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Bioengineered 2015N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto...
N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N(6)-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N(6)-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N(6)-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N(6)-amine group on NAD.
Topics: Alcohol Dehydrogenase; Animals; Cattle; Glutamate Dehydrogenase; L-Lactate Dehydrogenase; NAD; Oxidoreductases; Rabbits; Sepharose; Sugar Alcohol Dehydrogenases
PubMed: 25611453
DOI: 10.1080/21655979.2014.1004020 -
Open Veterinary Journal 2022Lactate dehydrogenase (LDH) isoenzymes may be useful in the differential diagnosis of pleural effusion (PE) and ascitic fluid (AF) etiologies in cats since tissue damage...
BACKGROUND
Lactate dehydrogenase (LDH) isoenzymes may be useful in the differential diagnosis of pleural effusion (PE) and ascitic fluid (AF) etiologies in cats since tissue damage induces their release, changing the pattern of their activity.
AIM
This study aimed to determine the diagnostic utility of measuring LDH levels and isoenzyme activities in PE or AF in cats with malignancy.
METHODS
LDH levels and isoenzyme activities in the serum, PE, and AF were compared among cats in the malignant, infectious, and non-malignant, non-infectious groups. A receiver operating characteristic (ROC) analysis was performed to assess the accuracy in diagnosing feline malignancy.
RESULTS
Significant differences in LDH level and LDH isoenzyme activities in the PE and AF were observed among the three groups. The combination of LDH level and LDH-1 activity in PE or AF had the highest area under the ROC (AUC) values for discriminating malignant effusion from non-malignant effusion. The AUC of the combination of LDH level and LDH-1 activity in PE or AF was 0.874. The sensitivity and specificity of using the combination of LDH level (cut-off: <2,269 U/l) and LDH-1 activity (cut-off: <4.8%) in PE or AF for predicting malignancy with the highest AUC value were 94.4% and 72.7%, respectively.
CONCLUSION
Our results suggest that the combination of LDH level and LDH-1 activity in PE or AF is a potential factor for diagnosing malignancy. Considering that LDH isoenzymes can be measured inexpensively and easily, LDH tests can be readily accommodated in veterinary clinical practice.
Topics: Cats; Animals; Isoenzymes; Ascitic Fluid; Pleural Effusion; Pleural Effusion, Malignant; L-Lactate Dehydrogenase; Cat Diseases
PubMed: 36589389
DOI: 10.5455/OVJ.2022.v12.i5.19 -
BMC Oral Health Nov 2020Increased levels of lactate dehydrogenase (LDH) as a tumor marker have been reported in malignant and some premalignant oral lesions such as oral lichen planus (OLP) and...
BACKGROUND
Increased levels of lactate dehydrogenase (LDH) as a tumor marker have been reported in malignant and some premalignant oral lesions such as oral lichen planus (OLP) and oral lichenoid reactions (OLRs). This study aimed to assess the level of total LDH in the saliva and serum of patients with oral squamous cell carcinoma (OSCC), OLP and OLRs.
METHODS
In this case-control study, the participants were divided into four groups (n = 25) of healthy controls, OLP, OLRs, and OSCC. The serum and stimulated/unstimulated salivary levels of LDH were spectrophotometrically measured using standard LDH kits (Pars Azmoun). One-way ANOVA, Chi-square test, Pearson's correlation test, and receiver operating characteristic (ROC) analysis were applied to analyze the data.
RESULTS
The serum and salivary levels of LDH in OSCC patients were significantly higher than that the corresponding values in other groups (P = 0.0001). The serum level of LDH in OLR group was significantly higher than that in the control and OLP groups (P = 0.0001), but the difference in salivary level of LDH was not significant. The ROC analysis showed that both the serum and salivary levels of LDH had significant diagnostic ability for detection of OSCC and OLRs. Significant associations were noted between the serum and salivary levels of LDH.
CONCLUSIONS
Patients with OSCC and OLRs had higher serum levels of LDH than OLP and control groups. Further prospective longitudinal studies are required to assess the tissue level of LDH and monitor the transformation of OLRs because they have low rate of malignant transformation compared with other oral premalignant lesions.
Topics: Carcinoma, Squamous Cell; Case-Control Studies; Head and Neck Neoplasms; Humans; L-Lactate Dehydrogenase; Lichen Planus, Oral; Mouth Neoplasms; Saliva; Squamous Cell Carcinoma of Head and Neck
PubMed: 33167957
DOI: 10.1186/s12903-020-01306-0 -
Biomarkers in Medicine Oct 2022The enzyme lactate dehydrogenase (LDH) is a good marker of general hyperinflammation correlated with mortality for COVID-19, and is therefore used in prognosis tools.... (Randomized Controlled Trial)
Randomized Controlled Trial
The enzyme lactate dehydrogenase (LDH) is a good marker of general hyperinflammation correlated with mortality for COVID-19, and is therefore used in prognosis tools. In a current COVID-19 clinical randomized trial (CRT), the blood level of LDH was selected as an inclusion criterion. However, LDH decreased during the pandemic; hence, the impact of this decrease on the prognostic value of LDH for mortality was evaluated. Data on LDH levels in 843 patients were obtained and analyzed. Relative risk, standard error and receiver operating characteristic curves were calculated for two cutoff values. Relative risk lost validity and the area under the curve narrowed by trimester during the pandemic. The progressive decrease in LDH impacted the capacity to predict mortality in COVID-19. More studies are needed to validate this finding and its implications.
