in the future. PMID:1084804

  17. Combination of International Scoring System 3, High Lactate Dehydrogenase, and t(4;14) and/or del(17p) Identifies Patients With Multiple Myeloma (MM) Treated With Front-Line Autologous Stem-Cell Transplantation at High Risk of Early MM Progression–Related Death

    PubMed Central

    Moreau, Philippe; Cavo, Michele; Sonneveld, Pieter; Rosinol, Laura; Attal, Michel; Pezzi, Annalisa; Goldschmidt, Hartmut; Lahuerta, Juan Jose; Marit, Gerald; Palumbo, Antonio; van der Holt, Bronno; Bladé, Joan; Petrucci, Maria Teresa; Neben, Kai; san Miguel, Jesus; Patriarca, Francesca; Lokhorst, Henk; Zamagni, Elena; Hulin, Cyrille; Gutierrez, Norma; Facon, Thierry; Caillot, Denis; Benboubker, Lotfi; Harousseau, Jean-Luc; Leleu, Xavier; Avet-Loiseau, Hervé; Mary, Jean-Yves

    2014-01-01

    Purpose To construct and validate among patients with multiple myeloma (MM) who were treated with intensive therapy a prognostic index of early MM progression–related death. Patients and Methods Patient-level data from the Intergroupe Francophone du Myélome (IFM) 2005-01 trial (N = 482) were used to construct the prognostic index. The event was MM progression–related death within 2 years from treatment initiation. The index was validated using data from three other trials: the Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) 26866138-MMY-3006 trial (N = 480), the Programa para el Estudio de la Terapéutica en Hemopatía Maligna (PETHEMA)–GEMMENOS65 trial (N = 390), and the Hemato-Oncologie voor Volwassenen Nederland (HOVON) –65/German-Speaking Myeloma Multicenter Group (GMMG) –HD4 trial (N = 827). Results The risk of early MM progression–related death was related to three independent prognostic variables: lactate dehydrogenase (LDH) higher than than normal, International Staging System 3 (ISS3), and adverse cytogenetics [t(4;14) and/or del(17p)]. These three variables enabled the definition of an ordinal prognostic classification composed of four scores (0 to 3). Patients with a score of 3, defined by the presence of t(4;14) and/or del(17p) in addition to ISS3 and/or high LDH, comprised 5% (20 of 387 patients) to 8% (94 of 1,139 patients) of the patients in the learning and validation samples, respectively, and they had a very poor prognosis. When applied to the population of 855 patients who had received bortezomib-based induction therapy in the four trials, the prognostic classification was also able to segregate patients into four categories, with a very poor prognosis attributed to patients with a score of 3. Conclusion Our model allows the simple definition of a subgroup of MM patients at high risk of early MM progression–related death despite the use of the most modern and effective strategies. PMID:24888806

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

    PubMed Central

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

    2004-01-01

    Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export. PMID:15128549

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

    ERIC Educational Resources Information Center

    Meany, J. E.

    2007-01-01

    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…

  20. [Autoimmune hemolytic anemia with normal serum lactate dehydrogenase level].

    PubMed

    Mizuno, Hideaki; Hangaishi, Akira; Saika, Makoto; Morioka, Takehiko; Ando, Yayoi; Kida, Michiko; Usuki, Kensuke

    2015-11-01

    We herein report two cases of AIHA (autoimmune hemolytic anemia), a 25-year-old woman and a 77-year-old man, who presented with normal serum LDH values. Though in these two cases, low hemoglobin and haptoglobin, high total bilirubin and positive direct Coombs' test results led to the diagnosis of AIHA, both patients had normal LDH levels (218 and 187 IU/l). Both cases were successfully treated with prednisone. In the diagnosis of AIHA, elevated LDH is usually used as a marker of hemolysis. However, medical records of 24 AIHA patients collected in our institute from January 2001 to August 2012 revealed LDH levels to have been normal in 25% of these cases. This report indicates the importance of obtaining complete information about the blood testing of patients and taking these data into account when considering the diagnosis of AIHA. PMID:26666722

  1. The Partial Purification and Characterization of Lactate Dehydrogenase.

    ERIC Educational Resources Information Center

    Wolf, Edward C.

    1988-01-01

    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)

  2. Multichannel Simultaneous Determination of Activities of Lactate Dehydrogenase

    SciTech Connect

    Ma, L.

    2000-09-12

    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.

  3. Native myosin from adult rabbit skeletal muscle: isoenzymes and states of aggregation.

    PubMed

    Morel, J E; D'hahan, N; Taouil, K; Francin, M; Aguilar, A; Dalbiez, J P; Merah, Z; Grussaute, H; Hilbert, B; Ollagnon, F; Selva, G; Piot, F

    1998-04-21

    The globular heads of skeletal muscle myosin have been shown to exist as isoenzymes S1 (A1) and S1 (A2), and there are also isoforms of the heavy chains. Using capillary electrophoresis, we found two dominant isoenzymes of the whole native myosin molecule, in agreement with what has previously been found by various techniques for native and nondenatured myosin from adult rabbits. Findings about possible states of aggregation of myosin and its heads are contradictory. By analytical ultracentrifugation, we confirmed the existence of a tail-tail dimer. By laser light scattering, we found a head-head dimer in the presence of MgATP. Capillary electrophoresis coupled with analytical ultracentrifugation and laser light scattering led us to refine these results. We found tail-tail dimers in a conventional buffer. We found tail-tail and head-head dimers in the presence of 0.5 mM MgATP and pure head-head dimers in the presence of 6 mM MgATP. All the dimers were homodimers. Naming the dominant isoenzymes of myosin a and b, we observed tail-tail dimers with isoenzyme a (TaTa) and with isoenzyme b (TbTb) and also head-head dimers with isoenzyme a (HaHa) and with isoenzyme b (HbHb). PMID:9548927

  4. Fine separation and characterization of Candida rugosa lipase isoenzymes.

    PubMed

    Xin, Jia-Ying; Xiao-Xue Hu, Yi Xu; Cui, Jun-Ru; Li, Shu-Ben; Xia, Chun-Gu; Zhu, Li-Min

    2002-01-01

    Commercial Candida rugosa lipase has been separated into two distinct fractions (CRLA and CRLB) by anion-exchange chromatography. As analyzed on SDS-polyacrylamide gel electrophoresis, CRLA and CRLB are homogenous. At high ionic strength, CRLA and CRLB have similar hydrophobicity and UV spectra, suggesting that the open extent of the large hydrophobic pockets of CRLA and CRLB may be similar. At low ionic strength, using "hydrophobic interfacial affinity chromatography", both CRLA and CRLB have been separated into four isofractions. They have different hydrophobicity and UV spectra, suggesting that the open extent of the large hydrophobic pocket of the four forms may be different. Further, the conversion of CRL isoenzymes in the process of organic solvent treatment and ester hydrolysis were examined. The results clearly showed not only that CRLB had been converted to CRLA, but also that CRLA sub-fractions with different open extent of large hydrophobic pocket had been converted PMID:12362407

  5. Physiology of lactation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The breast changes in size, shape, and function during puberty, pregnancy, and lactation. The physiology of lactation is reviewed here. The breast is composed of fat and connective tissue that supports a tubuloalveolar structure. During development, anatomic changes involving new lobule formation an...

  6. Metabolic control analysis of L-lactate synthesis pathway in Rhizopus oryzae As 3.2686.

    PubMed

    Ke, Wei; Chang, Shu; Chen, Xiaoju; Luo, Shuizhong; Jiang, Shaotong; Yang, Peizhou; Wu, Xuefeng; Zheng, Zhi

    2015-11-01

    The relationship between the metabolic flux and the activities of the pyruvate branching enzymes of Rhizopus oryzae As 3.2686 during L-lactate fermentation was investigated using the perturbation method of aeration. The control coefficients for five enzymes, pyruvate dehydrogenase (PDH), pyruvate carboxylase (PC), pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH), were calculated. Our results indicated significant correlations between PDH and PC, PDC and LDH, PDC and ADH, LDH and ADH, and PDC and PC. It also appeared that PDH, PC, and LDH strongly controlled the L-lactate flux; PDH and ADH strongly controlled the ethanol flux; while PDH and PC strongly controlled the acetyl coenzyme A flux and the oxaloacetate flux. Further, the flux control coefficient curves indicated that the control of the system gradually transferred from PDC to PC during the steady state. Therefore, PC was the key rate-limiting enzyme that controls the flux distribution. PMID:26288952

  7. Lactate Racemization as a Rescue Pathway for Supplying d-Lactate to the Cell Wall Biosynthesis Machinery in Lactobacillus plantarum

    PubMed Central

    Goffin, Philippe; Deghorain, Marie; Mainardi, Jean-Luc; Tytgat, Isabelle; Champomier-Vergès, Marie-Christine; Kleerebezem, Michiel; Hols, Pascal

    2005-01-01

    Lactobacillus plantarum is a lactic acid bacterium that produces d- and l-lactate using stereospecific NAD-dependent lactate dehydrogenases (LdhD and LdhL, respectively). However, reduction of glycolytic pyruvate by LdhD is not the only pathway for d-lactate production since a mutant defective in this activity still produces both lactate isomers (T. Ferain, J. N. Hobbs, Jr., J. Richardson, N. Bernard, D. Garmyn, P. Hols, N. E. Allen, and J. Delcour, J. Bacteriol. 178:5431-5437, 1996). Production of d-lactate in this species has been shown to be connected to cell wall biosynthesis through its incorporation as the last residue of the muramoyl-pentadepsipeptide peptidoglycan precursor. This particular feature leads to natural resistance to high concentrations of vancomycin. In the present study, we show that L. plantarum possesses two pathways for d-lactate production: the LdhD enzyme and a lactate racemase, whose expression requires l-lactate. We report the cloning of a six-gene operon, which is involved in lactate racemization activity and is positively regulated by l-lactate. Deletion of this operon in an L. plantarum strain that is devoid of LdhD activity leads to the exclusive production of l-lactate. As a consequence, peptidoglycan biosynthesis is affected, and growth of this mutant is d-lactate dependent. We also show that the growth defect can be partially restored by expression of the d-alanyl-d-alanine-forming Ddl ligase from Lactococcus lactis, or by supplementation with various d-2-hydroxy acids but not d-2-amino acids, leading to variable vancomycin resistance levels. This suggests that L. plantarum is unable to efficiently synthesize peptidoglycan precursors ending in d-alanine and that the cell wall biosynthesis machinery in this species is specifically dedicated to the production of peptidoglycan precursors ending in d-lactate. In this context, the lactate racemase could thus provide the bacterium with a rescue pathway for d-lactate production upon

  8. Plant Formate Dehydrogenase

    SciTech Connect

    John Markwell

    2005-01-10

    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.

  9. Laboratory Prototype of Bioreactor for Oxidation of Toxic D-Lactate Using Yeast Cells Overproducing D-Lactate Cytochrome c Oxidoreductase

    PubMed Central

    Karkovska, Maria

    2016-01-01

    D-lactate is a natural component of many fermented foods like yogurts, sour milk, cheeses, and pickles vegetable products. D-lactate in high concentrations is toxic for children and people with short bowel syndrome and provokes encephalopathy. These facts convincingly demonstrate a need for effective tools for the D-lactate removal from some food products. The main idea of investigation is focused on application of recombinant thermotolerant methylotrophic yeast Hansenula polymorpha “tr6,” overproducing D-lactate: cytochrome c oxidoreductase (EC 1.1.2.4, D-lactate cytochrome c oxidoreductase, D-lactate dehydrogenase (cytochrome), DLDH). In addition to 6-fold overexpression of DLDH under a strong constitutive promoter (prAOX), the strain of H. polymorpha “tr6” (gcr1 catX/Δcyb2, prAOX_DLDH) is characterized by impairment in glucose repression of AOX promoter, devoid of catalase and L-lactate-cytochrome c oxidoreductase activities. Overexpression of DLDH coupling with the deletion of L-lactate-cytochrome c oxidoreductase activity opens possibility for usage of the strain as a base for construction of bioreactor for removing D-lactate from fermented products due to oxidation to nontoxic pyruvate. A laboratory prototype of column-type bioreactor for removing a toxic D-lactate from model solution based on permeabilized cells of the H. polymorpha “tr6” and alginate gel was constructed and efficiency of this process was tested. PMID:27446952

  10. Laboratory Prototype of Bioreactor for Oxidation of Toxic D-Lactate Using Yeast Cells Overproducing D-Lactate Cytochrome c Oxidoreductase.

    PubMed

    Karkovska, Maria; Smutok, Oleh; Gonchar, Mykhailo

    2016-01-01

    D-lactate is a natural component of many fermented foods like yogurts, sour milk, cheeses, and pickles vegetable products. D-lactate in high concentrations is toxic for children and people with short bowel syndrome and provokes encephalopathy. These facts convincingly demonstrate a need for effective tools for the D-lactate removal from some food products. The main idea of investigation is focused on application of recombinant thermotolerant methylotrophic yeast Hansenula polymorpha "tr6," overproducing D-lactate: cytochrome c oxidoreductase (EC 1.1.2.4, D-lactate cytochrome c oxidoreductase, D-lactate dehydrogenase (cytochrome), DLDH). In addition to 6-fold overexpression of DLDH under a strong constitutive promoter (prAOX), the strain of H. polymorpha "tr6" (gcr1 catX/Δcyb2, prAOX_DLDH) is characterized by impairment in glucose repression of AOX promoter, devoid of catalase and L-lactate-cytochrome c oxidoreductase activities. Overexpression of DLDH coupling with the deletion of L-lactate-cytochrome c oxidoreductase activity opens possibility for usage of the strain as a base for construction of bioreactor for removing D-lactate from fermented products due to oxidation to nontoxic pyruvate. A laboratory prototype of column-type bioreactor for removing a toxic D-lactate from model solution based on permeabilized cells of the H. polymorpha "tr6" and alginate gel was constructed and efficiency of this process was tested. PMID:27446952

  11. Recommended nomenclature for the vertebrate alcohol dehydrogenase gene family.

    PubMed

    Duester, G; Farrés, J; Felder, M R; Holmes, R S; Höög, J O; Parés, X; Plapp, B V; Yin, S J; Jörnvall, H

    1999-08-01

    The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Studies on 19 vertebrate animals have identified ADH orthologs across several species, and this has now led to questions of how best to name ADH proteins and genes. Seven distinct classes of vertebrate ADH encoded by non-orthologous genes have been defined based upon sequence homology as well as unique catalytic properties or gene expression patterns. Each class of vertebrate ADH shares <70% sequence identity with other classes of ADH in the same species. Classes may be further divided into multiple closely related isoenzymes sharing >80% sequence identity such as the case for class I ADH where humans have three class I ADH genes, horses have two, and mice have only one. Presented here is a nomenclature that uses the widely accepted vertebrate ADH class system as its basis. It follows the guidelines of human and mouse gene nomenclature committees, which recommend coordinating names across species boundaries and eliminating Roman numerals and Greek symbols. We recommend that enzyme subunits be referred to by the symbol "ADH" (alcohol dehydrogenase) followed by an Arabic number denoting the class; i.e. ADH1 for class I ADH. For genes we recommend the italicized root symbol "ADH" for human and "Adh" for mouse, followed by the appropriate Arabic number for the class; i.e. ADH1 or Adh1 for class I ADH genes. For organisms where multiple species-specific isoenzymes exist within a class, we recommend adding a capital letter after the Arabic number; i.e. ADH1A, ADH1B, and ADH1C for human alpha, beta, and gamma class I ADHs, respectively. This nomenclature will accommodate newly discovered members of the vertebrate ADH family, and will facilitate functional and evolutionary studies. PMID:10424757

  12. 21 CFR 862.1215 - Creatine phosphokinase/creatine kinase or isoenzymes test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1215 Creatine phosphokinase/creatine kinase or isoenzymes test...

  13. 21 CFR 862.1215 - Creatine phosphokinase/creatine kinase or isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1215 Creatine phosphokinase/creatine kinase or isoenzymes test...

  14. 21 CFR 862.1215 - Creatine phosphokinase/creatine kinase or isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1215 Creatine phosphokinase/creatine kinase or isoenzymes test...

  15. 21 CFR 862.1215 - Creatine phosphokinase/creatine kinase or isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1215 Creatine phosphokinase/creatine kinase or isoenzymes test...

  16. 21 CFR 862.1215 - Creatine phosphokinase/creatine kinase or isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1215 Creatine phosphokinase/creatine kinase or isoenzymes test...

  17. 21 CFR 862.1360 - Gamma-glutamyl transpeptidase and isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... transpeptidase and isoenzymes measurements are used in the diagnosis and treatment of liver diseases such as alcoholic cirrhosis and primary and secondary liver tumors. (b) Classification. Class I (general...

  18. 21 CFR 862.1360 - Gamma-glutamyl transpeptidase and isoenzymes test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... transpeptidase and isoenzymes measurements are used in the diagnosis and treatment of liver diseases such as alcoholic cirrhosis and primary and secondary liver tumors. (b) Classification. Class I (general...

  19. 21 CFR 862.1360 - Gamma-glutamyl transpeptidase and isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... transpeptidase and isoenzymes measurements are used in the diagnosis and treatment of liver diseases such as alcoholic cirrhosis and primary and secondary liver tumors. (b) Classification. Class I (general...

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

    PubMed Central

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

    2004-01-01

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

  1. [Pyruvate dehydrogenase deficiency and cerebral malformations].

    PubMed

    Eirís, J; Alvarez-Moreno, A; Briones, P; Alonso-Alonso, C; Castro-Gago, M

    1996-10-01

    Pyruvate dehydrogenase (PDH) deficiency is a major cause of primary lactic acidosis and severe global developmental delay. A deficiency of PDH E1 alpha, a subunit of the PDH complex is a prominent cause of congenital lactic acidosis. The E1 alpha cDNA and corresponding genomic DNA have been located in the short arm of the X-chromosome (Xp22-1). A isolated 'cerebral' lactic acidosis with cerebral dysgenesis is a recognized pattern of presentation of PDH deficiency. Here, we report clinical features, magnetic resonance, and biochemical studies of two females aged 6 months (case 1) and 26 months (case 2). Both had severe development delay, minor dysmorphic features, microcephaly, severe hypoplasia of the corpus callosum, cerebral atrophy, ventricular dilatation and increase in serum lactate levels without systemic acidosis. Urinary organic acid profile was compatible with PDH deficiency. Increased CSF lactate and pyruvate levels and reduced total PDH and PDH E1 activities in muscle and fibroblasts were observed in case 1. Otherwise, decreased total PDH activity in muscle but not in fibroblasts was seen in case 2. The PDH E1á gene was sequenced in the case 1 and a deletion in exon 7 was demonstrated. Dysmorphism with severe cerebral malformations in female patients merits a metabolic evaluation, including determination of lactate and pyruvate levels in CSF. PMID:8983728

  2. Comparisons of subunit 5A and 5B isoenzymes of yeast cytochrome c oxidase

    PubMed Central

    Dodia, Raksha; Meunier, Brigitte; Kay, Christopher W. M.; Rich, Peter R.

    2014-01-01

    Subunit 5 of Saccharomyces cerevisiae cytochrome c oxidase (CcO) is essential for assembly and has two isoforms, 5A and 5B. 5A is expressed under normoxic conditions, whereas 5B is expressed at very low oxygen tensions. As a consequence, COX5A-deleted strains (Δcox5A) have no or only low levels of CcO under normoxic conditions rendering them respiratory deficient. Previous studies have reported that respiratory growth could be restored by combining Δcox5A with mutations of ROX1 that encodes a repressor of COX5B expression. In these mutants, 5B isoenzyme expression level was 30–50% of wild-type (5A isoenzyme) and exhibited a maximum catalytic activity up to 3-fold faster than that of 5A isoenzyme. To investigate the origin of this effect, we constructed a mutant strain in which COX5B replaced COX5A downstream of the COX5A promoter. This strain expressed wild-type levels of the 5B isoenzyme, without the complication of additional effects caused by mutation of ROX1. When produced this way, the isoenzymes displayed no significant differences in their maximum catalytic activities or in their affinities for oxygen or cytochrome c. Hence the elevated activity of the 5B isoenzyme in the rox1 mutant is not caused simply by exchange of isoforms and must arise from an additional effect that remains to be resolved. PMID:25241981

  3. Homofermentative Production of d- or l-Lactate in Metabolically Engineered Escherichia coli RR1

    PubMed Central

    Chang, Dong-Eun; Jung, Heung-Chae; Rhee, Joon-Shick; Pan, Jae-Gu

    1999-01-01

    We investigated metabolic engineering of fermentation pathways in Escherichia coli for production of optically pure d- or l-lactate. Several pta mutant strains were examined, and a pta mutant of E. coli RR1 which was deficient in the phosphotransacetylase of the Pta-AckA pathway was found to metabolize glucose to d-lactate and to produce a small amount of succinate by-product under anaerobic conditions. An additional mutation in ppc made the mutant produce d-lactate like a homofermentative lactic acid bacterium. When the pta ppc double mutant was grown to higher biomass concentrations under aerobic conditions before it shifted to the anaerobic phase of d-lactate production, more than 62.2 g of d-lactate per liter was produced in 60 h, and the volumetric productivity was 1.04 g/liter/h. To examine whether the blocked acetate flux could be reoriented to a nonindigenous l-lactate pathway, an l-lactate dehydrogenase gene from Lactobacillus casei was introduced into a pta ldhA strain which lacked phosphotransacetylase and d-lactate dehydrogenase. This recombinant strain was able to metabolize glucose to l-lactate as the major fermentation product, and up to 45 g of l-lactate per liter was produced in 67 h. These results demonstrate that the central fermentation metabolism of E. coli can be reoriented to the production of d-lactate, an indigenous fermentation product, or to the production of l-lactate, a nonindigenous fermentation product. PMID:10103226

  4. Phenolic compounds as antioxidants: carbonic anhydrase isoenzymes inhibitors.

    PubMed

    Gülçin, Ilhami; Beydemir, Şükrü

    2013-03-01

    Antioxidant compounds can scavenge free radicals and increase shelf life by retarding the process of lipid peroxidation, which is one of the major reasons for deterioration of food, medicine and pharmaceutical products during processing and storage. An antioxidant molecule has been defined as any substance when found in low concentrations compared to that of an oxidizable substrate significantly delays or inhibits the oxidation. The major antioxidant compounds are especially phenolics and flavonoids, which are responsible for their health benefits. Carbonic anhydrase (EC 4.2.1.1., CA) is a pH regulatory/metabolic enzyme in all life kingdoms, being found in organisms all over the phylogenetic tree. It catalyzes the hydration of carbon dioxide (CO(2)) to bicarbonate (HCO(3) -) and the corresponding dehydration of HCO(3) -in acidic medium with regeneration of CO(2). Also, CA isoforms are found in a variety of tissues where they participate in several important biological processes such as acid-base balance, respiration, carbon dioxide and ion transport, bone resorption, ureagenesis, gluconeogenesis, lipogenesis and electrolyte secretion. On the other hand, the phenyl moiety of phenol was found to lay in the hydrophobic part of the CA active site, where CO(2), the physiologic substrate of the CAs, binds in the precatalytic complex, explaining thus the behavior of phenol as a unique CO(2) competitive inhibitor. This review consists of two main sections. The first section is devoted to main phenolic antioxidant compounds in the foodstuffs and beverages. The second general section is about some definitions of CA inhibitory effects of the main phenolic compounds used for antioxidant activity. The phenolic compounds and acids had marked especially CA I and CA II inhibitory effects and might be used as leads for generating CA isoenzyme inhibitors. This class of compounds may lead to isoform-selective inhibitors targeting just one or few of the medicinally relevant CAs. In

  5. Evidences of basal lactate production in the main white adipose tissue sites of rats. Effects of sex and a cafeteria diet.

