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Clinical Biochemistry May 2021Lactate dehydrogenase (LDH) is a key enzyme that functions as a marker of cell damage. Its activity can be measured by a variety of laboratory methods. To eliminate...
BACKGROUND
Lactate dehydrogenase (LDH) is a key enzyme that functions as a marker of cell damage. Its activity can be measured by a variety of laboratory methods. To eliminate inter-method bias and enhance equivalence among different measurement procedures, LDH detection needs to be standardized.
METHODS
Optimized sequences coding for lactate dehydrogenase subunit A (LDH-A) and subunit B (LDH-B) were synthesized and cloned into the pRSFDuet vector, and then the constructed 6His-LDHA-pRSFDuet, 6His-LDHB-pRSFDuet, and 6His-LDHA-LDHB-pRSFDuet plasmids were transformed into Escherichia coli BL21 (DE3) for expression. The enzyme activity and specific activity of recombinant LDHs were detected. Electrophoresis of LDH isoenzymes was performed. The stability of recombinant LDHs and serum LDH was evaluated. Commutability of recombinant LDH-B was studied by the IFCC reference method and six routine methods.
RESULTS
Three plasmids were constructed and three highly concentrated recombinant LDH isoenzymes were obtained. The specific activities of LDH-A, LDH-AB, and LDH-B were 18.08 U/mg, 21.74 U/mg, and 14.18 U/mg, respectively. There was a desirable linear correlation between the activities of recombinant LDH isoenzymes and their protein concentrations. Electrophoresis of LDH isoenzymes showed that the recombinant LDH-B corresponded to LDH1 and it demonstrated good stability at 4 °C and 25 °C for 5 weeks. LDH-B formulations in saline-bovine serum albumin solution and human serum matrix were commutable for six routine methods.
CONCLUSION
Human recombinant LDH-B has great potential to become an improved and less expensive standard or reference material in external quality assessment for clinical LDH measurement.
Topics: Clinical Enzyme Tests; Humans; L-Lactate Dehydrogenase; Lactate Dehydrogenases; Recombinant Proteins; Reference Standards
PubMed: 33617848
DOI: 10.1016/j.clinbiochem.2021.02.002 -
ELife Jun 2022Lactate oxidation with NAD as electron acceptor is a highly endergonic reaction. Some anaerobic bacteria overcome the energetic hurdle by flavin-based electron...
Lactate oxidation with NAD as electron acceptor is a highly endergonic reaction. Some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) using a lactate dehydrogenase (Ldh) in concert with the electron-transferring proteins EtfA and EtfB. The electron cryo-microscopically characterized (Ldh-EtfAB) complex of at 2.43 Å resolution consists of a mobile EtfAB shuttle domain located between the rigid central Ldh and the peripheral EtfAB base units. The FADs of Ldh and the EtfAB shuttle domain contact each other thereby forming the D (dehydrogenation-connected) state. The intermediary Glu37 and Glu139 may harmonize the redox potentials between the FADs and the pyruvate/lactate pair crucial for FBEC. By integrating Alphafold2 calculations a plausible novel B (bifurcation-connected) state was obtained allowing electron transfer between the EtfAB base and shuttle FADs. Kinetic analysis of enzyme variants suggests a correlation between NAD binding site and D-to-B-state transition implicating a 75° rotation of the EtfAB shuttle domain. The FBEC inactivity when truncating the ferredoxin domain of EtfA substantiates its role as redox relay. Lactate oxidation in Ldh is assisted by the catalytic base His423 and a metal center. On this basis, a comprehensive catalytic mechanism of the FBEC process was proposed.
Topics: Electron Transport; Electrons; Kinetics; L-Lactate Dehydrogenase; Lactates; NAD; Oxidation-Reduction
PubMed: 35748623
DOI: 10.7554/eLife.77095 -
World Journal of Microbiology &... Nov 2020Lactate are proved to be attractive electron donor for the production of n-caproic acid (CA) that is a high value-added fuel precursor and chemical feedstock, but little...
Lactate are proved to be attractive electron donor for the production of n-caproic acid (CA) that is a high value-added fuel precursor and chemical feedstock, but little is known about molecular mechanism of lactate transformation. In the present study, the gene for L-lactate dehydrogenase (LDH, EC.1.1.1.27) from a Ruminococcaceae strain CPB6 was cloned and expressed in Escherichia coli BL21 (DE3) with plasmid pET28a. The recombinant LDH exhibited molecular weight of 36-38 kDa in SDS-PAGE. The purified LDH was found to have the maximal oxidation activity of 29.6 U/mg from lactate to pyruvate at pH 6.5, and the maximal reduction activity of 10.4 U/mg from pyruvate to lactate at pH 8.5, respectively. Strikingly, its oxidative activity predominates over reductive activity, leading to a 17-fold increase for the utilization of lactate in E. coli/pET28a-LDH than E. coli/pET28a. The CPB6 LDH gene encodes a 315 amino acid protein sharing 42.19% similarity with Clostridium beijerinckii LDH, and lower similarity with LDHs of other organisms. Significant difference were observed between the CPB6 LDH and C. beijerinckii and C. acetobutylicum LDH in the predicted tertiary structure and active center. Further, X-ray crystal structure analysis need to be performed to verify the specific active center of the CPB6 LDH and its role in the conversion of lactate into CA.