Topics: Humans; COVID-19; L-Lactate Dehydrogenase; Pandemics; Prognosis; Retrospective Studies; ROC Curve
PubMed: 36052694
DOI: 10.2217/bmm-2022-0364 -
PloS One 2016Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role of lactate dehydrogenase (LDH), a...
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.
Topics: Animals; Cell Differentiation; Cells, Cultured; Gene Expression; Glucose; Glycolysis; Immunoblotting; Isoenzymes; L-Lactate Dehydrogenase; Lactates; Macrophage Colony-Stimulating Factor; Male; Mice, Inbred C57BL; NFATC Transcription Factors; Osteoclasts; Oxygen Consumption; RANK Ligand; RNA Interference; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction
PubMed: 27077737
DOI: 10.1371/journal.pone.0153886 -
International Journal of Molecular... May 2022Accelerated glycolysis leads to secretion and accumulation of lactate and protons in the tumor environment and determines the efficacy of adoptive T cell and checkpoint...
Accelerated glycolysis leads to secretion and accumulation of lactate and protons in the tumor environment and determines the efficacy of adoptive T cell and checkpoint inhibition therapy. Here, we analyzed effects of lactic acid on different human CD4 T cell subsets and aimed to increase CD4 T cell resistance towards lactic acid. In all CD4 T cell subsets analyzed, lactic acid inhibited metabolic activity (glycolysis and respiration), cytokine secretion, and cell proliferation. Overexpression of the lactate-metabolizing isoenzyme LDHB increased cell respiration and mitigated lactic acid effects on intracellular cytokine production. Strikingly, LDHB-overexpressing cells preferentially migrated into HCT116 tumor spheroids and displayed higher expression of cytotoxic effector molecules. We conclude, that LDHB overexpression might be a promising strategy to increase the efficacy of adoptive T cell transfer therapy.
Topics: Cell Line, Tumor; Cytokines; Glycolysis; Humans; L-Lactate Dehydrogenase; Lactate Dehydrogenases; Lactic Acid; Neoplasms; T-Lymphocytes
PubMed: 35682650
DOI: 10.3390/ijms23115970 -
Molecular Biology and Evolution Oct 2023Lactate dehydrogenase (LDH, EC.1.1.127) is an important enzyme engaged in the anaerobic metabolism of cells, catalyzing the conversion of pyruvate to lactate and NADH to...
Lactate dehydrogenase (LDH, EC.1.1.127) is an important enzyme engaged in the anaerobic metabolism of cells, catalyzing the conversion of pyruvate to lactate and NADH to NAD+. LDH is a relevant enzyme to investigate structure-function relationships. The present work provides the missing link in our understanding of the evolution of LDHs. This allows to explain (i) the various evolutionary origins of LDHs in eukaryotic cells and their further diversification and (ii) subtle phenotypic modifications with respect to their regulation capacity. We identified a group of cyanobacterial LDHs displaying eukaryotic-like LDH sequence features. The biochemical and structural characterization of Cyanobacterium aponinum LDH, taken as representative, unexpectedly revealed that it displays homotropic and heterotropic activation, typical of an allosteric enzyme, whereas it harbors a long N-terminal extension, a structural feature considered responsible for the lack of allosteric capacity in eukaryotic LDHs. Its crystallographic structure was solved in 2 different configurations typical of the R-active and T-inactive states encountered in allosteric LDHs. Structural comparisons coupled with our evolutionary analyses helped to identify 2 amino acid positions that could have had a major role in the attenuation and extinction of the allosteric activation in eukaryotic LDHs rather than the presence of the N-terminal extension. We tested this hypothesis by site-directed mutagenesis. The resulting C. aponinum LDH mutants displayed reduced allosteric capacity mimicking those encountered in plants and human LDHs. This study provides a new evolutionary scenario of LDHs that unifies descriptions of regulatory properties with structural and mutational patterns of these important enzymes.
Topics: Humans; Lactate Dehydrogenases; L-Lactate Dehydrogenase
PubMed: 37797308
DOI: 10.1093/molbev/msad223 -
MBio Apr 2018Enterococci are important human commensals and significant opportunistic pathogens. Biofilm-related enterococcal infections, such as endocarditis, urinary tract...