    PubMed

    Arriarán, Sofía; Agnelli, Silvia; Sabater, David; Remesar, Xavier; Fernández-López, José Antonio; Alemany, Marià

    2015-01-01

    Female and male adult Wistar rats were fed standard chow or a simplified cafeteria diet for one month. Then, the rats were killed and the white adipose tissue (WAT) in four sites: perigonadal, retroperitoneal, mesenteric and subcutaneous (inguinal) were sampled and frozen. The complete WAT weight in each site was measured. Gene expression analysis of key lipid and glucose metabolism enzymes were analyzed, as well as tissue and plasma lactate and the activity of lactate dehydrogenase. Lactate gradients between WAT and plasma were estimated. The influence of sex and diet (and indirectly WAT mass) on lactate levels and their relationships with lactate dehydrogenase activity and gene expressions were also measured. A main conclusion is the high production of lactate by WAT, practically irrespective of site, diet or sex. Lactate production is a direct correlate of lactate dehydrogenase activity in the tissue. Furthermore, lactate dehydrogenase activity is again directly correlated with the expression of the genes Ldha and Ldhb for this enzyme. In sum, the ability to produce lactate by WAT is not directly dependent of WAT metabolic state. We postulate that, in WAT, a main function of the lactate dehydrogenase path may be that of converting excess available glucose to 3C fragments, as a way to limit tissue self-utilization as substrate, to help control glycaemia and/or providing short chain substrates for use as energy source elsewhere. More information must be gathered before a conclusive role of WAT in the control of glycaemia, and the full existence of a renewed glucose-lactate-fatty acid cycle is definitely established. PMID:25741703

  6. Evidences of Basal Lactate Production in the Main White Adipose Tissue Sites of Rats. Effects of Sex and a Cafeteria Diet

    PubMed Central

    Arriarán, Sofía; Agnelli, Silvia; Sabater, David; Remesar, Xavier; Fernández-López, José Antonio; Alemany, Marià

    2015-01-01

    Female and male adult Wistar rats were fed standard chow or a simplified cafeteria diet for one month. Then, the rats were killed and the white adipose tissue (WAT) in four sites: perigonadal, retroperitoneal, mesenteric and subcutaneous (inguinal) were sampled and frozen. The complete WAT weight in each site was measured. Gene expression analysis of key lipid and glucose metabolism enzymes were analyzed, as well as tissue and plasma lactate and the activity of lactate dehydrogenase. Lactate gradients between WAT and plasma were estimated. The influence of sex and diet (and indirectly WAT mass) on lactate levels and their relationships with lactate dehydrogenase activity and gene expressions were also measured. A main conclusion is the high production of lactate by WAT, practically irrespective of site, diet or sex. Lactate production is a direct correlate of lactate dehydrogenase activity in the tissue. Furthermore, lactate dehydrogenase activity is again directly correlated with the expression of the genes Ldha and Ldhb for this enzyme. In sum, the ability to produce lactate by WAT is not directly dependent of WAT metabolic state. We postulate that, in WAT, a main function of the lactate dehydrogenase path may be that of converting excess available glucose to 3C fragments, as a way to limit tissue self-utilization as substrate, to help control glycaemia and/or providing short chain substrates for use as energy source elsewhere. More information must be gathered before a conclusive role of WAT in the control of glycaemia, and the full existence of a renewed glucose-lactate-fatty acid cycle is definitely established. PMID:25741703

  7. Pyruvate Dehydrogenase and Pyruvate Dehydrogenase Kinase Expression in Non Small Cell Lung Cancer and Tumor-Associated Stroma1

    PubMed Central

    Koukourakis, Michael I; Giatromanolaki, Alexandra; Sivridis, Efthimios; Gatter, Kevin C; Harris, Adrian L; “Tumor and Angiogenesis Research Group”

    2005-01-01

    Abstract Pyruvate dehydrogenase (PDH) catalyzes the conversion of pyruvate to acetyl-coenzyme A, which enters into the Krebs cycle, providing adenosine triphosphate (ATP) to the cell. PDH activity is under the control of pyruvate dehydrogenase kinases (PDKs). Under hypoxic conditions, conversion of pyruvate to lactate occurs, a reaction catalyzed by lactate dehydrogenase 5 (LDH5). In cancer cells, however, pyruvate is transformed to lactate occurs, regardless of the presence of oxygen (aerobic glycolysis/Warburg effect). Although hypoxic intratumoral conditions account for HIF1α stabilization and induction of anaerobic metabolism, recent data suggest that high pyruvate concentrations also result in HIF1α stabilization independently of hypoxia. In the present immunohistochemical study, we provide evidence that the PDH/PDK pathway is repressed in 73% of non small cell lung carcinomas, which may be a key reason for HIF1α stabilization and “aerobic glycolysis.” However, about half of PDH-deficient carcinomas are not able to switch on the HIF pathway, and patients harboring these tumors have an excellent postoperative outcome. A small subgroup of clinically aggressive tumors maintains a coherent PDH and HIF/LDH5 expression. In contrast to cancer cells, fibroblasts in the tumor-supporting stroma exhibit an intense PDH but reduced PDK1 expression favoring maximum PDH activity. This means that stroma may use lactic acid produced by tumor cells, preventing the creation of an intolerable intratumoral acidic environment at the same time. PMID:15736311

  8. Capsaicin: a potent inhibitor of carbonic anhydrase isoenzymes.

    PubMed

    Arabaci, Betul; Gulcin, Ilhami; Alwasel, Saleh

    2014-01-01

    Carbonic anhydrase (CA, EC 4.2.1.1) is a zinc containing metalloenzyme that catalyzes the rapid and reversible conversion of carbon dioxide (CO2) and water (H2O) into a proton (H+) and bicarbonate (HCO3-) ion. On the other hand, capsaicin is the main component in hot chili peppers and is used extensively used in spices, food additives and drugs; it is responsible for their spicy flavor and pungent taste. There are sixteen known CA isoforms in humans. Human CA isoenzymes I, and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. In this study, the inhibition properties of capsaicin against the slow cytosolic isoform hCA I, and the ubiquitous and dominant rapid cytosolic isozymes hCA II were studied. Both CA isozymes were inhibited by capsaicin in the micromolar range. This naturally bioactive compound has a Ki of 696.15 µM against hCA I, and of 208.37 µM against hCA II. PMID:25014536

  9. [Systematics and genegeography of Juniperus communis inferred from isoenzyme data].

    PubMed

    Khantemirova, E V; Berkutenko, A N; Semerikov, V L

    2012-09-01

    Using isoenzyme analysis, 35 populations of Juniperus communis L. from various parts of the Russian species range and by one population from Sweden and Alaska were studied. The total sample size was 1200 plants. As a result, the existence ofJ. communis var. oblonga in North Caucasus and J. communis var. depressa in North America was confirmed, but genetic differences between J. communis var. communis and J. communis var. saxatilis were not detected in the main part of the Russian species range (European part of Russia, Ural, Siberia). These populations proved to be genetically uniform with the same predominant allelic frequencies, which may evidence recent settling of this species from one of Central or East European refugium. J. communis var. saxatilis from northeastern Russia inhabiting the region behind Verkhoyansk mountain and Russian Far East showed considerable differentiation in frequencies of alleles at three loci and geographical subdivision. These populations also exhibit high intrapopulation variation. This can be connected with the refugium in this territory. The origin of this group is probably connected with migrations from Central Asia (Tibet) in the direction to northeastern Russia along mountains connecting Central and North Asia. It is also assumed that migrations of this species previously proceeded across the Beringian land bridge. PMID:23113335

  10. [Psychopharmacotherapy in pregnancy and lactation. 2: Lactation].

    PubMed

    Lanczik, M; Knoche, M; Fritze, J

    1998-01-01

    Whilst the incidence of psychiatric disorders decreases during pregnancy, the risk during the postpartum period increases significantly, often leading to the necessity of psychopharmacological intervention during the puerperium, and subsequently during lactation and breast-feeding. The necessity for lithium prophylaxis in manic-depressive women after childbirth has been identified, and it is recommended that weaning rather than omission of psychopharmacological treatment is preferable during the puerperium. PMID:9522328

  11. Bioconversion of methane to lactate by an obligate methanotrophic bacterium

    PubMed Central

    Henard, Calvin A.; Smith, Holly; Dowe, Nancy; Kalyuzhnaya, Marina G.; Pienkos, Philip T.; Guarnieri, Michael T.

    2016-01-01

    Methane is the second most abundant greenhouse gas (GHG), with nearly 60% of emissions derived from anthropogenic sources. Microbial conversion of methane to fuels and value-added chemicals offers a means to reduce GHG emissions, while also valorizing this otherwise squandered high-volume, high-energy gas. However, to date, advances in methane biocatalysis have been constrained by the low-productivity and limited genetic tractability of natural methane-consuming microbes. Here, leveraging recent identification of a novel, tractable methanotrophic bacterium, Methylomicrobium buryatense, we demonstrate microbial biocatalysis of methane to lactate, an industrial platform chemical. Heterologous overexpression of a Lactobacillus helveticus L-lactate dehydrogenase in M. buryatense resulted in an initial titer of 0.06 g lactate/L from methane. Cultivation in a 5 L continuously stirred tank bioreactor enabled production of 0.8 g lactate/L, representing a 13-fold improvement compared to the initial titer. The yields (0.05 g lactate/g methane) and productivity (0.008 g lactate/L/h) indicate the need and opportunity for future strain improvement. Additionally, real-time analysis of methane utilization implicated gas-to-liquid transfer and/or microbial methane consumption as process limitations. This work opens the door to develop an array of methanotrophic bacterial strain-engineering strategies currently employed for biocatalytic sugar upgrading to “green” chemicals and fuels. PMID:26902345

  12. Bioconversion of methane to lactate by an obligate methanotrophic bacterium.

    PubMed

    Henard, Calvin A; Smith, Holly; Dowe, Nancy; Kalyuzhnaya, Marina G; Pienkos, Philip T; Guarnieri, Michael T

    2016-01-01

    Methane is the second most abundant greenhouse gas (GHG), with nearly 60% of emissions derived from anthropogenic sources. Microbial conversion of methane to fuels and value-added chemicals offers a means to reduce GHG emissions, while also valorizing this otherwise squandered high-volume, high-energy gas. However, to date, advances in methane biocatalysis have been constrained by the low-productivity and limited genetic tractability of natural methane-consuming microbes. Here, leveraging recent identification of a novel, tractable methanotrophic bacterium, Methylomicrobium buryatense, we demonstrate microbial biocatalysis of methane to lactate, an industrial platform chemical. Heterologous overexpression of a Lactobacillus helveticus L-lactate dehydrogenase in M. buryatense resulted in an initial titer of 0.06 g lactate/L from methane. Cultivation in a 5 L continuously stirred tank bioreactor enabled production of 0.8 g lactate/L, representing a 13-fold improvement compared to the initial titer. The yields (0.05 g lactate/g methane) and productivity (0.008 g lactate/L/h) indicate the need and opportunity for future strain improvement. Additionally, real-time analysis of methane utilization implicated gas-to-liquid transfer and/or microbial methane consumption as process limitations. This work opens the door to develop an array of methanotrophic bacterial strain-engineering strategies currently employed for biocatalytic sugar upgrading to "green" chemicals and fuels. PMID:26902345

  13. A novel mode of lactate metabolism in strictly anaerobic bacteria.

    PubMed

    Weghoff, Marie Charlotte; Bertsch, Johannes; Müller, Volker

    2015-03-01

    Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E0 ' = -190 mV excludes direct NAD(+) reduction (E0 ' = -320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron-transferring flavoprotein (Etf) that exhibited NAD(+) reduction only when reduced ferredoxin (Fd(2-) ) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin-based electron confurcation to drive endergonic lactate oxidation with NAD(+) as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E0 ' ≈ -500 mV) to NAD(+) according to: lactate + Fd(2-)  + 2 NAD(+)  → pyruvate + Fd + 2 NADH. The reduced Fd(2-) is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical ( Δ μ ˜ Na + ) and finally redox energy (Fd(2-) from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes. PMID:24762045

  14. Bioconversion of methane to lactate by an obligate methanotrophic bacterium

    DOE PAGESBeta

    Henard, Calvin A.; Smith, Holly; Dowe, Nancy; Kalyuzhnaya, Marina G.; Pienkos, Philip T.; Guarnieri, Michael T.

    2016-02-23

    Methane is the second most abundant greenhouse gas (GHG), with nearly 60% of emissions derived from anthropogenic sources. Microbial conversion of methane to fuels and value-added chemicals offers a means to reduce GHG emissions, while also valorizing this otherwise squandered high-volume, high-energy gas. However, to date, advances in methane biocatalysis have been constrained by the low-productivity and limited genetic tractability of natural methane-consuming microbes. Here, leveraging recent identification of a novel, tractable methanotrophic bacterium, Methylomicrobium buryatense, we demonstrate microbial biocatalysis of methane to lactate, an industrial platform chemical. Heterologous overexpression of a Lactobacillus helveticus L-lactate dehydrogenase in M. buryatense resultedmore » in an initial titer of 0.06 g lactate/L from methane. Cultivation in a 5 L continuously stirred tank bioreactor enabled production of 0.8 g lactate/L, representing a 13-fold improvement compared to the initial titer. The yields (0.05 g lactate/g methane) and productivity (0.008 g lactate/L/h) indicate the need and opportunity for future strain improvement. Additionally, real-time analysis of methane utilization implicated gas-to-liquid transfer and/or microbial methane consumption as process limitations. This work opens the door to develop an array of methanotrophic bacterial strain-engineering strategies currently employed for biocatalytic sugar upgrading to “green” chemicals and fuels.« less

  15. Mixed glucose and lactate uptake by Corynebacterium glutamicum through metabolic engineering.

    PubMed

    Neuner, Andreas; Heinzle, Elmar

    2011-03-01

    The Corynebacterium glutamicum ATCC 13032 lysC(fbr) strain was engineered to grow fast on racemic mixtures of lactate and to secrete lysine during growth on lactate as well as on mixtures of lactate and glucose. The wild-type C. glutamicum only grows well on L-lactate. Overexpression of D-lactate dehydrogenase (dld) achieved by exchanging the native promoter of the dld gene for the stronger promoter of the sod gene encoding superoxide dismutase in C. glutamicum resulted in a duplication of biomass yield and faster growth without any secretion of lysine. Elementary mode analysis was applied to identify potential targets for lysine production from lactate as well as from mixtures of lactate and glucose. Two targets for overexpression were pyruvate carboxylase and malic enzyme. The overexpression of these genes using again the sod promoter resulted in growth-associated production of lysine with lactate as sole carbon source with a carbon yield of 9% and a yield of 15% during growth on a lactate-glucose mixture. Both substrates were taken up simultaneously with a slight preference for lactate. As surmised from the elementary mode analysis, deletion of glucose-6-phosphate isomerase resulted in a decreased production of lysine on the mixed substrate. Elementary mode analysis together with suitable objective functions has been found a very useful tool guiding the design of strains producing lysine on mixed substrates. PMID:21370474

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

    SciTech Connect

    Husic, D.W.

    1986-01-01

    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.

  17. Alkaline phosphatase isoenzymes in mouse plasma detected by polyacrylamide-gel disk electrophoresis.

    PubMed

    Hatayama, Kazuhisa; Nishihara, Yoshito; Kimura, Sayaka; Goto, Ken; Nakamura, Daichi; Wakita, Atsushi; Urasoko, Yoshinaka

    2011-04-01

    Plasma alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. In the present study, we assessed the usefulness of a commercially available polyacrylamide-gel (PAG) disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co., Ltd., Tokyo, Japan) for identifying plasma ALP isoenzymes in mice of the Crlj:CD1 strain (ICR mice), which are commonly used in toxicity studies. We also examined age-related changes in plasma ALP isoenzymes in ICR mice. Electrophoresis was performed according to the manufacturer's instructions. In order to identify the origin of each ALP isoenzyme, in addition to plasma samples, tissue ALP extracts from the liver, bone and small intestine were treated with neuraminidase, anti-small intestinal ALP antibody, ALP inhibitor levamisole and/or wheat germ agglutinin (WGA). The kit revealed that main plasma ALP isoenzyme in intact ICR mice was bone-derived one, and it tended to decrease with age. On the other hand, liver-derived ALP isoenzyme greatly increased in plasma of cholestasis model mice induced by bile duct ligation. This model mouse had also a large molecular ALP detected in the stacking gel. This ALP was thought to be of intestinal origin because its activity remained even after levamisole inhibition. In addition, a minimum sample volume for sufficient resolution of plasma ALP isoenzymes was only 14µl. The results of this study suggest that the present method is a useful tool for detecting plasma ALP isoenzymes in mice and that pre-treatment of plasma with neuraminidase and concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity. PMID:21467748

  18. Hyperpolarized 13C NMR observation of lactate kinetics in skeletal muscle.

    PubMed

    Park, Jae Mo; Josan, Sonal; Mayer, Dirk; Hurd, Ralph E; Chung, Youngran; Bendahan, David; Spielman, Daniel M; Jue, Thomas

    2015-10-01

    The production of glycolytic end products, such as lactate, usually evokes a cellular shift from aerobic to anaerobic ATP generation and O2 insufficiency. In the classical view, muscle lactate must be exported to the liver for clearance. However, lactate also forms under well-oxygenated conditions, and this has led investigators to postulate lactate shuttling from non-oxidative to oxidative muscle fiber, where it can serve as a precursor. Indeed, the intracellular lactate shuttle and the glycogen shunt hypotheses expand the vision to include a dynamic mobilization and utilization of lactate during a muscle contraction cycle. Testing the tenability of these provocative ideas during a rapid contraction cycle has posed a technical challenge. The present study reports the use of hyperpolarized [1-(13)C]lactate and [2-(13)C]pyruvate in dynamic nuclear polarization (DNP) NMR experiments to measure the rapid pyruvate and lactate kinetics in rat muscle. With a 3 s temporal resolution, (13)C DNP NMR detects both [1-(13)C]lactate and [2-(13)C]pyruvate kinetics in muscle. Infusion of dichloroacetate stimulates pyruvate dehydrogenase activity and shifts the kinetics toward oxidative metabolism. Bicarbonate formation from [1-(13)C]lactate increases sharply and acetyl-l-carnitine, acetoacetate and glutamate levels also rise. Such a quick mobilization of pyruvate and lactate toward oxidative metabolism supports the postulated role of lactate in the glycogen shunt and the intracellular lactate shuttle models. The study thus introduces an innovative DNP approach to measure metabolite transients, which will help delineate the cellular and physiological role of lactate and glycolytic end products. PMID:26347554

  19. Separation of dehydrogenases on polyaminomethylstyrene.

    PubMed

    Schöpp, W; Meinert, S; Thyfronitou, J; Aurich, H

    1975-01-29

    The binding of dehydrogenases, especially alcohol dehydrogenase, and other proteins to several ion exchangers and hydrophobic polymers was investigated. Quantitative parameters for the stability of the polymer-protein complexes (obtained form double reciprocal plots) indicate a high but different affinity of many proteins for polyaminomethylstyrene. The chromatography of a mixture of five dehydrogenases and human serum albumin on polyaminomethylstyrene is described. PMID:237012

  20. Human soluble guanylate cyclase: functional expression and revised isoenzyme family.

    PubMed Central

    Zabel, U; Weeger, M; La, M; Schmidt, H H

    1998-01-01

    Soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to the second messenger cGMP, functions as the receptor for nitric oxide (NO) and nitrovasodilator drugs. Three distinct cDNA species of each subunit (alpha1-alpha3, beta1-beta3) have been reported from various species. From human sources, none of these have been expressed as functionally active enzyme. Here we describe the expression of human alpha/beta heterodimeric sGC in Sf9 cells yielding active recombinant enzyme that was stimulated by the nitrovasodilator sodium nitroprusside or the NO-independent activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1). At the protein level, both alpha and beta subunits were detected in human tissues, suggesting co-expression also in vivo. Moreover, resequencing of the human cDNA clones [originally termed alpha3 and beta3; Giuili, Scholl, Bulle and Guellaen (1992) FEBS Lett. 304, 83-88] revealed several sequencing errors in human alpha3; correction of these eliminated major regions of divergence from rat and bovine alpha1. As human beta3 also displays more than 98% similarity to rat and bovine beta1 at the amino acid level, alpha3 and beta3 represent the human homologues of rat and bovine alpha1 and beta1, and the isoenzyme family is decreased to two isoforms for each subunit (alpha1, alpha2; beta1, beta2). Having access to the human key enzyme of NO signalling will now permit the study of novel sGC-modulating compounds with therapeutic potential. PMID:9742212

  1. Creatine kinase activity and isoenzymes in lung, colon and liver carcinomas.

    PubMed Central

    Joseph, J.; Cardesa, A.; Carreras, J.