Topics: Bacterial Proteins; Cloning, Molecular; Clostridiales; Crystallography, X-Ray; Escherichia coli; Hydrogen-Ion Concentration; L-Lactate Dehydrogenase; Lactic Acid; Models, Molecular; Molecular Weight; Plasmids; Protein Structure, Tertiary; Pyruvic Acid; Recombinant Proteins
PubMed: 33170386
DOI: 10.1007/s11274-020-02958-4 -
Cellular and Molecular Life Sciences :... Jul 2022Once considered a waste product of anaerobic cellular metabolism, lactate has been identified as a critical regulator of tumorigenesis, maintenance, and progression. The...
Targeting lactate dehydrogenase B-dependent mitochondrial metabolism affects tumor initiating cells and inhibits tumorigenesis of non-small cell lung cancer by inducing mtDNA damage.
Once considered a waste product of anaerobic cellular metabolism, lactate has been identified as a critical regulator of tumorigenesis, maintenance, and progression. The putative primary function of lactate dehydrogenase B (LDHB) is to catalyze the conversion of lactate to pyruvate; however, its role in regulating metabolism during tumorigenesis is largely unknown. To determine whether LDHB plays a pivotal role in tumorigenesis, we performed 2D and 3D in vitro experiments, utilized a conventional xenograft tumor model, and developed a novel genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC), in which we combined an LDHB deletion allele with an inducible model of lung adenocarcinoma driven by the concomitant loss of p53 (also known as Trp53) and expression of oncogenic KRAS (G12D) (KP). Here, we show that epithelial-like, tumor-initiating NSCLC cells feature oxidative phosphorylation (OXPHOS) phenotype that is regulated by LDHB-mediated lactate metabolism. We show that silencing of LDHB induces persistent mitochondrial DNA damage, decreases mitochondrial respiratory complex activity and OXPHOS, resulting in reduced levels of mitochondria-dependent metabolites, e.g., TCA intermediates, amino acids, and nucleotides. Inhibition of LDHB dramatically reduced the survival of tumor-initiating cells and sphere formation in vitro, which can be partially restored by nucleotide supplementation. In addition, LDHB silencing reduced tumor initiation and growth of xenograft tumors. Furthermore, we report for the first time that homozygous deletion of LDHB significantly reduced lung tumorigenesis upon the concomitant loss of Tp53 and expression of oncogenic KRAS without considerably affecting the animal's health status, thereby identifying LDHB as a potential target for NSCLC therapy. In conclusion, our study shows for the first time that LDHB is essential for the maintenance of mitochondrial metabolism, especially nucleotide metabolism, demonstrating that LDHB is crucial for the survival and proliferation of NSCLC tumor-initiating cells and tumorigenesis.
Topics: Animals; Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; DNA, Mitochondrial; Homozygote; Humans; Isoenzymes; L-Lactate Dehydrogenase; Lactates; Lung Neoplasms; Mice; Mitochondria; Neoplastic Stem Cells; Nucleotides; Proto-Oncogene Proteins p21(ras); Sequence Deletion
PubMed: 35877003
DOI: 10.1007/s00018-022-04453-5 -
Oncoimmunology 2020Lactate dehydrogenase (LDH) levels are inversely related with response to checkpoint inhibitors. Elevated LDH levels are the product of enhanced glycolytic activity of... (Review)
Review
Lactate dehydrogenase (LDH) levels are inversely related with response to checkpoint inhibitors. Elevated LDH levels are the product of enhanced glycolytic activity of the tumor and tumor necrosis due to hypoxia, the latter being associated with high tumor burden. In this review, we elucidate the effects of glycolysis and hypoxia on antitumor immunity and set forth ways to improve response to immunotherapy in cancer patients with elevated LDH levels. We discuss the current knowledge on combining immunotherapy with glycolysis inhibitors, anti-acidifying drugs, anti-angiogenic or cytoreductive therapy.
Topics: Biomarkers; Glycolysis; Humans; Immunotherapy; L-Lactate Dehydrogenase; Neoplasms
PubMed: 32158624
DOI: 10.1080/2162402X.2020.1731942 -
Biotechnology and Applied Biochemistry Dec 2022This study presents that covalent immobilization technique has been utilized for the immobilization of l-lactate dehydrogenase (l-LDH) from porcine on mesoporous silica....
This study presents that covalent immobilization technique has been utilized for the immobilization of l-lactate dehydrogenase (l-LDH) from porcine on mesoporous silica. To develop mesoporous silica as support material for use in l-LDH immobilization, the particle surfaces were functionalized with 3-aminopropyltrimethoxysilane and further conjugated with glutaraldehyde. The effect of some parameters such as glutaraldehyde concentration, immobilization pH, initial enzyme concentration, and immobilization time was investigated and the optimum conditions for these parameters were determined as 1% (w/v), pH 8.0, 1 mg/ml, and 120 min, respectively. The maximum working pH and temperature for the oxidation of lactate to pyruvate reaction were determined as 10.0 and 35°C for free and 9.0 and 40°C for immobilized l-LDH, respectively. The kinetic parameters (K and V ) of l-LDH for the oxidation of lactate to pyruvate reaction were examined as 1.02 mM and 7.58 U/mg protein for free and 0.635 mM and 1.7 U/mg protein for immobilized l-LDH, respectively. Moreover, the immobilized l-LDH was 1.3-fold more stable than free l-LDH at 25°C according to calculated t values. The immobilized l-LDH retained 80% of its initial activity in a batch reactor after 14 reuses.