Enterococci are important human commensals and significant opportunistic pathogens. Biofilm-related enterococcal infections, such as endocarditis, urinary tract infections, wound and surgical site infections, and medical device-associated infections, often become chronic upon the formation of biofilm. The biofilm matrix establishes properties that distinguish this state from free-living bacterial cells and increase tolerance to antimicrobial interventions. The metabolic versatility of the enterococci is reflected in the diversity and complexity of environments and communities in which they thrive. Understanding metabolic factors governing colonization and persistence in different host niches can reveal factors influencing the transition to biofilm pathogenicity. Here, we report a form of iron-dependent metabolism for where, in the absence of heme, extracellular electron transfer (EET) and increased ATP production augment biofilm growth. We observe alterations in biofilm matrix depth and composition during iron-augmented biofilm growth. We show that the gene encoding l-lactate dehydrogenase is required for iron-augmented energy production and biofilm formation and promotes EET. Bacterial metabolic versatility can often influence the outcome of host-pathogen interactions, yet causes of metabolic shifts are difficult to resolve. The bacterial biofilm matrix provides the structural and functional support that distinguishes this state from free-living bacterial cells. Here, we show that the biofilm matrix can immobilize iron, providing access to this growth-promoting resource which is otherwise inaccessible in the planktonic state. Our data show that in the absence of heme, l-lactate dehydrogenase promotes EET and uses matrix-associated iron to carry out EET. Therefore, the presence of iron within the biofilm matrix leads to enhanced biofilm growth.
Topics: Biofilms; Electron Transport; Energy Metabolism; Enterococcus faecalis; Iron; L-Lactate Dehydrogenase
PubMed: 29636430
DOI: 10.1128/mBio.00626-17 -
Advances in Experimental Medicine and... 2021The Warburg effect, representing enhanced glycolysis and lactate production in adequately oxygenated cancer cells, has been widely regarded to cause increased...
The Warburg effect, representing enhanced glycolysis and lactate production in adequately oxygenated cancer cells, has been widely regarded to cause increased extracellular acidification. Converting pyruvate to lactate by lactate dehydrogenase A (LDHA) is the last step of glycolysis. Here, we report an interesting counterintuitive observation that inhibition of LDHA resulted in enhanced glycolysis in MDA-MB-231 breast cancer cells. The cells were treated with FX11 (7-benzyl-2,3-dihydroxy-6-methyl-4-propylnaphthalene-1-carboxylic acid), a specific LDHA inhibitor. Seahorse assay reported dose-dependent increases in both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Independent biochemical measurements also confirmed the increase of lactate production under FX11 treatment. The reasons and mechanism of these observations of elevated ECAR and lactate production in the MDA-MB-231 breast cancer cells under FX11 treatment remain to be investigated.
Topics: Cell Line, Tumor; Glycolysis; Hydrogen-Ion Concentration; Isoenzymes; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Lactic Acid
PubMed: 33966212
DOI: 10.1007/978-3-030-48238-1_26 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Sep 2023Reducing lactate accumulation has always been a goal of the mammalian cell biotechnology industry. When animal cells are cultured , the accumulation of lactate is mainly...
Reducing lactate accumulation has always been a goal of the mammalian cell biotechnology industry. When animal cells are cultured , the accumulation of lactate is mainly the combined result of two metabolic pathways. On one hand, glucose generates lactate under the function of lactate dehydrogenase A (LDHA); on the other hand, lactate can be oxidized to pyruvate by LDHB or LDHC and re-enter the TCA cycle. This study comprehensively evaluated the effects of LDH manipulation on the growth, metabolism and human adenovirus (HAdV) production of human embryonic kidney 293 (HEK-293) cells, providing a theoretical basis for engineering the lactate metabolism in mammalian cells. By knocking out gene and overexpression of and genes, the metabolic efficiency of HEK-293 cells was effectively improved, and HAdV production was significantly increased. Compared with the control cell, LDH manipulation promoted cell growth, reduced the accumulation of lactate and ammonia, significantly enhanced the efficiency of substrate and energy metabolism of cells, and significantly increased the HAdV production capacity of HEK-293 cells. Among these LDH manipulation measures, gene overexpression performed the best, with the maximum cell density increased by about 38.7%. The yield of lactate to glucose and ammonia to glutamine decreased by 33.8% and 63.3%, respectively; and HAdV titer increased by at least 16 times. In addition, the ATP production rate, ATP/O ratio, ATP/ADP ratio and NADH content of the modified cell lines were increased to varying degrees, and the energy metabolic efficiency was significantly improved.
Topics: Animals; Humans; L-Lactate Dehydrogenase; Lactic Acid; Adenoviruses, Human; Ammonia; HEK293 Cells; Glucose; Adenosine Triphosphate; Kidney; Mammals
PubMed: 37805860
DOI: 10.13345/j.cjb.220893