    1997-01-01

    We have compared the levels of creatine kinase (CK) activity and the distribution of CK isoenzymes determined by agarose gel electrophoresis in normal colon, liver and lung tissues, and in colon, liver and lung adenocarcinomas, lung squamous cell carcinomas and lung carcinoids. Colon and lung adenocarcinomas, and squamous cell carcinomas presented lower CK activity than the normal tissues and no differences were found between hepatocarcinoma and normal liver tissue. In contrast, lung carcinoids had higher CK activity than normal lung tissue. Type BB-CK was the predominant isoenzyme in normal lung, colon and liver tissues. Type MM isoenzyme was detected in normal lung and type MB-CK was found in normal colon. In most lung tumours the CK isoenzyme electrophoretic pattern did not change. However, no type BB-CK was detected in some hepatocarcinomas, type MM-CK decreased in lung carcinoids and type MB isoenzyme was not observed in colon adenocarcinomas. It is concluded that in most tumours there is a decrease in the expression of type B- and type M-CK subunits, whereas in lung carcinoid the expression of type B-CK activity increases. Thus, the increase in type BB-CK observed in the serum of patients with lung and colon adenocarcinomas is probably due mainly to enhanced enzyme release as a result of tumour cell necrosis. Images Figure 1 Figure 2 Figure 3 PMID:9303358

  2. Human gastric alcohol dehydrogenase activity: effect of age, sex, and alcoholism.

    PubMed Central

    Seitz, H K; Egerer, G; Simanowski, U A; Waldherr, R; Eckey, R; Agarwal, D P; Goedde, H W; von Wartburg, J P

    1993-01-01

    As various isoenzymes of gastric alcohol dehydrogenase exist and as the effect of sex and age on these enzymes is unknown, this study measured the activity of gastric alcohol dehydrogenase at high and low ethanol concentrations in endoscopic biopsy specimens from a total of 290 patients of various ages and from 10 patients with chronic alcoholism. Gastric alcohol dehydrogenase was also detected by immunohistological tests in biopsy specimens from 40 patients by the use of a polyclonal rabbit antibody against class I alcohol dehydrogenase. A significant correlation was found between the immunohistological reaction assessed by the intensity of the colour reaction in the biopsy specimen and the activity of alcohol dehydrogenase measured at 580 mM ethanol. While alcohol dehydrogenase activity measured at 16 mM ethanol was not significantly affected by age and sex, both factors influenced alcohol dehydrogenase activity measured at 580 mM ethanol. Young women below 50 years of age had significantly lower alcohol dehydrogenase activities in the gastric corpus and antrum when compared with age matched controls (SEM) (6.4 (0.7) v 8.8 (0.6) nmol/min/mg protein; p < 0.001 and 6.0 (1.3) v 9.5 (1.3) nmol/min/mg protein; p < 0.001). Over 50 years of age this sex difference was no longer detectable, as high Km gastric alcohol dehydrogenase activity decreases with age only in men and not in women. In addition, extremely low alcohol dehydrogenase activities have been found in gastric biopsy specimens from young male alcoholics (2.2 (0.5) nmol/min/mg protein), which returned to normal after two to three weeks of abstinence. The activity of alcohol dehydrogenase in the human stomach measured at 580 mM ethanol is decreased in young women, in elderly men, and in the subject with alcoholism. This decrease in alcohol dehydrogenase activity may contribute to the reduced first pass metabolism of ethanol associated with raised ethanol blood concentrations seen in these people. Images Figure

  3. Lactate: Friend or Foe.

    PubMed

    Hall, Mederic M; Rajasekaran, Sathish; Thomsen, Timothy W; Peterson, Andrew R

    2016-03-01

    Lactic acid has played an important role in the traditional theory of muscle fatigue and limitation of endurance exercise performance. It has been called a waste product of anaerobic metabolism and has been believed to be responsible for the uncomfortable "burn" of intense exercise and directly responsible for the metabolic acidosis of exercise, leading to decreased muscle contractility and ultimately cessation of exercise. Although this premise has been commonly taught, it is not supported by the scientific literature and has led to a great deal of confusion among the sports medicine and exercise science communities. This review will provide the sports medicine clinician with an understanding of contemporary lactate theories, including lactate's role in energy production, its contributions to metabolic acidosis, and its function as an energy substrate for a variety of tissues. Lactate threshold concepts will also be discussed, including a practical approach to understanding prediction of performance and monitoring of training progress based on these parameters. PMID:26972271

  4. Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production

    PubMed Central

    Maleki, Susan; Mærk, Mali; Valla, Svein

    2015-01-01

    The biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacterium Pseudomonas fluorescens relies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated in P. fluorescens. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption of zwf-1 resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption of gcd increased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell. PMID:25746989

  5. Optical detection of single cell lactate release for cancer metabolic analysis.

    PubMed

    Zheng, Xin Ting; Yang, Hong Bin; Li, Chang Ming

    2010-06-15

    Sensitive detection of extracellular lactate concentrations at a single cell level is of importance for studying the metabolic alterations in tumor progression. A unique nanoscale optical fiber lactate sensor was developed to monitor the extracellular lactate concentrations of cancer cells by immobilizing its nanotip with lactate dehydrogenases, which could catalyze lactate conversion to generate NADH for sensitive fluorescence detection. The results demonstrate that the fabricated nanosensor can successfully detect the extracellular lactate concentrations for single HeLa, MCF-7, and human fetal osteoblast (hFOB) cells, showing that the cancer cells have distinctly higher extracellular lactate concentrations than normal cells as that predicted by Warburg effect. The nanosensor was also employed to investigate the effect of a monocarboxylate transporter inhibitor on the lactate efflux from cancer cells. Different lactate efflux inhibition profiles were obtained for HeLa and MCF-7 cell lines. This work demonstrates that the nanosensor has potential for evaluating the effect of metabolic agents on cancer metabolism and survival. PMID:20469833

  6. Affinity chromatography of nicotinamide–adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide

    PubMed Central

    Barry, Standish; O'Carra, Pádraig

    1973-01-01

    1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD+ through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD+ (probably through the 8 position of the adenine residue) to a number of different spacer-arm–agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD+ derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD+. Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD+-binding site of this enzyme. Problems

  7. Lactate does not activate NF-κB in oxidative tumor cells

    PubMed Central

    Van Hée, Vincent F.; Pérez-Escuredo, Jhudit; Cacace, Andrea; Copetti, Tamara; Sonveaux, Pierre

    2015-01-01

    The lactate anion is currently emerging as an oncometabolite. Lactate, produced and exported by glycolytic and glutaminolytic cells in tumors, can be recycled as an oxidative fuel by oxidative tumors cells. Independently of hypoxia, it can also activate transcription factor hypoxia-inducible factor-1 (HIF-1) in tumor and endothelial cells, promoting angiogenesis. These protumoral activities of lactate depend on lactate uptake, a process primarily facilitated by the inward, passive lactate-proton symporter monocarboxylate transporter 1 (MCT1); the conversion of lactate and NAD+ to pyruvate, NADH and H+ by lactate dehydrogenase-1 (LDH-1); and a competition between pyruvate and α-ketoglutarate that inhibits prolylhydroxylases (PHDs). Endothelial cells do not primarily use lactate as an oxidative fuel but, rather, as a signaling agent. In addition to HIF-1, lactate can indeed activate transcription factor nuclear factor-κB (NF-κB) in these cells, through a mechanism not only depending on PHD inhibition but also on NADH alimenting NAD(P)H oxidases to generate reactive oxygen species (ROS). While NF-κB activity in endothelial cells promotes angiogenesis, NF-κB activation in tumor cells is known to stimulate tumor progression by conferring resistance to apoptosis, stemness, pro-angiogenic and metastatic capabilities. In this study, we therefore tested whether exogenous lactate could activate NF-κB in oxidative tumor cells equipped for lactate signaling. We report that, precisely because they are oxidative, HeLa and SiHa human tumor cells do not activate NF-κB in response to lactate. Indeed, while lactate-derived pyruvate is well-known to inhibit PHDs in these cells, we found that NADH aliments oxidative phosphorylation (OXPHOS) in mitochondria rather than NAD(P)H oxidases in the cytosol. These data were confirmed using oxidative human Cal27 and MCF7 tumor cells. This new information positions the malate-aspartate shuttle as a key player in the oxidative metabolism

  8. Elevated plasma citrulline: look for dihydrolipoamide dehydrogenase deficiency.

    PubMed

    Haviv, Ruby; Zeharia, Avraham; Belaiche, Corinne; Haimi Cohen, Yishai; Saada, Ann

    2014-02-01

    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 μM, range 59-282 μM) (normal range 1-45 μM) 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

  9. 21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alkaline phosphatase or isoenzymes test system. 862.1050 Section 862.1050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  10. 21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alkaline phosphatase or isoenzymes test system. 862.1050 Section 862.1050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  11. 21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alkaline phosphatase or isoenzymes test system. 862.1050 Section 862.1050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  12. Acetylcholinesterase and carbonic anhydrase isoenzymes I and II inhibition profiles of taxifolin.

    PubMed

    Gocer, Hulya; Topal, Fevzi; Topal, Meryem; Küçük, Murat; Teke, Dilek; Gülçin, İlhami; Alwasel, Saleh H; Supuran, Claudiu T

    2016-06-01

    Taxifolin, also known as dihydroquercetin, is a flavonoid commonly found in plants. Carbonic anhydrase (CA, EC 4.2.1.1) plays an important role in many critical physiological events including carbon dioxide (CO2)/bicarbonate ([Formula: see text]) respiration and pH regulation. There are 16 known CA isoforms in humans, of which human hCA isoenzymes I and II (hCA I and II) are ubiquitous cytosolic isoforms. In this study, the inhibition properties of taxifolin against the slow cytosolic isoenzyme hCA I, and the ubiquitous and dominant rapid cytosolic isoenzyme hCA II were studied. Taxifolin, as a naturally bioactive flavonoid, has a Ki of 29.2 nM against hCA I, and 24.2 nM against hCA II. For acetylcholinesterase enzyme (AChE) inhibition, Ki parameter of taxifolin was determined to be 16.7 nM. These results clearly show that taxifolin inhibited both CA isoenzymes and AChE at the nM levels. PMID:25893707

  13. Chitinase isoenzyme profiles in seedlings of Fusarium resistant and susceptible corn

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant chitinases have been implicated in both antagonistic and beneficial interactions with microorganisms. In an effort to better understand communication between Zea mays and its fungal environment, we are developing a knowledge base of corn chitinase isoenzymes. Of specific interest is the iden...

  14. Inhibition of Pig Phosphoenolpyruvate Carboxykinase Isoenzymes by 3-Mercaptopicolinic Acid and Novel Inhibitors.

    PubMed

    Hidalgo, Jorge; Latorre, Pedro; Carrodeguas, José Alberto; Velázquez-Campoy, Adrián; Sancho, Javier; López-Buesa, Pascual

    2016-01-01

    There exist two isoforms of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in pig populations that differ in a single amino acid (Met139Leu). The isoenzymes have different kinetic properties, affecting more strongly the Km and Vmax of nucleotides. They are associated to different phenotypes modifying traits of considerable economic interest. In this work we use inhibitors of phosphoenolpyruvate carboxykinase activity to search for further differences between these isoenzymes. On the one hand we have used the well-known inhibitor 3-mercaptopicolinic acid. Its inhibition patterns were the same for both isoenzymes: a three-fold decrease of the Ki values for GTP in 139Met and 139Leu (273 and 873 μM, respectively). On the other hand, through screening of a chemical library we have found two novel compounds with inhibitory effects of a similar magnitude to that of 3-mercaptopicolinic acid but with less solubility and specificity. One of these novel compounds, (N'1-({5-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]-2-thienyl}methylidene)-2,4-dichlorobenzene-1-carbohydrazide), exhibited significantly different inhibitory effects on either isoenzyme: it enhanced threefold the apparent Km value for GTP in 139Met, whereas in 139Leu, it reduced it from 99 to 69 μM. The finding of those significant differences in the binding of GTP reinforces the hypothesis that the Met139Leu substitution affects strongly the nucleotide binding site of PEPCK-C. PMID:27391465

  15. Inhibition of Pig Phosphoenolpyruvate Carboxykinase Isoenzymes by 3-Mercaptopicolinic Acid and Novel Inhibitors

    PubMed Central

    Hidalgo, Jorge; Latorre, Pedro; Carrodeguas, José Alberto; Velázquez-Campoy, Adrián; Sancho, Javier; López-Buesa, Pascual

    2016-01-01

    There exist two isoforms of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in pig populations that differ in a single amino acid (Met139Leu). The isoenzymes have different kinetic properties, affecting more strongly the Km and Vmax of nucleotides. They are associated to different phenotypes modifying traits of considerable economic interest. In this work we use inhibitors of phosphoenolpyruvate carboxykinase activity to search for further differences between these isoenzymes. On the one hand we have used the well-known inhibitor 3-mercaptopicolinic acid. Its inhibition patterns were the same for both isoenzymes: a three-fold decrease of the Ki values for GTP in 139Met and 139Leu (273 and 873 μM, respectively). On the other hand, through screening of a chemical library we have found two novel compounds with inhibitory effects of a similar magnitude to that of 3-mercaptopicolinic acid but with less solubility and specificity. One of these novel compounds, (N'1-({5-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]-2-thienyl}methylidene)-2,4-dichlorobenzene-1-carbohydrazide), exhibited significantly different inhibitory effects on either isoenzyme: it enhanced threefold the apparent Km value for GTP in 139Met, whereas in 139Leu, it reduced it from 99 to 69 μM. The finding of those significant differences in the binding of GTP reinforces the hypothesis that the Met139Leu substitution affects strongly the nucleotide binding site of PEPCK-C. PMID:27391465

  16. Alcohol's effect on lactation.

    PubMed

    Mennella, J

    2001-01-01

    Although pregnant women are discouraged from drinking alcohol because of alcohol's detrimental effect on fetal development, the lore of many cultures encourages lactating women to drink alcohol to optimize breast milk production and infant nutrition. In contrast to this folklore, however, studies demonstrate that maternal alcohol consumption may slightly reduce milk production. Furthermore, some of the alcohol consumed by a lactating woman is transferred to her milk and thus consumed by the infant. This alcohol consumption may adversely affect the infant's sleep and gross motor development and influence early learning about alcohol. Based on this science, it would seem that the recommendation for a nursing mother to drink a glass of beer or wine shortly before nursing may actually be counterproductive. PMID:11810962

  17. Efficiency of superoxide anions in the inactivation of selected dehydrogenases

    NASA Astrophysics Data System (ADS)

    Rodacka, Aleksandra; Serafin, Eligiusz; Puchala, Mieczyslaw

    2010-09-01

    The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as rad OH and ONOO -. In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH). We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion. Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.

  18. Cyanobacterial NADPH dehydrogenase complexes

    SciTech Connect

    Ogawa, Teruo; Mi, Hualing

    2007-07-01

    Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO2 uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO2 uptake. Other than CO2 uptake, chloroplastic NDH-1 complex has similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described forcomparison.

  19. Effect of municipal waste water effluent upon the expression of Glutathione S-transferase isoenzymes of brine shrimp Artemia.

    PubMed

    Grammou, Athina; Papadimitriou, Chrisa; Samaras, Peter; Vasara, Eleni; Papadopoulos, Athanasios I

    2011-06-01

    Multiple isoenzymes of the detoxification enzyme family Glutathione S-transferase are expressed in the brine shrimp Artemia. The number of the major ones detected in crude extract by means of chromatofocusing varied between three and four, depending on the age. Two isoenzymes, one alkaline and one neutral (with corresponding isoelectric points of 8.5 and 7.2) appear to be dominant in all three developmental stages studied, (24, 48, and 72 h after hatching). Culturing Artemia for 48 h after hatching, in artificial sea water prepared by municipal wastewater effluent resulted to significant alterations of the isoenzyme profile. In comparison to organisms cultured for the same period of time in artificial sea water prepared by filtered tap water, the expression of the alkaline isoenzyme decreased by 62% while that of the neutral isoenzyme increased by 58%. Furthermore, the enzyme activity of the major isoenzyme of the acidic area increased by more than two folds. It is worth mentioning that although the specific activity of the total enzyme in the whole body homogenate was elevated, no statistically significant alteration of the Km value was observed. These findings suggest that study of the isoenzyme profile of Glutathione S-transferase may offer high sensitivity in detecting environmental pollution and needs to be further investigated. PMID:21429555

  20. [Comparison of urinary and seminal N-acetyl-beta-glucosaminidase isoenzymes].

    PubMed

    Yasumoto, R; Asakawa, M; Yamamoto, M; Doi, Y; Kawashima, H; Umeda, M; Tanabe, S; Nishisaka, N; Kakinoki, K; Maekawa, M

    1987-11-01

    N-acetyl-beta-glucosaminidase (NAG) and its isoenzymes were measured in the urine and seminal plasma of healthy volunteers. Urinary NAG level was 2.62 +/- 1.30 U/L (mean +/- standard deviation), 1.99 +/- 0.77 U/g creatinine and seminal NAG level was 2370 +/- 1007 U/l. Urinary NAG level was 2.59 +/- 1.44 U/l, 1.93 +/- 0.80 U/g creatinine in males and 2.67 +/- 1.15 U/l, 2.08 +/- 0.78 U/g creatinine in females, and there was no significant sex difference. NAG isoenzymes in the urine and seminal plasma were divided into two major peaks, A and B. The A:B ratio was 78.0 +/- 6.5: 21.9 +/- 6.5 in the urine and 24.7 +/- 3.2: 75.2 +/- 3.2 in the seminal plasma, and was significantly different. Urinary NAG isoenzyme was 80.3 +/- 6.7: 19.6 +/- 6.7 in males and 74.1 +/- 4.3: 25.9 +/- 4.3 in females, and there was no significant difference between the sexes. These results indicated that urinary and seminal NAG can be differentiated by measuring the isoenzymes. Furthermore, the comparison of seminal NAG isoenzymes before and after vasectomy indicated that seminal NAG may be affected not only by the sperm but also by the prostatic fluid. PMID:3445866

  1. The phosphoglycerate kinase isoenzymes have distinct roles in the regulation of carbohydrate metabolism in Trypanosoma cruzi.

    PubMed

    Barros-Álvarez, Ximena; Cáceres, Ana J; Michels, Paul A M; Concepción, Juan Luis; Quiñones, Wilfredo

    2014-08-01

    The glycolytic enzyme phosphoglycerate kinase (PGK) is present in Trypanosoma cruzi as three isoenzymes, two of them located inside glycosomes (PGKA and PGKC) and another one in the cytosol (PGKB). The three isoenzymes are expressed at all stages of the life cycle of the parasite. A heterologous expression system for PGKA (rPGKA) was developed and the substrate affinities of the natural and recombinant PGKA isoenzyme were determined. Km values measured for 3-phosphoglycerate (3PGA) were 174 and 850 μM, and for ATP 217 and 236 μM, for the natural and recombinant enzyme, respectively. No significant differences were found between the two forms of the enzyme. The rPGKA was inhibited by Suramin with Ki values of 10.08 μM and 12.11 μM for ATP and 3PGA, respectively, and the natural enzyme was inhibited at similar values. A site-directed mutant was created in which the 80 amino acids PGKA sequence, present as a distinctive insertion in the N-terminal domain, was deleted. This internally truncated PGKA showed the same Km values and specific activity as the full-length rPGKA. The natural PGKC isoenzyme was purified from epimastigotes and separated from PGKA through molecular exclusion chromatography and its kinetic characteristics were determined. The Km value obtained for 3PGA was 192 μM, and 10 μM for ATP. Contrary to PGKA, the activity of PGKC is tightly regulated by ATP (substrate inhibition) with a Ki of 270 μM, suggesting a role for this isoenzyme in regulating metabolic fluxes inside the glycosomes. PMID:24858924

  2. Re-design of Saccharomyces cerevisiae flavocytochrome b2: introduction of L-mandelate dehydrogenase activity.

    PubMed

    Sinclair, R; Reid, G A; Chapman, S K

    1998-07-01

    Flavocytochrome b2 from Saccharomyces cerevisiae is an l-lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, l-mandelate. Using protein engineering methods we have altered the active site of flavocytochrome b2 and successfully introduced substantial mandelate dehydrogenase activity into the enzyme. Changes to Ala-198 and Leu-230 have significant effects on the ability of the enzyme to utilize l-mandelate as a substrate. The double mutation of Ala-198-->Gly and Leu-230-->Ala results in an enzyme with a kcat value (25 degrees C) with L-mandelate of 8.5 s-1, which represents an increase of greater than 400-fold over the wild-type enzyme. Perhaps more significantly, the mutant enzyme has a catalytic efficiency (as judged by kcat/Km values) that is 6-fold higher with l-mandelate than it is with L-lactate. Closer examination of the X-ray structure of S. cerevisiae flavocytochrome b2 led us to conclude that one of the haem propionate groups might interfere with the binding of L-mandelate at the active site of the enzyme. To test this idea, the activity with l-mandelate of the independently expressed flavodehydrogenase domain (FDH), was examined and found to be higher than that seen with the wild-type enzyme. In addition, the double mutation of Ala-198-->Gly and Leu-230-->Ala introduced into FDH produced the greatest mandelate dehydrogenase activity increase, with a kcat value more than 700-fold greater than that seen with the wild-type holoenzyme. In addition, the enzyme efficiency (kcat/Km) of this mutant enzyme was more than 20-fold greater with L-mandelate than with l-lactate. We have therefore succeeded in constructing an enzyme which is now a better mandelate dehydrogenase than a lactate dehydrogenase. PMID:9639570

  3. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

    ... control the activity of the complex: pyruvate dehydrogenase phosphatase turns on (activates) the complex, while pyruvate dehydrogenase ... binding protein (the PDHX gene), and pyruvate dehydrogenase phosphatase (the PDP1 gene) have been identified in people ...

  4. L-lactate production from seaweed hydrolysate of Laminaria japonica using metabolically engineered Escherichia coli.

    PubMed

    Mazumdar, Suman; Bang, Junho; Oh, Min-Kyu

    2014-02-01

    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

  5. Blocking Lactate Export by Inhibiting the Myc Target MCT1 Disables Glycolysis and Glutathione Synthesis

    PubMed Central

    Doherty, Joanne R.; Yang, Chunying; Scott, Kristen E. N.; Cameron, Michael D.; Fallahi, Mohammad; Li, Weimin; Hall, Mark A.; Amelio, Antonio L.; Mishra, Jitendra K.; Li, Fangzheng; Tortosa, Mariola; Genau, Heide Marika; Rounbehler, Robert J.; Lu, Yunqi; Dang, Chi. V.; Kumar, K. Ganesh; Butler, Andrew A.; Bannister, Thomas D.; Hooper, Andrea T.; Unsal-Kacmaz, Keziban; Roush, William R.; Cleveland, John L.

    2014-01-01

    Myc oncoproteins induce genes driving aerobic glycolysis, including lactate dehydrogenase-A that generates lactate. Here we report that Myc controls transcription of the lactate transporter SLC16A1/MCT1, and that elevated MCT1 levels are manifest in premalignant and neoplastic Eμ-Myc transgenic B cells and in human malignancies with MYC or MYCN involvement. Notably, disrupting MCT1 function leads to an accumulation of intracellular lactate that rapidly disables tumor cell growth and glycolysis, provoking marked alterations in glycolytic intermediates, and reductions in glucose transport, and in levels of ATP, NADPH and glutathione. Reductions in glutathione then lead to increases in hydrogen peroxide, mitochondrial damage and, ultimately, cell death. Finally, forcing glycolysis by metformin treatment augments this response and the efficacy of MCT1 inhibitors, suggesting an attractive combination therapy for MYC/MCT1-expressing malignancies. PMID:24285728

  6. Hormonal contraception and lactation.

    PubMed

    Kelsey, J J

    1996-12-01

    Hormonal contraceptive measures can be used immediately postpartum if the patient so desires. Progestin-only contraceptives are preferable to estrogen-containing methods if initiated during the first six months after delivery. Progestin only contraceptives do not appear to affect milk volume, composition, or to cause deleterious effects in the infant. Ideally for women who desire a form of contraception in addition to lactation-induced amenorrhea, progestin-only methods should be started at six weeks postpartum if the woman is fully breastfeeding. Since contraception protection is provided by lactation amenorrhea, the six week delay will decrease infant exposure to exogenous hormones and decrease the incidence of irregular postpartum bleeding. Milk volume may decrease with the use of estrogen; however, no detrimental effects have been shown on infant growth or development. For women who are planning to gradually wean their infant, use of COCs may provide an easier transition to bottle-feeding. COCs should be used with caution by women who are not able to obtain supplemental milk. A decrease in milk volume can lead to earlier discontinuation of the hormonal contraceptive in an attempt to increase milk quantity. Supplementation is often needed, and then the woman ovulates again, possibly resulting in an unintended pregnancy. Many women are motivated immediately postpartum to accept contraception. For other women, lack of access to health care may provide barriers in obtaining adequate contraception later. In either case, there are adequate data to show no detriments of starting progestin-only contraceptives within days of delivery. Therefore, the best method for the patient should be employed to ensure adequate contraception while preserving optimal lactation. PMID:9025449

  7. Uranyl nitrate inhibits lactate gluconeogenesis in isolated human and mouse renal proximal tubules: A {sup 13}C-NMR study

    SciTech Connect

    Renault, Sophie; Faiz, Hassan; Gadet, Rudy; Ferrier, Bernard; Martin, Guy; Baverel, Gabriel; Conjard-Duplany, Agnes

    2010-01-01

    As part of a study on uranium nephrotoxicity, we investigated the effect of uranyl nitrate in isolated human and mouse kidney cortex tubules metabolizing the physiological substrate lactate. In the millimolar range, uranyl nitrate reduced lactate removal and gluconeogenesis and the cellular ATP level in a dose-dependent fashion. After incubation in phosphate-free Krebs-Henseleit medium with 5 mM L-[1-{sup 13}C]-, or L-[2-{sup 13}C]-, or L-[3-{sup 13}C]lactate, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods. In the presence of 3 mM uranyl nitrate, glucose production and the intracellular ATP content were significantly reduced in both human and mouse tubules. Combination of enzymatic and NMR measurements with a mathematical model of lactate metabolism revealed an inhibition of fluxes through lactate dehydrogenase and the gluconeogenic enzymes in the presence of 3 mM uranyl nitrate; in human and mouse tubules, fluxes were lowered by 20% and 14% (lactate dehydrogenase), 27% and 32% (pyruvate carboxylase), 35% and 36% (phosphoenolpyruvate carboxykinase), and 39% and 45% (glucose-6-phosphatase), respectively. These results indicate that natural uranium is an inhibitor of renal lactate gluconeogenesis in both humans and mice.

  8. NADP-dependent aromatic alcohol dehydrogenase in polyploid wheats and their diploid relatives. On the origin and phylogeny of polyploid wheats.

    PubMed

    Jaaska, V

    1978-09-01

    The three major isoenzymes of the NADP-dependent aromatic alcohol dehydrogenase (ADH-B), distinguished in polyploid wheats by means of polyacrylamide gel electrophoresis, are shown to be coded by homoeoalleles of the locus Adh-2 on short arms of chromosomes of the fifth homoeologous group. Essentially codominant expression of the Adh-2 homoeolleles of composite genomes was observed in young seedlings of hexaploid wheats (T. aestivum s.l.) and tetraploid wheats of the emmer group (T. turgidum s.l.), whereas only the isoenzyme characteristic of the A genome is present in the seedlings of the timopheevii-group tetraploids (T. timopheevii s.str. and T. araraticum).The slowest-moving B(3) isoenzyme of polyploid wheats, coded by the homoeoallele of the B genome, is characteristic of the diploid species Aegilops speltoides S.l., including both its awned and awnless forms, but was not encountered in Ae. bicornis, Ae. sharonensis and Ae. longissima. The last two diploids, as well as Ae. tauschii, Ae. caudata, Triticum monococcum s.str., T. boeoticum s.l. (incl. T. thaoudar) and T. urartu all shared a common isoenzyme coinciding electrophoretically with the band B(2) controlled by the A and D genome homoeoalleles in polyploid wheats. Ae. bicomis is characterized by the slowest isoenzyme, B(4), not found in wheats and in the other diploid Aegilops species studied.Two electrophoretic variants of ADH-B, B(1) and B(2), considered to be alloenzymes of the A genome homoeoallele, were observed in T. dicoccoides, T. dicoccon, T. turgidum. s.str. and T. spelta, whereas B(2) was characteristic of T. timopheevii s.l. and only B(1) was found in the remaining taxa of polyploid wheats. The isoenzyme B(1), not encountered among diploid species, is considered to be a mutational derivative which arose on the tetraploid level from its more ancestral form B(2) characteristic of diploid wheats.The implication of the ADH-B isoenzyme data to the problems of wheat phylogeny and gene evolution is

  9. Isolation, purification, and characterization of thermophilic T80 isoenzyme of xylose isomerase from the xerophyte Cereus pterogonus.

    PubMed

    Ravikumar, Sambandam; Shyamala, Sivalingam; Muthuraman, Pandurangan; Srikumar, Kotteazeth

    2011-01-01

    A thermostable isoenzyme (T(80)) of xylose isomerase from the eukaryote xerophyte Cereus pterogonus was purified to homogeneity by precipitation with ammonium sulfate and column chromatography on Dowex-1 ion exchange, with Sephadex G-100 gel filtration, resulting in an approximately 25.55-fold increase in specific activity and a final yield of approximately 17.9%. Certain physiochemical and kinetic properties (K(m) and V(max)) of the T(80) xylose isomerase isoenzyme were investigated. The molecular mass of the purified T(80) isoenzyme was 68 kD determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyclonal antibodies against the purified T(80) isoenzyme recognized a single polypeptide band on Western blots. The activation energy required for the thermal denaturation of the isoenzyme was determined to be 61.84 KJ mol(-1). The use of differential scanning calorimetry established the melting temperature of the CPXI isoenzyme to be 80°C, but when studied with added metal ions, melting temperature increases to more than the normal. Fluorescence spectroscopy of T(80) isoenzymes yielded an emission peak with λ(em) at 320 nm and 340 nm, respectively, confirming the presence of Trp residue in these proteins. Electron paramagnetic resonance (EPR) analysis at liquid nitrogen temperature established the presence of Mn(2+) and Co(2+) associated with each isoenzyme. These enzyme species exhibited different thermal and pH stabilities compared to their mesophilic counterparts and offered greater efficiency in functioning as a potential alternate catalytic converter of glucose in the production of high-fructose corn syrup (HFCS) for the sweetener industry and for ethanol production. PMID:21442549

  10. The role of two isoenzymes of alpha-amylase of Araucaria araucana (Araucariaceae) on the digestion of starch granules during germination.

    PubMed

    Waghorn, Juana J; del Pozo, Talía; Acevedo, Elba A; Cardemil, Liliana A

    2003-03-01

    Starch is the principal reserve of Araucaria araucana seeds, and it is hydrolysed during germination mainly by alpha-amylase. There are several alpha-amylase isoenzymes whose patterns change in the embryo and in the megagametophyte from the one observed in quiescent seeds (T(0)) to a different one observed 90 h after imbibition (T(90)). The objective of this research was to study the roles of two purified alpha-amylase isoenzymes by in vitro digestion of starch granules extracted from the tissues at two times of imbibition: one is abundant in quiescent seeds and the other is abundant after 90 h of imbibition. The isoenzymes digested the starch granules of their own stage of germination better, since the isoenzyme T(0) digested starch granules mainly from quiescent seeds, while the isoenzyme T(90) digested starch mainly at 90 h of imbibition. The sizes of the starch granule and the tissue from which these granules originated make a difference to digestion by the isoenzymes. Embryonic isoenzyme T(0) digested large embryonic starch granules better than small and medium-sized granules, and better than those isolated from megagametophytes. Similarly isoenzyme T(90) digested small embryonic starch granules better than medium-sized and large granules, and better than those isolated from megagametophytes. However, a mixture of partially purified megagametophytic isoenzymes T(0) and T(90) digested the megagametophytic granules better than those isolated from embryos. Studies of in vitro sequential digestion of starch granules with these isoenzymes corroborated their specificity. The isoenzyme T(90) digested starch granules previously digested by the isoenzyme T(0). This suggests that in vivo these two isoenzymes may act sequentially in starch granule digestion. PMID:12598561

  11. Testis-specific expression of a functional retroposon encoding glucose-6-phosphate dehydrogenase in the mouse

    SciTech Connect

    Hendriksen, P.J.M. |; Hoogerbrugge, J.W.; Baarends, W.M.

    1997-05-01

    The X-chromosomal gene glucose-6-phosphate dehydrogenase (G6pd) is known to be expressed in most cell types of mammalian species. In the mouse, we have detected a novel gene, designated G6pd-2, encoding a G6PD isoenzyme. G6pd-2 does not contain introns and appears to represent a retroposed gene. This gene is uniquely transcribed in postmeiotic spermatogenic cells in which the X-encoded G6pd gene is not transcribed. Expression of the G6pd-2 sequence in a bacterial system showed that the encoded product is an active enzyme. Zymogramic analysis demonstrated that recombinant G6PD-2, but not recombinant G6PD-1 (the X-chromosome-encoded G6PD), formed tetramers under reducing conditions. Under the same conditions, G6PD tetramers were also found in extracts of spermatids and spermatozoa, indicating the presence of G6pd-2-encoded isoenzyme in these cell types. G6pd-2 is one of the very few known expressed retroposons encoding a functional protein, and the presence of this gene is probably related to X chromosome inactivation during spermatogenesis. 62 refs., 7 figs.

  12. Serum hydroxybutyrate dehydrogenase (HBD) assays in the clinical laboratory

    PubMed Central

    Montazemi, K.; Lines, J. G.

    1972-01-01

    Measurement of serum hydroxybutyrate dehydrogenase (HBD) activity for suspected myocardial infarction will nowadays usually be carried out with a commercially available test kit. Four such kits have been compared and evaluated: British Drug Houses HBDH set, Boehringer Corporation LDH-1-Iso-enzyme `α-HBDH' kit, Calbiochem α-HBDH Statpack, and Eskalab α-HBDH reagent tablets. Of the 100 sera examined, 60 were from patients believed to have suffered a recent myocardial infarction, and 40 samples were from patients aged between 45 and 65 years who had no known hepatic, renal, or cardiac pathology, these being used to determine the normal range for each kit. Under standardized conditions the activity found differed from kit to kit; the clinical value of the various assay systems in terms of their ability to discriminate between normal and pathological sera is assessed; and finally the cost and the convenience of use of each kit procedure are discussed. Theoretical and experimental evidence is provided in support of an assay temperature of 30°C, and a plea is made for international agreement on enzyme assay conditions. PMID:5017443

  13. Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes.

    PubMed

    Cuba, C A; Torno, C O; Ledesma, O; Visciarelli, E; Garcia, S; Prat, M I; Costamagna, R; Barbieri, L; Evans, D A

    1996-01-01

    Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and species-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly species-specific Monoclonal Antibody (Clone: B-18, Leishmania-(Viannia) braziliensis marker) disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosine as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme. PMID:9293087

  14. Acetohydroxy acid synthase isoenzymes of Escherichia coli K12 and Salmonella typhimurium.

    PubMed

    de Felice, M; Lago, C T; Squires, C H; Calvo, J M

    1982-01-01

    In Escherichia coli K12 and in Salmonella typhimurium the first step common to the biosynthesis of isoleucine, leucine and valine is catalyzed by an intriguing system of isoenzymes. Two of these are normally expressed, while the genetic determinant for a third one is transcribed, but not translated as an active polypeptide. We analyze here the significance of this system in the light of the most recent results. PMID:6805381

  15. Regional and systemic oxygen delivery/uptake relations and lactate flux in hyperdynamic, endotoxin-treated dogs.

    PubMed

    Curtis, S E; Cain, S M

    1992-02-01

    Pathologic oxygen supply dependency (PO2SD) may be etiologic in multisystem organ failure (MSOF) and has been related to mortality in sepsis. Although elevated lactate levels are generally assumed to be a marker of anaerobiosis in these patients, endotoxin may increase serum lactate by inactivation of pyruvate dehydrogenase (PDH), unrelated to tissue PO2. We hypothesized that regional lactate flux may correlate poorly with local oxygen delivery in sepsis. This study examined both the whole-body (WB) and regional (isolated hind limb L and gut G) responses to endotoxin infusion in terms of oxygen delivery, oxygen uptake, and lactate flux in 12 pentobarbital-anesthetized dogs. To separate hypoxia-induced lactate production from that related to inactivation of PDH by endotoxin, half the dogs received dichloroacetate (DCA), a PDH activator. After endotoxin and volume resuscitation, each animal had low systemic vascular resistance with normal to high cardiac output. Despite adequate oxygen delivery to WB, L, and G, arterial lactate levels rose significantly. A 30-min hypoxic challenge (12% FIO2) did not increase lactate levels but did increase WB O2 uptake. DCA normalized lactate levels without influencing oxygen delivery and uptake relations. These data show that lactate levels in endotoxic states may be a poor marker of tissue hypoxia and may be more related to PDH activity. PMID:1736740

  16. Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes.

    PubMed

    Brocca, Stefania; Secundo, Francesco; Ossola, Mattia; Alberghina, Lilia; Carrea, Giacomo; Lotti, Marina

    2003-10-01

    The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site. In the active enzyme conformation, the open lid participates in the substrate-binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain-length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium. PMID:14500889

  17. Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes

    PubMed Central

    Brocca, Stefania; Secundo, Francesco; Ossola, Mattia; Alberghina, Lilia; Carrea, Giacomo; Lotti, Marina

    2003-01-01

    The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site. In the active enzyme conformation, the open lid participates in the substrate-binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain-length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium. PMID:14500889

  18. Liquid-chromatographic separation and on-line bioluminescence detection of creatine kinase isoenzymes

    SciTech Connect

    Bostick, W.D.; Denton, M.S.; Dinsmore, S.R.

    1980-01-01

    Isoenzymes of creatine kinase were separated by anion-exchange chromatography, with use of an elution gradient containing lithium acetate (0.1 to 0.6 mol/L). A stream splitter was used to divert a 5% side stream of column effluent, which was subsequently mixed with the reagents necessary for bioluminescence assay of the separated isoenzymes. The use of the stream splitter greatly decreased the rate of consumption of reagent and, when combined with a peristaltic pumping system, permitted independent control of the side-stream flow rate. Thus both the residence interval in a delay coil in which the ATP reaction product is formed and the bioluminescence emission was monitored in a flow-through fluorometer without use of an external light source or filters. Separation and detection of the isoenzymes of creatine kinase were rapid, sensitive, and highly selective. The incremental decrease of bioluminescence response owing to inhibition by the ions in the eluent was less than 31% across the entire gradient.

  19. Discovery of a novel class of covalent inhibitor for aldehyde dehydrogenases

    SciTech Connect

    Khanna, Mary; Chen, Che-Hong; Kimble-Hill, Ann; Parajuli, Bibek; Perez-Miller, Samantha; Baskaran, Sulochanadevi; Kim, Jeewon; Dria, Karl; Vasiliou, Vasilis; Mochly-Rosen, Daria; Hurley, Thomas D.

    2012-10-23

    Human aldehyde dehydrogenases (ALDHs) comprise a family of 17 homologous enzymes that metabolize different biogenic and exogenic aldehydes. To date, there are relatively few general ALDH inhibitors that can be used to probe the contribution of this class of enzymes to particular metabolic pathways. Here, we report the discovery of a general class of ALDH inhibitors with a common mechanism of action. The combined data from kinetic studies, mass spectrometric measurements, and crystallographic analyses demonstrate that these inhibitors undergo an enzyme-mediated {beta}-elimination reaction generating a vinyl ketone intermediate that covalently modifies the active site cysteine residue present in these enzymes. The studies described here can provide the basis for rational approach to design ALDH isoenzyme-specific inhibitors as research tools and perhaps as drugs, to address diseases such as cancer where increased ALDH activity is associated with a cellular phenotype.

  20. Plasma lactic dehydrogenase activities in men during bed rest with exercise training

    NASA Technical Reports Server (NTRS)

    Greenleaf, J. E.; Juhos, L. T.; Young, H. L.

    1985-01-01

    Peak oxygen uptake and the activity of lactic dehydrogenase (LDH-T) and its five isoenzymes were measured by spectrophotometer in seven men before, during, and after bed rest and exercise training. Exercise training consisted of isometric leg exercises of 250 kcal/hr for a period of one hour per day. It is found that LDH-T was reduced by 0.05 percent in all three regimens by day 10 of bed rest, and that the decrease occurred at different rates. The earliest reduction in LDH-T activity in the no-exercise regimen was associated with a decrease in peak oxygen uptake of 12.3 percent. It is concluded that isometric (aerobic) muscular strength training appear to maintain skeletal muscle integrity better during bed rest than isotonic exercise training. Reduced hydrostatic pressure during bed rest, however, ultimately counteracts the effects of both moderate isometric and isotonic exercise training, and may result in decreased LDH-T activity.

  1. Alcohol Dehydrogenase from Methylobacterium organophilum

    PubMed Central

    Wolf, H. J.; Hanson, R. S.

    1978-01-01

    The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10−5 M for methanol and 8.2 × 10−5 M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum. Images PMID:80974

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

    SciTech Connect

    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

    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.

  3. Lactate and lactate clearance in acute cardiac care patients

    PubMed Central

    Lazzeri, Chiara; Picariello, Claudio; Dini, Carlotta Sorini; Gensini, Gian Franco; Valente, Serafina

    2012-01-01

    Hyperlactataemia is commonly used as a diagnostic and prognostic tool in intensive care settings. Recent studies documented that serial lactate measurements over time (or lactate clearance), may be clinically more reliable than lactate absolute value for risk stratification in different pathological conditions. While the negative prognostic role of hyperlactataemia in several critical ill diseases (such as sepsis and trauma) is well established, data in patients with acute cardiac conditions (i.e. acute coronary syndromes) are scarce and controversial. The present paper provides an overview of the current available evidence on the clinical role of lactic acid levels and lactate clearance in acute cardiac settings (acute coronary syndromes, cardiogenic shock, cardiac surgery), focusing on its prognostic role. PMID:24062898

  4. Migraine in pregnancy and lactation.

    PubMed

    David, Paru S; Kling, Juliana M; Starling, Amaal J

    2014-04-01

    Migraine headache is a significant health problem affecting women more than men. In women, the hormonal fluctuations seen during pregnancy and lactation can affect migraine frequency and magnitude. Understanding the evaluation of headache in pregnancy is important, especially given the increased risk of secondary headache conditions. Pregnancy and lactation can complicate treatment options for women with migraine because of the risk of certain medications to the fetus. This review includes details of the workup and then provides treatment options for migraine during pregnancy and lactation. PMID:24604057

  5. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    PubMed Central

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated. PMID:24949265

  6. Lactate levels with glioblastoma multiforme.

    PubMed

    Kahlon, Arunpreet Singh; Alexander, Mariam; Kahlon, Arundeep; Wright, Jonathan

    2016-07-01

    A 37-year-old woman with known glioblastoma multiforme was admitted for treatment of new deep vein thrombosis. Anion gap and plasma lactate levels were found to be elevated. Magnetic resonance imaging of the brain showed a stable, advanced glioblastoma multiforme. All causes of lactic acidosis, including infections and medications, were ruled out. Aggressive tumors have been shown to produce lactate levels in minute quantities in their microenvironment, which helps them metastasize and evade immune response and even radiation. PMID:27365883

  7. Lactate levels with glioblastoma multiforme

    PubMed Central

    Kahlon, Arunpreet Singh; Alexander, Mariam; Kahlon, Arundeep

    2016-01-01

    A 37-year-old woman with known glioblastoma multiforme was admitted for treatment of new deep vein thrombosis. Anion gap and plasma lactate levels were found to be elevated. Magnetic resonance imaging of the brain showed a stable, advanced glioblastoma multiforme. All causes of lactic acidosis, including infections and medications, were ruled out. Aggressive tumors have been shown to produce lactate levels in minute quantities in their microenvironment, which helps them metastasize and evade immune response and even radiation. PMID:27365883

  8. The separation, partial purification and some properties of isoenzymes of aldolase from guinea-pig cerebral cortex

    PubMed Central

    Nicholas, P. C.; Bachelard, H. S.

    1969-01-01

    1. Aldolase isoenzymes from guinea-pig cerebral cortex were partially purified and separated by ammonium sulphate fractionation and chromatography on DEAE-cellulose. 2. Each purified isoenzyme was shown to be virtually uncontaminated with other forms by starch-gel electrophoresis. The quantitative distribution of the isoenzymes was: I, 6·2%; II, 5·2%; III, 15·3%; IV, 25·7%; V, 33·3%. 3. The pH optima for the five separated isoenzymes were similar; all were in the range pH7·5–8·0. Values for pKa (6·31–6·55) and pKb (9·45–9·59) were calculated from the data and suggested the involvement of histidine and lysine residues. 4. The stabilities of the isoenzymes were shown to be I=II>III>IV>V at pH4·4 in order of decreasing stability and are discussed in terms of subunit structure. 5. The substrate activity ratios (fructose 1,6-diphosphate/fructose 1-phosphate) were measured and all were in the range 12–44. ImagesPLATE 1 PMID:5821724

  9. Functional surface engineering of quantum dot hydrogels for selective fluorescence imaging of extracellular lactate release.

    PubMed

    Zhang, Xiaomeng; Ding, Shushu; Cao, Sumei; Zhu, Anwei; Shi, Guoyue

    2016-06-15

    Selective and sensitive detection of extracellular lactate is of fundamental significance for studying the metabolic alterations in tumor progression. Here we report the rational design and synthesis of a quantum-dot-hydrogel-based fluorescent probe for biosensing and bioimaging the extracellular lactate. By surface engineering the destabilized quantum dot sol with Nile Blue, the destabilized Nile-Blue-functionalized quantum dot sol cannot only self-assemble forming quantum dot hydrogel but also monitor lactate in the presence of nicotinamide adenine dinucleotide cofactor and lactate dehydrogenase through fluorescence resonance energy transfer. Notably, the surface engineered quantum dot hydrogel show high selectivity toward lactate over common metal ions, amino acids and other small molecules that widely coexist in biological system. Moreover, the destabilized Nile-Blue-functionalized quantum dots can encapsulate isolated cancer cells when self-assembled into a hydrogel and thus specifically detect and image the extracellular lactate metabolism. By virtue of these properties, the functionalized quantum dot hydrogel was further successfully applied to monitor the effect of metabolic agents. PMID:26852200

  10. Lactating Adenoma of the Breast.

    PubMed

    Barco Nebreda, Israel; Vidal, M Carmen; Fraile, Manel; Canales, Lydia; González, Clarisa; Giménez, Nuria; García-Fernández, Antonio

    2016-08-01

    Lactating adenoma is an uncommon breast palpable lesion occurring in pregnancy or lactation. Although it is a benign condition, it often requires core biopsy or even surgery to exclude malignancy. As with other solid lesions in pregnancy and lactation, lactating adenoma needs an accurate evaluation in order to ensure its benign nature. Work-up must include both imaging and histologic findings. Ultrasound evaluation remains the first step in assessing the features of the lesion. Some authors consider magnetic resonance imaging as a useful tool in cases of inconclusive evaluation after ultrasound and histologic exam in an attempt to avoid surgery. Most lactating adenomas resolve spontaneously, whereas others persist or even increase in size and must be removed. The authors present a case of a 35-year-old woman at 6 months postpartum with a lactating adenoma in her right breast. After surgical removal, breastfeeding was perfectly continued within the next 24 hours, which highlights the fact that breast surgery is most often compatible with breastfeeding. PMID:27197575

  11. Size-dependent effects of nanoparticles on the activity of cytochrome P450 isoenzymes

    SciTech Connect

    Froehlich, Eleonore; Kueznik, Tatjana; Samberger, Claudia; Roblegg, Eva; Wrighton, Christopher

    2010-02-01

    Nanoparticles are known to be able to interfere with cellular metabolism and to cause cytotoxicity and moreover may interfere with specific cellular functions. Serious effects on the latter include changes in liver cell function. The cytochrome P450 system is expressed in many cells but is especially important in hepatocytes and hormone-producing cells. The interaction of polystyrene nanoparticles with the most important drug-metabolizing cytochrome P450 isoenzymes, CYP3A4, CYP2D6, CYP2C9 and CYP2A1 expressed individually in insect cells (BACULOSOMES) was studied by the cleavage of substrates coupled to a fluorescent dye. The data obtained for individual isoenzymes were compared to metabolism in microsomes isolated from normal liver and from the hepatoma cell line H4-II-E-C3. Small (20-60 nm) carboxyl polystyrene particles but not larger (200 nm) ones reached high intracellular concentrations in the vicinity of the endoplasmic reticulum. These small particles inhibited the enzymatic activity of CYP450 isoenzymes in BACULOSOMES and substrate cleavage in normal liver microsomes. They moreover increased the effect of known inhibitors of the cytochrome P450 system (cimetidine, phenobarbital and paclitaxel). Substrate cleavage by the hepatoma cell line H4-II-E-C3 in contrast was undetectable, making this cell line unsuitable for this type of study. Our results thus demonstrate that nanoparticles can inhibit the metabolism of xenobiotics by the CYP450 system in model systems in vitro. Such inhibition could also potentially occur in vivo and possibly cause adverse effects in persons receiving medication.

  12. [Influences of uncommon isoenzymes on determination of alkaline phosphatase activity by dry-chemistry analyzers].

    PubMed

    Tozawa, T; Hashimoto, M

    2001-04-01

    Dry-chemistry(DC) analysis may be influenced by some matrix effects for measuring uncommon isoenzyme forms. Placental and intestinal alkaline phosphatase(AP) are overestimated by the VITROS DC, compared with results obtained with the wet-chemistry(WC) method of Bretaudiere, et al. using 2-amino-2-methyl-1-propanol (AMP) buffer, however, no such discrepancy between AP results in any DC method and that with a routine WC method recommended by Japanese Society of Clinical Chemistry in that 2-ethylaminoethanol(EAE) buffer is used, has been demonstrated. The type of buffer used affects differently the rates of AP isoenzymes activities. We therefore examined whether the presence of uncommon AP isoenzyme forms in serum caused aberrant DC results for AP in comparison with a routine WC method using EAE buffer. Here, serum samples with only liver AP and bone AP(n : 32); high-molecular-mass AP(n : 11); placental AP(n : 12); intestinal AP(n : 13) and immunoglobulin (Ig) bound AP(n : 12) were analyzed for total AP activity on three different DC analyzers: VITROS 700XR, FUJIDRYCHEM 5000, SPOTCHEM 4410 and a WC analyzer: HITACHI 7350. Values obtained in all of the DCs for sera containing only liver/bone AP agreed with those with the WC method. For sera containing placental AP, the VITROS values were higher than those with the WC method, while the FUJIDRYCHEM values and the SPOTCHEM values were lower. The VITROS values and the FUJIDRYCHEM values for sera containing intestinal AP were lower than those with the WC method, while the SPOTCHEM values were higher. All of the DCs did not affect high-molecular-mass AP and Ig bound liver/bone AP types of macro AP, but underestimated Ig bound intestinal type. Ig bound intestinal AP may be sieved by DC multilayer elements. PMID:11391954

  13. Nitric oxide isoenzymes regulate lipopolysaccharide-enhanced insulin transport across the blood-brain barrier.

    PubMed

    Banks, William A; Dohgu, Shinya; Lynch, Jessica L; Fleegal-DeMotta, Melissa A; Erickson, Michelle A; Nakaoke, Ryota; Vo, Than Q

    2008-04-01

    Insulin transported across the blood-brain barrier (BBB) has many effects within the central nervous system. Insulin transport is not static but altered by obesity and inflammation. Lipopolysaccharide (LPS), derived from the cell walls of Gram-negative bacteria, enhances insulin transport across the BBB but also releases nitric oxide (NO), which opposes LPS-enhanced insulin transport. Here we determined the role of NO synthase (NOS) in mediating the effects of LPS on insulin BBB transport. The activity of all three NOS isoenzymes was stimulated in vivo by LPS. Endothelial NOS and inducible NOS together mediated the LPS-enhanced transport of insulin, whereas neuronal NOS (nNOS) opposed LPS-enhanced insulin transport. This dual pattern of NOS action was found in most brain regions with the exception of the striatum, which did not respond to LPS, and the parietal cortex, hippocampus, and pons medulla, which did not respond to nNOS inhibition. In vitro studies of a brain endothelial cell (BEC) monolayer BBB model showed that LPS did not directly affect insulin transport, whereas NO inhibited insulin transport. This suggests that the stimulatory effect of LPS and NOS on insulin transport is mediated through cells of the neurovascular unit other than BECs. Protein and mRNA levels of the isoenzymes indicated that the effects of LPS are mainly posttranslational. In conclusion, LPS affects insulin transport across the BBB by modulating NOS isoenzyme activity. NO released by endothelial NOS and inducible NOS acts indirectly to stimulate insulin transport, whereas NO released by nNOS acts directly on BECs to inhibit insulin transport. PMID:18187549

  14. Hydrolysis of synthetic pyrophosphoric esters by an isoenzyme of apyrase from Solanum tuberosum.

    PubMed Central

    Del Campo, G; Puente, J; Valenzuela, M A; Traverso-Cori, A; Cori, O

    1977-01-01

    A highly purified isoenzyme of apyrase obtained from potatoes (Solanum tuberosum var. Pimpernel) exhibits a low specificity for the organic moiety of synthetic pyro- and triphosphates. Methyl di- and tri-phosphates were hydrolysed at higher rates than ADP and ATP, but their Km values were also higher. Steric hindrance at the carbon atom linked to the pyrophosphate chain decreases both binding and maximum rate, whereas length or polarity of the organic chain do not have systematic effects. t-Butyl diphosphate, inorganic pyrophosphate, adenosine 5'-[alpha,beta-methylene]triphosphate and adenosine 5'-[beta,gamma-methylene]triphosphate are competitive inhibitors of the hydrolysis of ATP and ADP. PMID:203267

  15. Method of empirical dependences in estimation and prediction of activity of creatine kinase isoenzymes in cerebral ischemia

    NASA Astrophysics Data System (ADS)

    Sergeeva, Tatiana F.; Moshkova, Albina N.; Erlykina, Elena I.; Khvatova, Elena M.

    2016-04-01

    Creatine kinase is a key enzyme of energy metabolism in the brain. There are known cytoplasmic and mitochondrial creatine kinase isoenzymes. Mitochondrial creatine kinase exists as a mixture of two oligomeric forms - dimer and octamer. The aim of investigation was to study catalytic properties of cytoplasmic and mitochondrial creatine kinase and using of the method of empirical dependences for the possible prediction of the activity of these enzymes in cerebral ischemia. Ischemia was revealed to be accompanied with the changes of the activity of creatine kinase isoenzymes and oligomeric state of mitochondrial isoform. There were made the models of multiple regression that permit to study the activity of creatine kinase system in cerebral ischemia using a calculating method. Therefore, the mathematical method of empirical dependences can be applied for estimation and prediction of the functional state of the brain by the activity of creatine kinase isoenzymes in cerebral ischemia.

  16. Depletion of lactate by dichloroacetate reduces cardiac efficiency after hemorrhagic shock.

    PubMed

    Barbee, R W; Kline, J A; Watts, J A

    2000-08-01

    We have demonstrated previously that dichloroacetate (DCA) treatment in rodents ameliorates, via activation of the pyruvate dehydrogenase complex, the cardiovascular depression observed after hemorrhagic shock. To explore the mechanism of this effect, we administered DCA in a large animal model of hemorrhagic shock. Mongrel hounds were anesthetized with 1.5% isoflurane and were measured for hemodynamics, myocardial contractility, and myocardial substrate utilization. They were hemorrhaged to a mean arterial pressure of 35 mm Hg for 90 min or until arterial lactate levels reached 7.0 mM (1137 +/- 47 mL or 49 +/- 2% total blood volume). Animals were chosen at random to receive DCA dissolved in water or an equal volume of saline at the onset of resuscitation. Two-thirds of the shed blood volume was returned immediately after giving an equivalent volume of saline. Two hours after the onset of resuscitation, mean arterial pressure was not different between DCA and control groups (79 +/- 3 vs. 82 +/- 3 mm Hg, respectively). Arterial lactate levels were significantly reduced by DCA (0.5 +/- 0.06 vs. 2.0 +/- 0.2 mM). However, DCA treatment was associated with a decreased stroke volume index (0.56 +/- 0.06 vs. 0.82 +/- 0.08 mL/kg/beat) and a decreased myocardial efficiency (19 vs. 41 L x mm Hg/mL/100 g tissue). During resuscitation by DCA, myocardial lactate consumption was reduced (21.4 +/- 3.7 vs. 70.7 +/- 16.3 micromole/min/100 g tissue) despite a three-fold increase in myocardial pyruvate dehydrogenase activity, while free fatty acid levels actually began to rise. Although increased lactate oxidation should be beneficial during resuscitation, we propose that DCA treatment led to a deprivation of myocardial lactate supply, which reduced net myocardial lactate oxidation, thus compromising myocardial function during resuscitation from hemorrhagic shock. PMID:10947168

  17. Fermentation and alternative respiration compensate for NADH dehydrogenase deficiency in a prokaryotic model of DJ-1-associated Parkinsonism.

    PubMed

    Messaoudi, Nadia; Gautier, Valérie; Dairou, Julien; Mihoub, Mouhad; Lelandais, Gaëlle; Bouloc, Philippe; Landoulsi, Ahmed; Richarme, Gilbert

    2015-11-01

    YajL is the closest prokaryotic homologue of Parkinson's disease-associated DJ-1, a protein of undefined function involved in the oxidative stress response. We reported recently that YajL and DJ-1 protect cells against oxidative stress-induced protein aggregation by acting as covalent chaperones for the thiol proteome, including the NuoG subunit of NADH dehydrogenase 1, and that NADH dehydrogenase 1 activity is negligible in the yajL mutant. We report here that this mutant compensates for low NADH dehydrogenase activity by utilizing NADH-independent alternative dehydrogenases, including pyruvate oxidase PoxB and d-amino acid dehydrogenase DadA, and mixed acid aerobic fermentations characterized by acetate, lactate, succinate and ethanol excretion. The yajL mutant has a low adenylate energy charge favouring glycolytic flux, and a high NADH/NAD ratio favouring fermentations over pyruvate dehydrogenase and the Krebs cycle. DNA array analysis showed upregulation of genes involved in glycolytic and pentose phosphate pathways and alternative respiratory pathways. Moreover, the yajL mutant preferentially catabolized pyruvate-forming amino acids over Krebs cycle-related amino acids, and thus the yajL mutant utilizes pyruvate-centred respiro-fermentative metabolism to compensate for the NADH dehydrogenase 1 defect and constitutes an interesting model for studying eukaryotic respiratory complex I deficiencies, especially those associated with Alzheimer's and Parkinson's diseases. PMID:26377309

  18. Digitisers: their use in the entry of orders, urinalysis results, and isoenzyme interpretation in a clinical biochemistry computer system.

    PubMed Central

    Loughlin, J F; Tuckerman, J F; Henderson, A R

    1984-01-01

    The processes of order entry, urinalysis result, and isoenzyme interpretation entry into a laboratory computer system is a time consuming activity. We have designed a series of forms for use with a digitising pad that allow us rapidly to enter orders, urinalysis results, and isoenzyme interpretative comments into our laboratory computer system. We have shown that digitiser entry is always significantly faster than manual entry. Although there are many devices available to facilitate computer entry, we believe that the digitiser technique is an attractive option because of its ease of use, speed, reliability, and low cost. Images PMID:6490955

  19. Cardiac responses to induced lactate oxidation: NMR analysis of metabolic equilibria.

    PubMed

    Lewandowski, E D; Damico, L A; White, L T; Yu, X

    1995-07-01

    The role of lactate as a source of pyruvate oxidation in supporting cardiac work, energetics, and formation of oxidative metabolites was examined in normal myocardium. 13C- and 31P-nuclear magnetic resonance (NMR) spectra were acquired from isolated rabbit hearts supplied 2.5 mM [3-13C]lactate or [3-13C]pyruvate with or without stimulation of pyruvate dehydrogenase (PDH) by dichloroacetate (DCA). Similar workloads determined by rate-pressure products were noted with pyruvate (21,700 +/- 2,400; mean +/- SE) and lactate (18,970 +/- 1,510). Oxygen consumption was similar in all four groups with means between 19.0 and 22.2 mumol.min-1.g dry weight-1 (SE = 1.6-2.0) as was the ratio of phosphocreatine to ATP with means between 1.8 and 2.1 (SE = 0.1-0.6). Intracellular pH, determined from 31P-NMR spectra, was essentially the same with pyruvate (7.06 +/- 0.02) and lactate (7.05 +/- 0.04). 13C enrichment of glutamate was higher with lactate (92%) than with pyruvate (70%). Pyruvate plus DCA induced no change in glutamate content at 9-10 mumol/g, but 13C enrichment increased to 83%, while lactate plus DCA maintained enrichment at 90%. Levels of alpha-ketoglutarate were lower with lactate (1.81 mumol/g) than with pyruvate (2.36 mumol/g). Lactate plus DCA elevated glutamate by 60% with a proportional increase in alpha-ketoglutarate. Thus the balance between glutamate and alpha-ketoglutarate was affected by substrate supply only and not by PDH activation. The results suggest that the equilibrium between alpha-ketoglutarate and glutamate is sensitive to cytosolic redox state, an important consideration for 13C-NMR analyses that rely on glutamate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7631845

  20. Lactate Utilization Is Regulated by the FadR-Type Regulator LldR in Pseudomonas aeruginosa

    PubMed Central

    Gao, Chao; Hu, Chunhui; Zheng, Zhaojuan; Jiang, Tianyi; Dou, Peipei; Zhang, Wen; Che, Bin; Wang, Yujiao; Lv, Min

    2012-01-01

    NAD-independent l-lactate dehydrogenase (l-iLDH) and NAD-independent d-lactate dehydrogenase (d-iLDH) activities are induced coordinately by either enantiomer of lactate in Pseudomonas strains. Inspection of the genomic sequences of different Pseudomonas strains revealed that the lldPDE operon comprises 3 genes, lldP (encoding a lactate permease), lldD (encoding an l-iLDH), and lldE (encoding a d-iLDH). Cotranscription of lldP, lldD, and lldE in Pseudomonas aeruginosa strain XMG starts with the base, C, that is located 138 bp upstream of the lldP ATG start codon. The lldPDE operon is located adjacent to lldR (encoding an FadR-type regulator, LldR). The gel mobility shift assays revealed that the purified His-tagged LldR binds to the upstream region of lldP. An XMG mutant strain that constitutively expresses d-iLDH and l-iLDH was found to contain a mutation in lldR that leads to an Ile23-to-serine substitution in the LldR protein. The mutated protein, LldRM, lost its DNA-binding activity. A motif with a hyphenated dyad symmetry (TGGTCTTACCA) was identified as essential for the binding of LldR to the upstream region of lldP by using site-directed mutagenesis. l-Lactate and d-lactate interfered with the DNA-binding activity of LldR. Thus, l-iLDH and d-iLDH were expressed when the operon was induced in the presence of l-lactate or d-lactate. PMID:22408166

  1. Nutritional aspects of human lactation*

    PubMed Central

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

    1975-01-01

    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

  2. Midtrimester abortion by ethacridine lactate.

    PubMed

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

    1982-07-01

    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

  3. Wounding Induces One of Two Isoenzymes of 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase in Solanum tuberosum L. 1

    PubMed Central

    Muday, Gloria K.; Herrmann, Klaus M.

    1992-01-01

    Potato (Solanum tuberosum L.) tubers contain two isoenzymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15), the enzyme that catalyzes the first step of aromatic amino acid biosynthesis. One of the isoenzymes is specifically activated by Mn2+, and the other requires Co2+, Mg2+, or another divalent cation for activity. Monospecific polyclonal antibodies against the Mn2+-activated isoenzyme do not cross-react with the other isoenzyme. Wounding of potato tubers induces the Mn2+-activated form but not the other. We conclude that two different genes encode two different isoenzymes that catalyze the first step in the shikimate pathway. ImagesFigure 1 PMID:16668667

  4. Activity determination, kinetic analyses and isoenzyme identification of gamma glutamyltransferase in human neutrophils.

    PubMed

    Sener, Azize; Yardimci, Turay

    2005-05-31

    Gamma-glutamyltransferase (GGT, EC 2.3.2.2) which hydrolyzes glutathione (GSH), is required for the maintenance of normal intracellular GSH concentration. GGT is a membrane enzyme present in leukocytes and platelets. Its activity has also been observed in human neutrophils. In this study, GGT was purified from Triton X-100 solubilized neutrophils and its kinetic parameters were determined. For kinetic analyses of transpeptidation reaction, gamma-glutamyl p-nitroanilide was used as the substrate and glycylglycine as the acceptor. Apparent K(m) values were determined as 1.8 mM for gamma-glutamyl p-nitroanilide and 16.9 mM for glycylglycine. The optimum pH of GGT activity was 8.2 and the optimum temperature was 37 degrees C. It had thermal stability with 58 % relative activity at 56 degrees C for 30 min incubation. L-serine, in the presence of borate, was detected as the competitive inhibitor. Bromcresol green inhibited neutrophil GGT activity as a noncompetitive inhibitor. The neutrophils seem to contain only the isoenzyme that is present in platelets. We characterized the kinetic properties and compared the type of the isoenzyme of neutrophil GGT with platelet GGT via polyacrylamide gel electrophoresis (PAGE) under a standard set of conditions. PMID:15943911

  5. Isoenzyme Multiplicity and Characterization of Recombinant Manganese Peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium

    PubMed Central

    Larrondo, Luis F.; Lobos, Sergio; Stewart, Phillip; Cullen, Dan; Vicuña, Rafael

    2001-01-01

    We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the α-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium. PMID:11319083

  6. Thermodynamic determination of beta-hexosaminidase isoenzymes in mononuclear and polymorphonuclear leukocyte populations.

    PubMed

    Casal, J Antonio; Chabás, Amparo; Tutor, J Carlos

    2003-01-30

    Isoenzymes of beta-hexosaminidase (Hex) were determined in mononuclear (MN) and polymorphonuclear (PMN) leukocytes, with a thermodynamic method using the chromogenic substrate sodio-3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-beta-D-glucosaminide. Imprecision was very satisfactory, and the results are very much in agreement with those obtained using the fluorogenic substrates 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide and 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide 6-sulfate. In 163 healthy individuals we found, for the proportion as a percentage of the Hex A isoenzyme, significantly higher values (P < 0.001) in PMN than in MN cells (71.56 +/- 0.30% vs. 54.28 +/- 0.24%), meaning that it would not appear advisable to use total leukocyte lysates for evaluating this variable. The method is fast, precise, and highly suitable for the biochemical diagnosis and heterozygote screening of GM2 gangliosidoses, and would be applicable in cases of thermolabile Hex B and for detecting the B1 variant. PMID:12503097

  7. Molecular genetics and pathophysiology of 17{beta}-hydroxysteroid dehydrogenase 3 deficiency

    SciTech Connect

    Andersson, S.; Geissler, W.M.; Wu, L.

    1996-01-01

    Autosomal recessive mutations in the 17{beta}-hydroxysteroid dehydrogenase 3 gene impair the formation of testosterone in the fetal testis and give rise to genetic males with female external genitalia. Such individuals are usually raised as females, but virilize at the time of expected puberty as the result of increases in serum testosterone. Here we describe mutations in 12 additional subjects/families with this disorder. The 14 mutations characterized to date include 10 missense mutations, 3 splice junction abnormalities, and 1 small deletion that results in a frame shift. Three of these mutations have occurred in more than 1 family. Complementary DNAs incorporating 9 of the 10 missense mutations have been constructed and expressed in reporter cells; 8 of the 9 missense mutations cause almost complete loss of enzymatic activity. In 2 subjects with loss of function, missense mutations testosterone levels in testicular venous blood were very low. Considered together, these findings strongly suggest that the common mechanism for testosterone formation in postpubertal subjects with this disorder is the conversion of circulating androstenedione to testosterone by one or more of the unaffected 17{beta}-hydroxysteroid dehydrogenase isoenzymes. 29 refs., 2 figs., 3 tabs.

  8. Antipsychotics in pregnancy and lactation

    PubMed Central

    Babu, Girish N.; Desai, Geetha; Chandra, Prabha S.

    2015-01-01

    Research on psychotropic medications during pregnancy and lactation is limited as often involves complex ethical issues. Information on safety of psychotropic drugs during these critical phases is either inconclusive or undetermined. Many women with severe mental illness have unplanned pregnancies and require antipsychotic medication during pregnancy and lactation. Multiple issues have to be considered while choosing safe treatments for pregnant and lactating women and the best approach is to individualize the treatment. Medication should be guided primarily by its safety data and by the psychiatric history of the patient. Important issues to be kept in mind include pre-pregnancy counseling for all women, including planning pregnancies; folate supplementation, discussion with patient and family regarding options, and active liaison with obstetricians, ultrasonologists and pediatricians. Whenever possible, non-pharmacological approaches should be used in addition. PMID:26330648

  9. Lactational infertility in family planning.

    PubMed

    Short, R V

    1993-04-01

    The contraceptive effect of breast-feeding is the single most important determinant of human population growth rates in traditional societies without access to modern forms of contraception; lactational amenorrhoea is Nature's contraceptive. Even today, breast-feeding still prevents more pregnancies than all modern forms of contraception in many developing countries. Afferent neural inputs from the nipple pass via the spinal cord to the hypothalamus, where they cause a local release of beta endorphin. This acts to depress GnRH secretion, thereby inhibiting pituitary gonadotrophin secretion, ovarian follicular development, ovulation and menstruation. The hypothalamic beta endorphin release also inhibits dopamine production, resulting in increased pituitary prolactin secretion. The higher the suckling frequency, the more beta endorphin that is released and hence the longer the duration of lactational amenorrhoea. Lactational amenorrhoea can be relied up to give over 98% contraceptive protection to breast-feeding women in the first 6 months postpartum, regardless of their nutritional status or the time of first supplement introduction to the baby. This is because the first postpartum menstruation usually precedes the first ovulation during these early months. Once menstruation has resumed, lactation's contraceptive effect can no longer be relied upon, even though the woman continues to breast-feed. In breast-feeding women whose amenorrhoea extends beyond 6 months, there is an increasing tendency for the first ovulation to precede the first menstruation, thereby decreasing the reliability of lactational amenorrhoea as a contraceptive. Nevertheless, many women who continue to breast-feed may still have up to 1-2 years of good contraceptive protection from prolonged lactational amenorrhoea.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8489757

  10. Development of an enzymatic chromatography strip with nicotinamide adenine dinucleotide-tetrazolium coupling reactions for quantitative l-lactate analysis.

    PubMed

    Kan, Shu-Chen; Chang, Wei-Feng; Lan, Min-Chi; Lin, Chia-Chi; Lai, Wei-Shiang; Shieh, Chwen-Jen; Hsiung, Kuang-Pin; Liu, Yung-Chuan

    2015-02-15

    In this study, a dry assay of l-lactate via the enzymatic chromatographic test (ECT) was developed. An l-lactate dehydrogenase plus a nicotinamide adenine dinucleotide (NADH) regeneration reaction were applied simultaneously. Various tetrazolium salts were screened to reveal visible color intensities capable of determining the lactate concentrations in the sample. The optimal analysis conditions were as follows. The diaphorase (0.5 μl, 2(-6)U/μl) was immobilized in the test line of the ECT strip. Nitrotetrazolium blue chloride (5 μl, 12 mM), l-lactate dehydrogenase (1 μl, 0.25U/μl), and NAD(+) (2μl, 1.5×10(-5)M) were added into the mobile phase (100 μl) composed of 0.1% (w/w) Tween 20 in 10mM phosphate buffer (pH 9.0), and the process was left to run for 10 min. This detection had a linear range of 0.039 to 5mM with a detection limit of 0.047 mM. This quantitative analysis process for l-lactate was easy to operate with good stability and was proper for the point-of-care testing applications. PMID:25454507

  11. Reduction of d-lactate content in sauerkraut using starter cultures of recombinant Leuconostoc mesenteroides expressing the ldhL gene.

    PubMed

    Jin, Qing; Li, Ling; Moon, Jin Seok; Cho, Seung Kee; Kim, Yu Jin; Lee, Soo Jin; Han, Nam Soo

    2016-05-01

    The d-form of lactate, which causes metabolic stress upon excessive dietary intake, is mainly produced by Leuconostoc sp., the predominant species in sauerkraut. To shift the metabolic flux of d-lactate from pyruvate to l-lactate, we expressed the l-lactate dehydrogenase (ldhL) gene in Leuconostoc mesenteroides ATCC 8293. The ldhL gene from Lactobacillus plantarum was introduced into L. mesenteroides using the shuttle vectors pLeuCM and pLeuCM42. To elevate the expression level of ldhL in L. mesenteroides, the nucleotides for pyruvate kinase promoter were fused to ldhL and cloned into above vectors to construct pLC18pkL and pLC42pkL. As results, introduction of pLC42pkL in L. mesenteroides significantly improved both l-LDH activity and l-lactate productivity during fermentation, decreasing the d-/l-lactate ratio. When used as a starter culture for sauerkraut fermentation, recombinant L. mesenteroides harboring pLC42pkL increased l-lactate concentration and decreased d-lactate concentration compared to the wild type strain. We newly developed a recombinant L. mesenteroides which has high l-lactate dehydrogenase activity and applied this strain to minimize the harmful effect of d-lactate during the sauerkraut fermentation. To the best of our knowledge, we demonstrate for the first time the effective use of recombinant Leuconostoc sp. for quality improvement of fermented foods. PMID:26472127

  12. Liver- and bone-derived isoenzymes of alkaline phosphatase in serum as determined by high-performance affinity chromatography.

    PubMed

    Anderson, D J; Branum, E L; O'Brien, J F

    1990-02-01

    To separate liver and bone alkaline phosphatase (ALP) isoenzymes in human serum, we used high-performance affinity chromatography (HPAC) on a column of wheat-germ lectin conjugated to 7-microns-diameter silica particles and an eluent containing N-acetyl-D-glucosamine (NAG). On-line spectrophotometric detection of ALP involved pumping diethanolamine-buffered p-nitrophenyl phosphate solution post-column. Bone and liver isoenzymes could be separated into two peaks with only 10% overlap when an exponential gradient was used. A linear-step gradient separated 80.9% of liver ALP and 91.6% of bone ALP in two distinct peaks. True bone and liver ALP peak areas for the linear-step gradient were determined by using correction factors, because each peak contained a co-eluted portion of the other ALP isoenzyme. The detection limit improved 10-fold over those of other techniques for ALP isoenzymes, owing to the relatively large sample that could be applied to the column. Correlation with a urea-inactivation procedure was reasonable for patients' serum samples (r = 0.98 and 0.79 for liver ALP and bone ALP, respectively). PMID:2302767

  13. Isoenzymes and soluble protein in Arthrospira from alkaline lakes in Erdos Plateau, China and in exotic species

    NASA Astrophysics Data System (ADS)

    Yang, Qian; Li, Shuyuan; Hu, Ruiping; Liu, Yan; Qiao, Chen

    2006-06-01

    The authors compared isoenzymes of five enzymes and soluble protein in Arthrospira platensis (A3) and A. erdosensis (A4) from alkaline lakes in Erdos Plateau, Nei Monggo (Inner Mongalia), China and exotic species of A. platensis (A1) from Chad and A. maxima (A2) from Mexico by means of polyacrylamide gel electrophoresis. The results showed that the isoenzymes of EST, POD and soluble protein were polymorphic. Monomorphism and polymorphism were found in isoenzymes of AMY, CAT and SOD, and monomorphism was found only in the introduced species. The isoenzymes and soluble protein of the local species are all polymorphic. The number of bands in these species were in the order of A3>A4>A1>A2. A2 is the most primary, A1 from Chad Lake is relatively primary, A3 and A4 are advanced species in evolution. Cluster analysis showed that the relation between the two introduced species are the closest to each other, and so too are the two local ones.

  14. 21 CFR 582.5311 - Ferrous lactate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1 § 582.5311 Ferrous lactate. (a) Product. Ferrous lactate. (b) Conditions of use. This...

  15. 21 CFR 184.1311 - Ferrous lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... prepared by reacting calcium lactate or sodium lactate with ferrous sulfate, direct reaction of lactic acid.... (1996), pp. 154 to 155, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1...

  16. 21 CFR 184.1311 - Ferrous lactate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... reacting calcium lactate or sodium lactate with ferrous sulfate, direct reaction of lactic acid with iron... to 155, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part...

  17. Wearable Sensor System Powered by a Biofuel Cell for Detection of Lactate Levels in Sweat

    PubMed Central

    Garcia, S. O.; Ulyanova, Y. V.; Figueroa-Teran, R.; Bhatt, K. H.; Singhal, S.; Atanassov, P.

    2016-01-01

    An NAD+-dependent enzymatic sensor with biofuel cell power source system for non-invasive monitoring of lactate in sweat was designed, developed, and tested. The sensor component, based on lactate dehydrogenase, showed linear current response with increasing lactate concentrations with limits of detection from 5 to 100 mM lactate and sensitivity of 0.2 µA.mM−1 in the presence of target analyte. In addition to the sensor patch a power source was also designed, developed and tested. The power source was a biofuel cell designed to oxidize glucose via glucose oxidase. The biofuel cell showed excellent performance, achieving over 80 mA at 0.4 V (16 mW) in a footprint of 3.5 × 3.5 × 0.7 cm. Furthermore, in order to couple the sensor to the power source, system electronic components were designed and fabricated. These consisted of an energy harvester (EH) and a micropotentiostat (MP). The EH was employed for harvesting power provided by the biofuel cell as well as up-converting the voltage to 3.0 V needed for the operation of the MP. The sensor was attached to MP for chronoamperometric detection of lactate. The Sensor Patch System was demonstrated under laboratory conditions. PMID:27375962

  18. The isoenzymes of carbonic anhydrase: tissue, subcellular distribution and functional significance, with particular reference to the intestinal tract

    PubMed Central

    Carter, M. J.; Parsons, D. S.

    1971-01-01

    1. The total carbonic anhydrase activity in some guinea-pig tissues has been measured using a pH-stat procedure. Stomach, gall bladder, proximal colon and caecum all possess more carbonic anhydrase activity per unit amount of protein than does whole blood. 2. The carbonic anhydrase activity of the small intestine is low. Reasons are given for supposing that activity found there is not entirely due to contamination by whole blood, and it is suggested that in this tissue the enzyme may be localized in some cell type other than the columnar absorbing cells. 3. Evidence is presented which indicates that heavy metals interfere with the activity of the enzyme as measured in tissue homogenates. 4. The distribution and concentration of the two major isoenzymes of carbonic anhydrase have been measured in different tissues. Blood and proximal colon contain both isoenzymes in comparable concentrations, the ratio of the concentration of the `low activity' isoenzyme to that of the `high activity' being about 2. The gastric mucosa contains much `high activity' carbonic anhydrase, but only a negligible amount of the `low activity' isoenzyme. In the caecal mucosa, the `low activity' isoenzyme is predominant, the ratio of its concentration to that of the `high activity' isoenzyme being about 9. It is also found that more than 1·5% of the protein in the caecal mucosa is accounted for as carbonic anhydrase enzymes. 5. It is found that some 45% of the total carbonic anhydrase activity of sucrose homogenates of the guinea-pig colon is bound to particles. The activity is located mainly in the nuclear and microvillous fraction and in the `high-speed supernatant' fraction. The form of enzyme bound is largely of the `high activity' variety. When the tissue is homogenized in potassium chloride solutions less than 4% of the total activity is recovered in particulate fractions. The amount of activity which is bound to particulate fractions increases as the ionic strength or pH of the

  19. The Occurrence of Glycolate Dehydrogenase and Glycolate Oxidase in Green Plants

    PubMed Central

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

    1973-01-01

    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

  20. Is lactation nature's contraceptive? Data from Samoa.

    PubMed

    Fitzgerald, M H

    1992-01-01

    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

  1. The origin and evolution of lactation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The presence of mammary glands are the defining morphological feature of mammals, and a successful lactation is crucial to mammalian reproductive strategies. Among mammalian species, the nature of lactation and the composition of milk vary greatly. The evolution of lactation and its diversity amon...

  2. 21 CFR 582.1207 - Calcium lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium lactate. 582.1207 Section 582.1207 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1207 Calcium lactate. (a) Product. Calcium lactate. (b) Conditions of use. This substance...

  3. Human liver alcohol dehydrogenase. 2. The primary structure of the gamma 1 protein chain.

    PubMed

    Bühler, R; Hempel, J; Kaiser, R; de Zalenski, C; von Wartburg, J P; Jörnvall, H

    1984-12-17

    The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE. PMID:6391921

  4. Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation.

    PubMed Central

    Muñoz, C; Guillén, F; Martínez, A T; Martínez, M J

    1997-01-01

    Two laccase isoenzymes produced by Pleurotus eryngii were purified to electrophoretic homogeneity (42- and 43-fold) with an overall yield of 56.3%. Laccases I and II from this fungus are monomeric glycoproteins with 7 and 1% carbohydrate content, molecular masses (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 65 and 61 kDa, and pIs of 4.1 and 4.2, respectively. The highest rate of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) oxidation for laccase I was reached at 65 degrees C and pH 4, and that for laccase II was reached at 55 degrees C and pH 3.5. Both isoenzymes are stable at high pH, retaining 60 to 70% activity after 24 h from pH 8 to 12. Their amino acid compositions and N-terminal sequences were determined, the latter strongly differing from those of laccases of other basidiomycetes. Antibodies against laccase I reacted with laccase II, as well as with laccases from Pleurotus ostreatus, Pleurotus pulmonarius, and Pleurotus floridanus. Different hydroxy- and methoxy-substituted phenols and aromatic amines were oxidized by the two laccase isoenzymes from P. eryngii, and the influence of the nature, number, and disposition of aromatic-ring substituents on kinetic constants is discussed. Although both isoenzymes presented similar substrate affinities, the maximum rates of reactions catalyzed by laccase I were higher than those of laccase II. In reactions with hydroquinones, semiquinones produced by laccase isoenzymes were in part converted into quinones via autoxidation. The superoxide anion radical produced in the latter reaction dismutated, producing hydrogen peroxide. In the presence of manganous ion, the superoxide union was reduced to hydrogen peroxide with the concomitant production of manganic ion. These results confirmed that laccase in the presence of hydroquinones can participate in the production of both reduced oxygen species and manganic ions. PMID:9172335

  5. Effects of protein-modifying reagents on an isoenzyme of potato apyrase

    PubMed Central

    Valenzuela, M. Antonieta; Del Campo, Guillermo; Marín, Eugenio; Traverso-Cori, Aída

    1973-01-01

    Treatment of an isoenzyme of potato apyrase of high adenosine triphosphatase/adenosine diphosphatase (ATPase/ADPase) ratio with iodine, N-acetylimidazole or tetranitromethane inactivates the ATPase activity of this enzyme faster than its ADPase activity. There was protection by substrates with the two last-named substances. This and the appearance of nitrotyrosine suggests the participation of tyrosyl residues in both enzymic activities of potato apyrase. The participation of thiol groups is excluded by the insensitivity of apyrase to p-chloromercuribenzoate. Also, 2-hydroxy-5-nitrobenzyl bromide or carboxymethylation produce the same rate of inactivation of ATPase and ADPase activities. Substrates protect both activities from inactivation. Hydrogen peroxide and photo-oxidation inactivate ATPase activity faster than ADPase activity. There is no protection by substrates. Analysis of pH effects on Vmax. and Km suggest different pK values for the amino acid residues at the ATP and ADP sites. PMID:4356057

  6. Metabolic indicators of drought stress tolerance in wheat: glutamine synthetase isoenzymes and Rubisco.

    PubMed

    Nagy, Zoltán; Németh, Edit; Guóth, Adrienn; Bona, Lajos; Wodala, Barnabás; Pécsváradi, Attila

    2013-06-01

    Drought stress has a considerable impact on the ecosystem and agriculture. Continuous water deficit induces early leaf senescence in plants. During this process, chloroplasts are degraded and photosynthesis drastically drops. The objective of this investigation was to look into the regulation of nitrogen and carbon metabolism during water deficit. Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) and the total protein contents inform us of the sink-source relation in plants. Glutamine synthetase (GS, EC 6.3.1.2) isoenzymes are good markers of plastid status (GS2) and the nitrogen metabolism (GS1). Tolerant and sensitive wheat (Triticum aestivum L.) genotypes were tested, which are widely used in agriculture. The amount of protein, Rubisco and GS isoforms in leaves were measured during the grain filling period, as indicative traits that ultimately determine the onset and stage of senescence. The symptoms of senescence first appeared on the oldest and finally on the youngest leaves. Drought stress disrupted the sequentiality of senescence in the sensitive varieties. An untimely senescence appeared in flag leaves, earlier than in the older leaves. Total protein and Rubisco contents decreased and the GS2 isoenzyme declined considerably in the youngest leaves. In the tolerant varieties, however, these physiological parameters did not change under drought, only the sequential senescence of leaf levels accelerated in some cases compared to the control, well-watered plants. Our results revealed that GS is a good indicator of drought stress, which can be applied for the characterization of wheat cultivars in terms of drought stress tolerance. PMID:23542183

  7. Phosphodiesterase isoenzyme families in human osteoarthritis chondrocytes – functional importance of phosphodiesterase 4

    PubMed Central

    Tenor, Hermann; Hedbom, Erik; Häuselmann, Hans-Jörg; Schudt, Christian; Hatzelmann, Armin

    2002-01-01

    We studied whether selective inhibitors of cyclic nucleotide hydrolysing phosphodiesterase (PDE) isoenzymes influence IL-1β-induced nitric oxide (NO) release from human articular chondrocytes. In addition, the pattern of PDE isoenzymes contributing to cyclic nucleotide hydrolysis in human chondrocytes was characterized.Chondrocytes were isolated from human osteoarthritic cartilage and cultured in alginate beads. IL-1β-induced chondrocyte products (nitric oxide and prostaglandin E2) were measured in culture supernatants after 48 h incubation time. PDE activities were assessed in chondrocyte lysates. Inducible nitric oxide synthase (iNOS) and PDE4A-D proteins were detected by immunoblotting.The selective PDE4 inhibitors Piclamilast and Roflumilast partially attenuated IL-1β-induced NO production whereas selective inhibitors of PDE2 (EHNA), PDE3 (Motapizone) or PDE5 (Sildenafil) were inactive. Indomethacin reversed the reduction of IL-1β-induced NO by PDE4 inhibitors. It was shown that autocrine prostaglandin E2 (PGE2) enabled PDE4 inhibitors to reduce IL-1β-induced NO in this experimental setting.Major PDE4 and PDE1 activities were identified in chondrocyte lysates whereas only minor activities of PDE2, 3 and 5 were found. IL-1β and cyclic AMP-mimetics upregulated PDE4 activity and this was associated with an augmentation of PDE4B2 protein.Based on the view that nitric oxide contributes to cartilage degradation in osteoarthritis our study suggests that PDE4 inhibitors may have chondroprotective effects. PMID:11834608

  8. Co-expression of glutaminase K and L isoenzymes in human tumour cells

    PubMed Central

    2004-01-01

    The pattern of expression of glutaminase isoenzymes in tumour cells has been investigated to clarify its role in the malignant transformation and the prospect of its use as a clinically relevant factor. Using leukaemia cells from medullar blood of human patients and several established human cancer cell lines, we have developed a competitive RT (reverse transcriptase)-PCR assay to quantify simultaneously K-type (kidney-type) and L-type (liver-type) glutaminase mRNAs. Co-expression of both transcripts and higher amounts of L-type mRNA were always found in all cancer cell types analysed. However, mature lymphocytes from the medullar blood of a patient suffering aplasia did not express the K-type transcript and showed a 15-fold increase of L-type transcript. Co-expression was also confirmed at the protein level using isoform-specific antibodies; nevertheless, it did not correlate with the relative abundance of glutaminase transcripts and strong K-type protein signals were detected. On the other hand, marked differences were found with regard to glutamate inhibition and phosphate activation of tumour glutaminase activity. Taken together, the protein data suggest that K isoform would account for the majority of glutaminase activity in these human tumour cells. The results confirm that simultaneous expression of both isoenzymes in human cancer cells is a more frequent event than previously thought. Furthermore, the present work and other previous data suggest that K isoform is up-regulated with increased rates of proliferation, whereas prevalence of the L isoform seems to be related with resting or quiescent cell states. PMID:15496140

  9. Inactivation of phosphoglycerate mutase and creatine kinase isoenzymes in human serum

    PubMed Central

    Durany, N; Carreras, J; Valentí, M; Cámara, J; Carreras, J

    2002-01-01

    Aims/Background: Total phosphoglycerate mutase (PGM) activity in serum has been shown to be increased in acute myocardial infarction with the same time course as creatine kinase (CK) activity. However, the increase in the muscle (MM) and in the cardiac (MB) PGM isoenzymes was not as high as expected. The present study was undertaken to characterise PGM inactivation by serum and to compare it with serum CK inactivation. Methods: The PGM and the CK activities of extracts of human heart, skeletal muscle, and brain were determined spectrophotometrically after incubation with different media, namely: plasma, whole serum, dialysed serum, heated serum, serum ultrafiltrate, urate solution, and buffer solution. Results: Type MM PGM was inactivated by plasma, whole serum, heated serum, dialysed serum, and serum ultrafiltrate. Inactivation in dialysed serum was reduced by EDTA and largely reversed by thiol agents. Inactivation in serum ultrafiltrate was not prevented by EDTA and only partially reversed by dithiothreitol. The muscle and type BB CK isoenzymes were inactivated in all the tested media. The incubation of human and rabbit skeletal muscle PGM and CK in urate solution showed that urate does not affect mutase activity under conditions that inactivate CK. Conclusions: These results confirm the mechanisms of CK inactivation proposed by others and show that the type M PGM subunit is inactivated by two different mechanisms, which appear to involve the thiol groups of the enzyme. One mechanism is caused by either a protein component or a protein bound serum component and involves calcium ions and/or another chelatable metal ion. The other mechanism is caused by a lower molecular weight serum component and is metal ion independent. PMID:12147715

  10. Bioluminescence assay of creatine kinase and its isoenzymes in serum and cerebrospinal fluid.

    PubMed

    Tarkkanen, P; Komor, S; Cornely, C; Hacke, W; Greiling, H

    1979-09-01

    We examined the sensitivity of bioluminescence for the determination of very low concentrations of creatine kinase brain-type subunit (CK-BB) in serum and in cerebrospinal fluid. To optimize the sensitivity of CK-isoenzyme assays and eliminate possible sources of error, we separated the isoenzyme fractions by using inhibiting anti-MM and precipitating anti-MM and anti-BB antibodies. The results with the bioluminescence assay correlated with spectrophotometric values such that r = 0.97 for the total CK activity and r = 0.98 for the CK-B activity. The reproducibility of the present method was comparable with the spectrophotometric method and was even better at low enzyme activities. The within-series precision for assay of total CK activity at 2 U/L corresponded to a CV of 9%; at 13 U/L the CV was 5.8%. All the assays were carried out at 25 degrees C. Even at this low temperature, CK activities as low as 0.2 U/L could be determined. In eight patients without any evidence of cerebral cell damage, total CK activity in cerebrospinal fluid was x = 1.05 +/- 0.6 U/L, and CK-BB activity was x = 0.7 +/- 0.4 U/L. In sera of these patients CK-BB activity was x = 0.6 +/- 0.5 U/L. Differences in CK and CK-BB activities in four patients with transient or progressive brain-cell damage are discussed. PMID:466791

  11. Lack of correlation between mRNA expression and enzymatic activity of the aspartate aminotransferase isoenzymes in various tissues of the rat.

    PubMed

    Abruzzese, F; Greco, M; Perlino, E; Doonan, S; Marra, E

    1995-06-12

    Little is known about control of expression of basal levels of the aspartate aminotransferases which are ubiquitous 'house keeping' enzymes in vertebrates. We have measured both mRNA and activity levels for both isoenzymes in various rat tissues as a function of age. Patterns of mRNA expression for the two isoenzymes were similar in a particular tissue about differed widely between tissues. Surprisingly, there was no simple correlation between mRNA levels and specific activities of the enzyme products. We conclude that translation for mRNA for these two isoenzymes is subject to tissue-specific, and in some cases age-related, regulation. PMID:7789537

  12. Formaldehyde dehydrogenase preparations from Methylococcus capsulatus (Bath) comprise methanol dehydrogenase and methylene tetrahydromethanopterin dehydrogenase.

    PubMed

    Adeosun, Ekundayo K; Smith, Thomas J; Hoberg, Anne-Mette; Velarde, Giles; Ford, Robert; Dalton, Howard

    2004-03-01

    In methylotrophic bacteria, formaldehyde is an important but potentially toxic metabolic intermediate that can be assimilated into biomass or oxidized to yield energy. Previously reported was the purification of an NAD(P)(+)-dependent formaldehyde dehydrogenase (FDH) from the obligate methane-oxidizing methylotroph Methylococcus capsulatus (Bath), presumably important in formaldehyde oxidation, which required a heat-stable factor (known as the modifin) for FDH activity. Here, the major protein component of this FDH preparation was shown by biophysical techniques to comprise subunits of 64 and 8 kDa in an alpha(2)beta(2) arrangement. N-terminal sequencing of the subunits of FDH, together with enzymological characterization, showed that the alpha(2)beta(2) tetramer was a quinoprotein methanol dehydrogenase of the type found in other methylotrophs. The FDH preparations were shown to contain a highly active NAD(P)(+)-dependent methylene tetrahydromethanopterin dehydrogenase that was the probable source of the NAD(P)(+)-dependent formaldehyde oxidation activity. These results support previous findings that methylotrophs possess multiple pathways for formaldehyde dissimilation. PMID:14993320

  13. Point of Care Measurement of Lactate.

    PubMed

    Di Mauro, Francesca Miranda; Schoeffler, Gretchen Lee

    2016-03-01

    Lactate is generated as a consequence of anaerobic glycolysis by all tissues of the body. Increased l-lactate, the isoform produced by most mammals, reflects increased anaerobic metabolism secondary to tissue hypoperfusion or tissue hypoxia in most clinical situations, and is called type A lactic acidosis. The utility of lactate measurement and serial lactate monitoring in veterinary patients has been demonstrated in multiple studies. Blood lactate concentration is significantly elevated in many disease processes including septic peritonitis, immune-mediated hemolytic anemia, Babesiosis, trauma, gastric dilation and volvulus, and intracranial disease. Lactate clearance can be used to assess response to fluid therapy, cardiovascular therapeutics, and blood product transfusion in patients affected by type A lactic acidosis. Lactate concentration in peritoneal, pericardial, and synovial fluid can also be used as a diagnostic tool. Point of care analyzers such as the Lactate Pro, Lactate Scout, Accutrend, iSTAT, and Lactate Plus have been shown to be accurate lactate measurement instruments in small animal patients. PMID:27451047

  14. Purification and properties of three (1-->3)-beta-D-glucanase isoenzymes from young leaves of barley (Hordeum vulgare).

    PubMed Central

    Hrmova, M; Fincher, G B

    1993-01-01

    Three (1-->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.39) isoenzymes GI, GII and GIII were purified from young leaves of barley (Hordeum vulgare) using (NH4)2SO4 fractional precipitation, ion-exchange chromatography, chromatofocusing and gel-filtration chromatography. The three (1-->3)-beta-D-glucanases are monomeric proteins of apparent M(r)32,000 with pI values in the range 8.8-10.3. N-terminal amino-acid-sequence analyses confirmed that the three isoenzymes represent the products of separate genes. Isoenzymes GI and GII are less stable at elevated temperatures and are active over a narrower pH range than is isoenzyme GIII, which is a glycoprotein containing 20-30 mol of hexose equivalents/mol of enzyme. The preferred substrate for the enzymes is laminarin from the brown alga Laminaria digitata, an essentially linear (1-->3)-beta-D-glucan with a low degree of glucosyl substitution at 0-6 and a degree of polymerization of approx. 25. The three enzymes are classified as endohydrolases, because they yield (1-->3)-beta-D-oligoglucosides with degrees of polymerization of 3-8 in the initial stages of hydrolysis of laminarin. Kinetic analyses indicate apparent Km values in the range 172-208 microM, kcat. constants of 36-155 s-1 and pH optima of 4.8. Substrate specificity studies show that the three isoenzymes hydrolyse substituted (1-->3)-beta-D-glucans with degrees of polymerization of 25-31 and various high-M(r), substituted and side-branched fungal (1-->3;1-->6)-beta-D-glucans. However, the isoenzymes differ in their rates of hydrolysis of a (1-->3;1-->6)-beta-D-glucan from baker's yeast and their specific activities against laminarin vary significantly. The enzymes do not hydrolyse (1-->3;1-->4)-beta-D-glucans, (1-->6)-beta-D-glucan, CM-cellulose, insoluble (1-->3)-beta-D-glucans or aryl beta-D-glycosides. Images Figure 3 Figure 6 PMID:8424790

  15. Cellobiose dehydrogenase in cellulose degradation

    SciTech Connect

    Eriksson, L.; Igarashi, Kiyohiko; Samejima, Masahiro

    1996-10-01

    Cellobiose dehydrogenase is produced by a variety of fungi. Although it was already discovered during the 70`s, it`s role in cellulose and lignin degradation is yet ambiguous. The enzyme contains both heme and FAD as prosthetic groups, and seems to have a domain specifically designed to bind the enzyme to cellulose. It`s affinity to amorphous cellulose is higher than to crystalline cellulose. We will report on the binding behavior of the enzyme, its usefulness in elucidation of cellulose structures and also, possibilities for applications such as its use in measuring individual and synergistic mechanisms for cellulose degradation by endo- and exo-glucanases.

  16. Short communication: Amino acid supplementation and stage of lactation alter apparent utilization of nutrients by blood neutrophils from lactating dairy cows in vitro.

    PubMed

    Garcia, M; Elsasser, T H; Juengst, L; Qu, Y; Bequette, B J; Moyes, K M

    2016-05-01

    Glutamine is the preferred AA used by polymorphonuclear leukocytes (PMN) during the inflammatory response. However, the effect of other AA on bovine PMN response during inflammation and how this is altered by stage of lactation has not been fully elucidated. The objective of this study was to determine the effect of additional AA supplementation (pool of AA excluding Gln) on AA profiles, gene expression, and inflammatory function of PMN from dairy cows in early and mid lactation in vitro. We used 18 Holstein cows for this study. Polymorphonuclear leukocytes were isolated. Working solutions of AA (0 or 4 mM) and LPS (0 or 50μg/mL) were added to cell populations suspended in RPMI and incubated for 2h at 37°C. We used a subset of samples for gene and protein expression. Concentrations of AA in medium were determined using gas chromatography-mass spectrometry with norleucine as an internal standard. Apparent AA and glucose utilization were calculated by subtracting the concentration after from that of before incubation. Data were analyzed as a randomized block design. Challenge with LPS increased the expression of proinflammatory genes and AA supplementation decreased both the expression of some proinflammatory genes and the media concentrations of tumor necrosis factor-α. Neither stage of lactation, LPS challenge, nor AA supplementation altered the chemotactic or phagocytic abilities of PMN in vitro. Polymorphonuclear leukocytes supplemented with AA had greater concentrations and apparent utilization of most of the supplemented AA, whereas the unsupplemented group had greater apparent utilization of glucose. Alanine was not provided in the media but was present in spent media, and Ile, Gly, and Pro were greater in spent media than in media before incubation indicating synthesis of these AA. Regarding expression of genes involved in nutrient metabolism, the expression of G6PD, coding for the enzyme glucose 6-phosphate dehydrogenase, was increased and that of PDHA1

  17. Lactate promotes glioma migration by TGF-β2–dependent regulation of matrix metalloproteinase-2

    PubMed Central

    Baumann, Fusun; Leukel, Petra; Doerfelt, Anett; Beier, Christoph P.; Dettmer, Katja; Oefner, Peter J.; Kastenberger, Michael; Kreutz, Marina; Nickl-Jockschat, Thomas; Bogdahn, Ulrich; Bosserhoff, Anja-Katrin; Hau, Peter

    2009-01-01

    Lactate dehydrogenase type A (LDH-A) is a key metabolic enzyme catalyzing pyruvate into lactate and is excessively expressed by tumor cells. Transforming growth factor-β2 (TGF-β2) is a key regulator of invasion in high-grade gliomas, partially by inducing a mesenchymal phenotype and by remodeling the extracellular matrix. In this study, we tested the hypothesis that lactate metabolism regulates TGF-β2–mediated migration of glioma cells. Small interfering RNA directed against LDH-A (siLDH-A) suppresses, and lactate induces, TGF-β2 expression, suggesting that lactate metabolism is strongly associated with TGF-β2 in glioma cells. Here we demonstrate that TGF-β2 enhances expression, secretion, and activation of matrix metalloproteinase-2 (MMP-2) and induces the cell surface expression of integrin αvβ3 receptors. In spheroid and Boyden chamber migration assays, inhibition of MMP-2 activity using a specific MMP-2 inhibitor and blocking of integrin αvβ3 abrogated glioma cell migration stimulated by TGF-β2. Furthermore, siLDH-A inhibited MMP2 activity, leading to inhibition of glioma migration. Taken together, we define an LDH-A–induced and TGF-β2–coordinated regulatory cascade of transcriptional regulation of MMP-2 and integrin αvβ3. This novel interaction between lactate metabolism and TGF-β2 might constitute a crucial mechanism for glioma migration. PMID:19033423

  18. NAD-dependent aromatic alcohol dehydrogenase in wheats (Triticum L.) and goatgrasses (Aegilops L.): evolutionary genetics.

    PubMed

    Jaaska, V

    1984-04-01

    Evolutionary electrophoretic variation of a NAD-specific aromatic alcohol dehydrogenase, AADH-E, in wheat and goatgrass species is described and discussed in comparison with a NAD-specific alcohol dehydrogenase (ADH-A) and a NADP-dependent AADH-B studied previously. Cultivated tetraploid emmer wheats (T. turgidum s. l.) and hexaploid bread wheats (T. aestivum s. l.) are all fixed for a heterozygous triplet, E(0.58)/E(0.64). The slowest isoenzyme, E(0.58), is controlled by a homoeoallelic gene on the chromosome arm 6AL of T. aestivum cv. 'Chinese Spring' and is inherent in all diploid wheats, T. monococcum s. Str., T. boeoticum s. l. and T. urartu. The fastest isoenzyme, E(0.64), is presumably controlled by the B- and D-genome homoeoalleles of the bread wheat and is the commonest alloenzyme of diploid goat-grasses, including Ae. speltaides and Ae. tauschii. The tetraploid T. timopheevii s. str. has a particular heterozygous triplet E(0.56)/E(0.71), whereas the hexaploid T. zhukovskyi exhibited polymorphism with electromorphs characteristic of T. timopheevii and T. monococcum. Wild tetraploid wheats, T. dicoccoides and T. araraticum, showed partially homologous intraspecific variation of AADH-E with heterozygous triplets E(0.58)/E(0.64) (the commonest), E(0.58)/E(0.71), E(0.45)/E(0.58), E(0.48)/E(0.58) and E(0.56)/E(0.58) recorded. Polyploid goatgrasses of the D-genome group, excepting Ae. cylindrica, are fixed for the common triplet E(0.58)/E(0.64). Ae. cylindrica and polyploid goatgrasses of the C(u)-genome group, excepting Ae. kotschyi, are homozygous for E(0.64). Ae. kotschyi is exceptional, showing fixed heterozygosity for both AADH-E and ADH-A with unique triplets E(0.56)/E(0.64) and A(0.49)/A(0.56). PMID:24258843

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

    PubMed Central

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

    2014-01-01

    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

  20. Lactate metabolism by pediococci isolated from cheese.

    PubMed

    Thomas, T D; McKay, L L; Morris, H A

    1985-04-01

    Pediococcus pentosaceus is commonly found among the adventitious microflora of Cheddar cheese. When this organism was incubated with L-(+)-lactate under anaerobic conditions, L-(+)-lactate was rapidly converted to D-(-)-lactate until racemic (DL) lactate was present. Under aerobic conditions this initial reaction was followed by a slower reaction resulting in the use of both lactate isomers and in the production of acetate and CO2. With intact cells the lactate oxidation system had an optimum pH of 5 to 6, depending on the initial lactate concentration. Cells grown anaerobically possessed lactate-oxidizing activity which increased two- to fourfold as sugar was exhausted from the medium. Aerobic growth further increased specific activities. Cheddar cheese was made with the deliberate addition of P. pentosaceus. When the resulting cheese was grated to expose a large surface area to O2, lactate was converted to acetate at a rate which depended on the density of pediococci in the cheese. The lactate oxidation system remained active in cheese which had been ripened for 6 months. PMID:4004222

  1. Study for identification of beneficial uses of Space (BUS). Volume 2: Technical report. Book 1: Development and business analysis of space processed isoenzymes

    NASA Technical Reports Server (NTRS)

    1975-01-01

    A separation method to provide reasonable yields of high specificity isoenzymes for the purpose of large scale, early clinical diagnosis of diseases and organic damage such as, myocardial infarction, hepatoma, muscular dystrophy, and infectous disorders is presented. Preliminary development plans are summarized. An analysis of required research and development and production resources is included. The costs of such resources and the potential profitability of a commercial space processing opportunity for electrophoretic separation of high specificity isoenzymes are reviewed.

  2. Betaine aldehyde dehydrogenase in sorghum.

    PubMed Central

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

    1996-01-01

    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

  3. Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I*

    PubMed Central

    Aro, Ellinoora; Salo, Antti M.; Khatri, Richa; Finnilä, Mikko; Miinalainen, Ilkka; Sormunen, Raija; Pakkanen, Outi; Holster, Tiina; Soininen, Raija; Prein, Carina; Clausen-Schaumann, Hauke; Aszódi, Attila; Tuukkanen, Juha; Kivirikko, Kari I.; Schipani, Ernestina; Myllyharju, Johanna

    2015-01-01

    Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α2β2 tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1−/−) leads to embryonic lethality in mouse, whereas P4ha1+/− mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2−/− mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1+/−;P4ha2−/− mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the Tm of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1+/−;P4ha2−/− mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2−/− mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype. PMID:26001784

  4. Creatine kinase isoenzymes specificities: histidine 65 in human CK-BB, a role in protein stability, not in catalysis.

    PubMed

    Mourad-Terzian, T; Steghens, J P; Min, K L; Collombel, C; Bozon, D

    2000-06-01

    Creatine kinases (CK) play a prominent role in cell energy distribution through an energy shuttle between mitochondria and other organelles. Human brain CK was cloned and overexpressed in COS-7 cells. We then deleted His-65 and/or Pro-66 situated near the center of a flexible loop as shown by X-ray crystallography on mitochondrial and cytosolic CK. The DeltaH65 mutant had nearly the same affinity for its substrates as wild isoenzyme, but its stability was very low. Unlike DeltaH65, DeltaH65P66 had a eightfold decreased affinity for creatine phosphate and was unable to dephosphorylate cyclocreatine phosphate. Our results demonstrate that, despite an overall similar shape of the proteins, this loop accounts for some subtle differences in isoenzyme functions. PMID:10854850

  5. Serum Lactate Dehydrogenase in Non-Hodgkin's Lymphoma: A Prognostic Indicator.

    PubMed

    Yadav, Charu; Ahmad, Afzal; D'Souza, Benedicta; Agarwal, Ashish; Nandini, M; Ashok Prabhu, K; D'Souza, Vivian

    2016-04-01

    Non-Hodgkin's lymphoma constitutes a group of disorders originating from the malignant transformation of lymphocytes and involving either the lymph nodes or extranodal sites. NHL commonly presents in the sixth to seventh decade of life with a male preponderance (50-75 %). Recent studies have shown importance of serum LDH in prognosis of NHL. Authors report a case of a 63 year old male presenting with complaints of fever and backache for past 4 months. General and systemic examination revealed bilateral axillary lymphadenopathy and splenomegaly respectively. Serum LDH level was highly elevated (3441 U/l). Excisional axillary and bone marrow biopsy were done before oncology referral. Complete workup revealed diffuse Non-Hodgkin's lymphoma with bone marrow infiltration. Patient died because of acute renal failure due to NHL and DM 2 (Type 2 diabetes mellitus). PMID:27069334

  6. Relationship between polymorphisms in lactate dehydrogenase B gene and milk characteristics in beef cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytochrome P450s are a group of heme-containing monooxygenases necessary for the oxidative metabolism of foreign biological substances. Our goal was to determine the frequency of single nucleotide polymorphism (SNP) 994 in the CYP3A28 sequence of three breed types of cattle. The distribution of geno...

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

    ERIC Educational Resources Information Center

    Anderson, Alexander J.

    1988-01-01

    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)

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

    ERIC Educational Resources Information Center

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

    2005-01-01

    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…

  9. [The contribution of different alpha-amylase isoenzymes of the commodity grain spring wheat in the formation of falling number values].

    PubMed

    Mamytova, N S; Kuzovlev, V A; Khakimzhanov, A A; Fursov, O V

    2014-01-01

    The participation of various isoenzymes of alpha-amylase in the formation of falling number values of the commodity grain of wheat grown in the Republic of Kazakhstan was investigated. It was found that active isoenzymes alpha-AMY1 and alpha-AMY2 of the embryonic shield were present in the grain with an index over 200. A significant decrease in the falling number depended mainly on the synthesis of alpha-AMY1 and alpha-AMY2 isoenzymes in the aleurone layer. In the grain, isoenzymes with high isoelectric points (p1 > or = 7.3) were found; these isoenzymes belong to alpha-amylase or late maturing or alpha-amylase of practically mature grains. It was discovered that the exogenous hormone (gibberellic acid) induced synthesis of alpha-amylase isoenzymes of scutellum, whole caryopses, and aleurone. It was shown that the impact of exogenous gibberellic acid on the activity and structure of alpha-amylase is reduced in grain with a low falling number. PMID:25707111

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

    PubMed Central

    Omlin, Teye; Langevin, Karolanne

    2014-01-01

    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 μmol·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

  11. Maternal flaxseed diet during lactation changes adrenal function in adult male rat offspring.

    PubMed

    Figueiredo, Mariana Sarto; da Conceição, Ellen Paula Santos; de Oliveira, Elaine; Lisboa, Patricia Cristina; de Moura, Egberto Gaspar

    2015-10-14

    Flaxseed (Linum usitatissimum L.) has been a focus of interest in the field of functional foods because of its potential health benefits. However, we hypothesised that maternal flaxseed intake during lactation could induce several metabolic dysfunctions in adult offspring. In the present study, we aimed to characterise the adrenal function of adult offspring whose dams were supplemented with whole flaxseed during lactation. At birth, lactating Wistar rats were divided into two groups: rats from dams fed the flaxseed diet (FLAX) with 25% of flaxseed and controls dams. Pups received standard diet after weaning and male offspring were killed at age 180 days old to collect blood and tissues. We evaluated body weight and food intake during development, corticosteronaemia, adrenal catecholamine content, hepatic cholesterol, TAG and glycogen contents, and the protein expression of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), 11-β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and adrenaline β2 receptor at postnatal day 180 (PN180). After weaning, pups from the FLAX group had a higher body weight (+10 %) and food intake (+10%). At PN180, the FLAX offspring exhibited higher serum corticosterone (+48%) and lower adrenal catecholamine ( - 23%) contents, lower glycogen ( - 30%), higher cholesterol (4-fold increase) and TAG (3-fold-increase) contents in the liver, and higher 11β-HSD1 (+62%) protein expression. Although the protein expression of hypothalamic CRH was unaffected, the FLAX offspring had lower protein expression of pituitary ACTH ( - 34%). Therefore, induction of hypercorticosteronaemia by dietary flaxseed during lactation may be due to an increased hepatic activation of 11β-HSD1 and suppression of ACTH. The changes in the liver fat content of the FLAX group are suggestive of steatosis, in which hypercorticosteronaemia may play an important role. Thus, it is recommended that lactating women restrict the intake of flaxseed during

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

    SciTech Connect

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

    2011-12-01

    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.

  13. Hyperpolarized (13)C MR imaging detects no lactate production in mutant IDH1 gliomas: Implications for diagnosis and response monitoring.

    PubMed

    Chaumeil, Myriam M; Radoul, Marina; Najac, Chloé; Eriksson, Pia; Viswanath, Pavithra; Blough, Michael D; Chesnelong, Charles; Luchman, H Artee; Cairncross, J Gregory; Ronen, Sabrina M

    2016-01-01

    Metabolic imaging of brain tumors using (13)C Magnetic Resonance Spectroscopy (MRS) of hyperpolarized [1-(13)C] pyruvate is a promising neuroimaging strategy which, after a decade of preclinical success in glioblastoma (GBM) models, is now entering clinical trials in multiple centers. Typically, the presence of GBM has been associated with elevated hyperpolarized [1-(13)C] lactate produced from [1-(13)C] pyruvate, and response to therapy has been associated with a drop in hyperpolarized [1-(13)C] lactate. However, to date, lower grade gliomas had not been investigated using this approach. The most prevalent mutation in lower grade gliomas is the isocitrate dehydrogenase 1 (IDH1) mutation, which, in addition to initiating tumor development, also induces metabolic reprogramming. In particular, mutant IDH1 gliomas are associated with low levels of lactate dehydrogenase A (LDHA) and monocarboxylate transporters 1 and 4 (MCT1, MCT4), three proteins involved in pyruvate metabolism to lactate. We therefore investigated the potential of (13)C MRS of hyperpolarized [1-(13)C] pyruvate for detection of mutant IDH1 gliomas and for monitoring of their therapeutic response. We studied patient-derived mutant IDH1 glioma cells that underexpress LDHA, MCT1 and MCT4, and wild-type IDH1 GBM cells that express high levels of these proteins. Mutant IDH1 cells and tumors produced significantly less hyperpolarized [1-(13)C] lactate compared to GBM, consistent with their metabolic reprogramming. Furthermore, hyperpolarized [1-(13)C] lactate production was not affected by chemotherapeutic treatment with temozolomide (TMZ) in mutant IDH1 tumors, in contrast to previous reports in GBM. Our results demonstrate the unusual metabolic imaging profile of mutant IDH1 gliomas, which, when combined with other clinically available imaging methods, could be used to detect the presence of the IDH1 mutation in vivo. PMID:27437179

  14. Differential effects of peroxynitrite on human mitochondrial creatine kinase isoenzymes. Inactivation, octamer destabilization, and identification of involved residues.

    PubMed

    Wendt, Silke; Schlattner, Uwe; Wallimann, Theo

    2003-01-10

    Creatine kinase isoenzymes are very susceptible to free radical damage and are inactivated by superoxide radicals and peroxynitrite. In this study, we have analyzed the effects of peroxynitrite on enzymatic activity and octamer stability of the two human mitochondrial isoenzymes (ubiquitous mitochondrial creatine kinase (uMtCK) and sarcomeric mitochondrial creatine kinase (sMtCK)), as well as of chicken sMtCK, and identified the involved residues. Inactivation by peroxynitrite was concentration-dependent and similar for both types of MtCK isoenzymes. Because peroxynitrite did not lower the residual activity of a sMtCK mutant missing the active site cysteine (C278G), oxidation of this residue is sufficient to explain MtCK inactivation. Mass spectrometric analysis confirmed oxidation of Cys-278 and further revealed oxidation of the C-terminal Cys-358, possibly involved in MtCK/membrane interaction. Peroxynitrite also led to concentration-dependent dissociation of MtCK octamers into dimers. In this study, ubiquitous uMtCK was much more stable than sarcomeric sMtCK. Mass spectrometric analysis revealed chemical modifications in peptide Gly-263-Arg-271 located at the dimer/dimer interface, including oxidation of Met-267 and nitration of Trp-268 and/or Trp-264, the latter being a very critical residue for octamer stability. These data demonstrate that peroxynitrite affects the octameric state of MtCK and confirms human sMtCK as the generally more susceptible isoenzyme. The results provide a molecular explanation of how oxidative damage can lead to inactivation and decreased octamer/dimer ratio of MtCK, as seen in neurodegenerative diseases and heart pathology, respectively. PMID:12401781

  15. Regulation by phosphodiesterase isoenzymes of non-adrenergic non-cholinergic contraction in guinea-pig isolated main bronchus.

    PubMed Central

    Spina, D.; Harrison, S.; Page, C. P.

    1995-01-01

    1. We have investigated the role of phosphodiesterase isoenzymes in modulating electric field stimulation (EFS), substance P and capsaicin-induced contraction of the guinea-pig isolated main bronchus. 2. Non-adrenergic non-cholinergic contractile responses were elicited by EFS (3 Hz, 20 s) in the guinea-pig isolated main bronchus in the presence of the non-selective muscarinic antagonist, atropine (0.1 microM), the non-selective beta-adrenoceptor antagonist, propranolol (1 microM), the neutral endopeptidase inhibitor, thiorphan (10 microM) and the cyclo-oxygenase inhibitor, indomethacin (5 microM). The type III, type III/IV, type IV and type V phosphodiesterase isoenzyme inhibitor, SKF 94836, benzafentrine, Ro-20-1724 and zaprinast respectively, significantly attenuated the contractile response to EFS. The IC50 (95% confidence limits) value for SKF 94836, benzafentrine, Ro-20-1724 and zaprinast was 8.3 microM (0.89-78); 0.7 microM (0.1-4.5); 0.5 microM (0.2-1.2) and 13 microM (2-87) respectively. 3. The phosphodiesterase isoenzyme inhibitors, SKF 94836, Ro-20-1724 and zaprinast, partially attenuated the contractile response to substance P (10 nM). Benzafentrine significantly inhibited the contractile response to substance P, yielding an IC50 value of 1.9 microM (0.9-3.8). 4. The phosphodiesterase isoenzyme inhibitor, Ro-20-1724 (0.1-100 microM) failed to reduce significantly the contractile potency of capsaicin (P > 0.05). In contrast, SKF 94836 (1 microM), benzafentrine (10 microM) and zaprinast (100 microM) significantly reduced the contractile potency of capsaicin (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8564269

  16. Dichloroacetate increases glucose use and decreases lactate in developing rat brain

    SciTech Connect

    Miller, A.L.; Hatch, J.P.; Prihoda, T.J. )

    1990-12-01

    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.

  17. Targeting metabolic flexibility by simultaneously inhibiting respiratory complex I and lactate generation retards melanoma progression

    PubMed Central

    Chaube, Balkrishna; Malvi, Parmanand; Singh, Shivendra Vikram; Mohammad, Naoshad; Meena, Avtar Singh; Bhat, Manoj Kumar

    2015-01-01

    Melanoma is a largely incurable skin malignancy owing to the underlying molecular and metabolic heterogeneity confounded by the development of resistance. Cancer cells have metabolic flexibility in choosing either oxidative phosphorylation (OXPHOS) or glycolysis for ATP generation depending upon the nutrient availability in tumor microenvironment. In this study, we investigated the involvement of respiratory complex I and lactate dehydrogenase (LDH) in melanoma progression. We show that inhibition of complex I by metformin promotes melanoma growth in mice via elevating lactate and VEGF levels. In contrast, it leads to the growth arrest in vitro because of enhanced extracellular acidification as a result of increased glycolysis. Inhibition of LDH or lactate generation causes decrease in glycolysis with concomitant growth arrest both in vitro and in vivo. Blocking lactate generation in metformin-treated melanoma cells results in diminished cell proliferation and tumor progression in mice. Interestingly, inhibition of either LDH or complex I alone does not induce apoptosis, whereas inhibiting both together causes depletion in cellular ATP pool resulting in metabolic catastrophe induced apoptosis. Overall, our study suggests that LDH and complex I play distinct roles in regulating glycolysis and cell proliferation. Inhibition of these two augments synthetic lethality in melanoma. PMID:26484566

  18. Genetic structure and phylogenetic relationships of Colombian Trypanosoma cruzi populations as determined by schizodeme and isoenzyme markers.

    PubMed

    Jaramillo, N; Moreno, J; Triana, O; Arcos-Burgos, M; Muñoz, S; Solari, A

    1999-12-01

    Twenty-four Trypanosoma cruzi stocks isolated from vectors and from human and Didelphis marsupialis hosts from highly separated sylvatic localities in Colombia were characterized by isoenzyme and schizodeme analyses. The stocks were collected primarily from sylvatic ecotopes representing areas of low, moderate, and high endemicity for Chagas' diseases in Colombia. Parasites were characterized mainly by schizodeme analysis with the restriction enzyme Eco RI and the isoenzyme analysis was performed at 10 genetic loci. These analyses demonstrated an agreement between the classifications based on the isoenzyme analysis and on the restriction fragment length polymorphism patterns obtained with the Colombian stocks. There is clear evidence of demic subdivision between the eastern (E) and western (W) stocks separated by the Andean Mountains and Magdalena River, which is likely due to the geographic isolation generated by these topographic features. Heterozygosity estimates indicate that the E group could be more ancient than the W group. As was postulated in a previous study, these results are also compatible with the existence of a clonal population structure in Colombian sylvatic T. cruzi. Evidence presented here failed to demonstrate a correlation between the degree of endemy and genetic clustering. Finally, schizodeme and isoenzymatic analyses comparing Colombian T. cruzi stocks with others from Chile confirm that Colombian isolates are genetically related to zymodeme 1 and distant from zymodeme 2. PMID:10674683

  19. 21 CFR 184.1207 - Calcium lactate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Calcium lactate. 184.1207 Section 184.1207 Food... GRAS § 184.1207 Calcium lactate. (a) Calcium lactate (C6H10CaO6.xH2O, where x is any integer up to 5, CAS Reg. No. 814-80-2) is prepared commercially by the neutralization of lactic acid with...

  20. 21 CFR 184.1207 - Calcium lactate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Calcium lactate. 184.1207 Section 184.1207 Food... Specific Substances Affirmed as GRAS § 184.1207 Calcium lactate. (a) Calcium lactate (C6H10CaO6.xH2O, where... lactic acid with calcium carbonate or calcium hydroxide. (b) The ingredient meets the specifications...

  1. 21 CFR 184.1207 - Calcium lactate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Calcium lactate. 184.1207 Section 184.1207 Food... Specific Substances Affirmed as GRAS § 184.1207 Calcium lactate. (a) Calcium lactate (C6H10CaO6.xH2O, where... lactic acid with calcium carbonate or calcium hydroxide. (b) The ingredient meets the specifications...

  2. 21 CFR 184.1207 - Calcium lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium lactate. 184.1207 Section 184.1207 Food and... Substances Affirmed as GRAS § 184.1207 Calcium lactate. (a) Calcium lactate (C6H10CaO6.xH2O, where x is any... calcium carbonate or calcium hydroxide. (b) The ingredient meets the specifications of the Food...

  3. 21 CFR 184.1639 - Potassium lactate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Potassium lactate. 184.1639 Section 184.1639 Food... Specific Substances Affirmed as GRAS § 184.1639 Potassium lactate. (a) Potassium lactate (C3H5O3K, CAS Reg. No. 996-31-6) is the potassium salt of lactic acid. It is a hydroscopic, white, odorless solid and...

  4. 21 CFR 184.1639 - Potassium lactate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Potassium lactate. 184.1639 Section 184.1639 Food... GRAS § 184.1639 Potassium lactate. (a) Potassium lactate (C3H5O3K, CAS Reg. No. 996-31-6) is the potassium salt of lactic acid. It is a hydroscopic, white, odorless solid and is prepared commercially...

  5. 21 CFR 184.1639 - Potassium lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium lactate. 184.1639 Section 184.1639 Food... Specific Substances Affirmed as GRAS § 184.1639 Potassium lactate. (a) Potassium lactate (C3H5O3K, CAS Reg. No. 996-31-6) is the potassium salt of lactic acid. It is a hydroscopic, white, odorless solid and...

  6. 21 CFR 184.1639 - Potassium lactate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Potassium lactate. 184.1639 Section 184.1639 Food... Specific Substances Affirmed as GRAS § 184.1639 Potassium lactate. (a) Potassium lactate (C3H5O3K, CAS Reg. No. 996-31-6) is the potassium salt of lactic acid. It is a hydroscopic, white, odorless solid and...

  7. 21 CFR 184.1639 - Potassium lactate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Potassium lactate. 184.1639 Section 184.1639 Food... Specific Substances Affirmed as GRAS § 184.1639 Potassium lactate. (a) Potassium lactate (C3H5O3K, CAS Reg. No. 996-31-6) is the potassium salt of lactic acid. It is a hydroscopic, white, odorless solid and...

  8. Cerebral Lactate Metabolism After Traumatic Brain Injury.

    PubMed

    Patet, Camille; Suys, Tamarah; Carteron, Laurent; Oddo, Mauro

    2016-04-01

    Cerebral energy dysfunction has emerged as an important determinant of prognosis following traumatic brain injury (TBI). A number of studies using cerebral microdialysis, positron emission tomography, and jugular bulb oximetry to explore cerebral metabolism in patients with TBI have demonstrated a critical decrease in the availability of the main energy substrate of brain cells (i.e., glucose). Energy dysfunction induces adaptations of cerebral metabolism that include the utilization of alternative energy resources that the brain constitutively has, such as lactate. Two decades of experimental and human investigations have convincingly shown that lactate stands as a major actor of cerebral metabolism. Glutamate-induced activation of glycolysis stimulates lactate production from glucose in astrocytes, with subsequent lactate transfer to neurons (astrocyte-neuron lactate shuttle). Lactate is not only used as an extra energy substrate but also acts as a signaling molecule and regulator of systemic and brain glucose use in the cerebral circulation. In animal models of brain injury (e.g., TBI, stroke), supplementation with exogenous lactate exerts significant neuroprotection. Here, we summarize the main clinical studies showing the pivotal role of lactate and cerebral lactate metabolism after TBI. We also review pilot interventional studies that examined exogenous lactate supplementation in patients with TBI and found hypertonic lactate infusions had several beneficial properties on the injured brain, including decrease of brain edema, improvement of neuroenergetics via a "cerebral glucose-sparing effect," and increase of cerebral blood flow. Hypertonic lactate represents a promising area of therapeutic investigation; however, larger studies are needed to further examine mechanisms of action and impact on outcome. PMID:26898683

  9. 21 CFR 184.1768 - Sodium lactate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium lactate. 184.1768 Section 184.1768 Food and... Substances Affirmed as GRAS § 184.1768 Sodium lactate. (a) Sodium lactate (C3H5O3Na, CAS Reg. No. 72-17-3) is the sodium salt of lactic acid. It is prepared commercially by the neutralization of lactic acid...

  10. 21 CFR 184.1768 - Sodium lactate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium lactate. 184.1768 Section 184.1768 Food and... Substances Affirmed as GRAS § 184.1768 Sodium lactate. (a) Sodium lactate (C3H5O3Na, CAS Reg. No. 72-17-3) is the sodium salt of lactic acid. It is prepared commercially by the neutralization of lactic acid...

  11. 21 CFR 184.1768 - Sodium lactate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium lactate. 184.1768 Section 184.1768 Food and... Substances Affirmed as GRAS § 184.1768 Sodium lactate. (a) Sodium lactate (C3H5O3Na, CAS Reg. No. 72-17-3) is the sodium salt of lactic acid. It is prepared commercially by the neutralization of lactic acid...

  12. 21 CFR 184.1768 - Sodium lactate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium lactate. 184.1768 Section 184.1768 Food and... Substances Affirmed as GRAS § 184.1768 Sodium lactate. (a) Sodium lactate (C3H5O3Na, CAS Reg. No. 72-17-3) is the sodium salt of lactic acid. It is prepared commercially by the neutralization of lactic acid...

  13. 21 CFR 184.1207 - Calcium lactate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... lactic acid with calcium carbonate or calcium hydroxide. (b) The ingredient meets the specifications of... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Calcium lactate. 184.1207 Section 184.1207 Food... Specific Substances Affirmed as GRAS § 184.1207 Calcium lactate. (a) Calcium lactate (C6H10CaO6.xH2O,...

  14. Lactation amenorrhea in Saudi women.

    PubMed Central

    Madani, K A; Khashoggi, R H; al-Nowaisser, A A; Nasrat, H A; Khalil, M H

    1994-01-01

    STUDY OBJECTIVE--The study aimed to investigate some aspects of breast feeding, namely-lactation amenorrhea, the average interval between pregnancies, and the extent of knowledge that an average Saudi woman has about breast feeding. DESIGN--This was a cross sectional study in which a pretested questionnaire was used to collect the information. SETTING--The study was conducted in the Taif area between January and April of 1990. Seventy nine primary health care centres participated. PARTICIPANTS--Altogether 1019 of 2400 women contacted who agreed to participate and met the criteria were studied. Eligible subjects were defined as Saudi women, between 16 and 40 years old, who came with their infants for vaccination, and had delivered between one week and 12 months previously. Each mother had at least one other child. MEASUREMENT AND MAIN RESULT--At birth, the percentage of infants who were initially breast fed was 98% but within three days of delivery over two thirds (68.9%) of the mothers gave other supplementary liquids to their infants. At the time of interview more than half (55.1%) of mothers had lactation amenorrhea. The mean (SD) lactation amenorrhea period and birth interval were 5.95 (5) and 26.8 (14.1) months, respectively. Mothers obtained information on breast feeding mainly from their doctors and television. Within families, husbands had the primary role in encouraging their wives to breast feed, followed by the mother and then by the mother in law. It was found that a high percentage (94.2%) of women had breast fed their previous child. CONCLUSION--The lack of adequate information on breast feeding and the short interval between births are local problems which should be considered by the health authorities. PMID:8051529

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

    PubMed

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

    2013-08-01

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

  16. Oxamate Improves Glycemic Control and Insulin Sensitivity via Inhibition of Tissue Lactate Production in db/db Mice

    PubMed Central

    Ye, Weiran; Zheng, Yijia; Zhang, Shanshan; Yan, Li; Cheng, Hua; Wu, Muchao

    2016-01-01

    Oxamate (OXA) is a pyruvate analogue that directly inhibits the lactate dehydrogenase (LDH)-catalyzed conversion process of pyruvate into lactate. Earlier and recent studies have shown elevated blood lactate levels among insulin-resistant and type 2 diabetes subjects and that blood lactate levels independently predicted the development of incident diabetes. To explore the potential of OXA in the treatment of diabetes, db/db mice were treated with OXA in vivo. Treatment of OXA (350–750 mg/kg of body weight) for 12 weeks was shown to decrease body weight gain and blood glucose and HbA1c levels and improve insulin secretion, the morphology of pancreatic islets, and insulin sensitivity in db/db mice. Meanwhile, OXA reduced the lactate production of adipose tissue and skeletal muscle and serum lactate levels and decreased serum levels of TG, FFA, CRP, IL-6, and TNF-α in db/db mice. The PCR array showed that OXA downregulated the expression of Tnf, Il6, leptin, Cxcr3, Map2k1, and Ikbkb, and upregulated the expression of Irs2, Nfkbia, and Pde3b in the skeletal muscle of db/db mice. Interestingly, LDH-A expression increased in the islet cells of db/db mice, and both treatment of OXA and pioglitazone decreased LDH-A expression, which might be related to the improvement of insulin secretion. Taken together, increased lactate production of adipose tissue and skeletal muscle may be at least partially responsible for insulin resistance and diabetes in db/db mice. OXA improved glycemic control and insulin sensitivity in db/db mice primarily via inhibition of tissue lactate production. Oxamic acid derivatives may be a potential drug for the treatment of type 2 diabetes. PMID:26938239

  17. Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

    PubMed Central

    Arrabal, Sergio; Lucena, Miguel Angel; Canduela, Miren Josune; Ramos-Uriarte, Almudena; Rivera, Patricia; Serrano, Antonia; Pavón, Francisco Javier; Decara, Juan; Vargas, Antonio; Baixeras, Elena; Martín-Rufián, Mercedes; Márquez, Javier; Fernández-Llébrez, Pedro; De Roos, Baukje; Grandes, Pedro; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2015-01-01

    Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB1 receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB1-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD. PMID:26671069

  18. Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle.

    PubMed

    Arrabal, Sergio; Lucena, Miguel Angel; Canduela, Miren Josune; Ramos-Uriarte, Almudena; Rivera, Patricia; Serrano, Antonia; Pavón, Francisco Javier; Decara, Juan; Vargas, Antonio; Baixeras, Elena; Martín-Rufián, Mercedes; Márquez, Javier; Fernández-Llébrez, Pedro; De Roos, Baukje; Grandes, Pedro; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2015-01-01

    Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg(-1), 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle--regulated by both diet and CB1 receptor activity--through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB1-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD. PMID:26671069

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

    PubMed Central

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

    2014-01-01

    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 μM 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

  20. Reconstruction of an acetogenic 2,3-butanediol pathway involving a novel NADPH-dependent primary-secondary alcohol dehydrogenase.

    PubMed

    Köpke, Michael; Gerth, Monica L; Maddock, Danielle J; Mueller, Alexander P; Liew, FungMin; Simpson, Séan D; Patrick, Wayne M

    2014-06-01

    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 μM 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