Topics: Swine; Animals; Enzymes, Immobilized; Enzyme Stability; L-Lactate Dehydrogenase; Silicon Dioxide; Glutaral; Hydrogen-Ion Concentration; Temperature; Lactates; Kinetics
PubMed: 34962677
DOI: 10.1002/bab.2304 -
Cancer Letters Apr 2024Lactate dehydrogenase A (LDHA) serves as a key regulator of the Warburg Effect by catalyzing the conversion of pyruvate to lactate in the final step of glycolysis. Both...
Lactate dehydrogenase A (LDHA) serves as a key regulator of the Warburg Effect by catalyzing the conversion of pyruvate to lactate in the final step of glycolysis. Both the expression level and enzyme activity of LDHA are upregulated in cancers, however, the underlying mechanism remains incompletely understood. Here, we show that LDHA is post-translationally palmitoylated by ZDHHC9 at cysteine 163, which promotes its enzyme activity, lactate production, and reduces reactive oxygen species (ROS) generation. Replacement of endogenous LDHA with a palmitoylation-deficient mutant leads to reduced pancreatic cancer cell proliferation, increased T-cell infiltration, and limited tumor growth; it also affects pancreatic cancer cell response to chemotherapy. Moreover, LDHA palmitoylation is upregulated in gemcitabine resistant pancreatic cancer cells. Clinically, ZDHHC9 is upregulated in pancreatic cancer and correlated with poor prognoses for patients. Overall, our findings identify ZDHHC9-mediated palmitoylation as a positive regulator of LDHA, with potentially significant implications for cancer etiology and targeted therapy for pancreatic cancer.
Topics: Humans; L-Lactate Dehydrogenase; Lipoylation; Cell Line, Tumor; Lactate Dehydrogenase 5; Pancreatic Neoplasms; Glycolysis; Cell Proliferation; Lactates
PubMed: 38331089
DOI: 10.1016/j.canlet.2024.216696 -
Aging Feb 2023The astrocyte-neuron lactate shuttle hypothesis posits that glial-generated lactate is transported to neurons to fuel metabolic processes required for long-term memory....
The astrocyte-neuron lactate shuttle hypothesis posits that glial-generated lactate is transported to neurons to fuel metabolic processes required for long-term memory. Although studies in vertebrates have revealed that lactate shuttling is important for cognitive function, it is uncertain if this form of metabolic coupling is conserved in invertebrates or is influenced by age. Lactate dehydrogenase (Ldh) is a rate limiting enzyme that interconverts lactate and pyruvate. Here we genetically manipulated expression of lactate dehydrogenase (dLdh) in neurons or glia to assess the impact of altered lactate metabolism on invertebrate aging and long-term courtship memory at different ages. We also assessed survival, negative geotaxis, brain neutral lipids (the core component of lipid droplets) and brain metabolites. Both upregulation and downregulation of dLdh in neurons resulted in decreased survival and memory impairment with age. Glial downregulation of dLdh expression caused age-related memory impairment without altering survival, while upregulated glial dLdh expression lowered survival without disrupting memory. Both neuronal and glial dLdh upregulation increased neutral lipid accumulation. We provide evidence that altered lactate metabolism with age affects the tricarboxylic acid (TCA) cycle, 2-hydroxyglutarate (2HG), and neutral lipid accumulation. Collectively, our findings indicate that the direct alteration of lactate metabolism in either glia or neurons affects memory and survival but only in an age-dependent manner.
Topics: Animals; L-Lactate Dehydrogenase; Drosophila melanogaster; Neuroglia; Neurons; Astrocytes; Memory Disorders; Lactic Acid; Lipids
PubMed: 36849157
DOI: 10.18632/aging.204565 -
Archives of Toxicology Apr 2021A fundamental metabolic feature of cancerous tissues is high glucose consumption. The rate of glucose consumption in a cancer cell can be 10-15 times higher than in... (Review)
Review
A fundamental metabolic feature of cancerous tissues is high glucose consumption. The rate of glucose consumption in a cancer cell can be 10-15 times higher than in normal cells. Isolation and cultivation of tumor cells in vitro highlight properties that are associated with intensive glucose utilization, the presence of minimal oxidative metabolism, an increase in lactate concentrations in the culture medium and a reduced rate of oxygen consumption. Although glycolysis is suggested as a general feature of malignant cells and recently identified as a possible contributing factor to tumor progression, several studies highlight distinct metabolic characteristics in some tumors, including a relative decrease in avidity compared to glucose and/or a glutamine dependency of lactate and even proliferative tumor cells. The aim of this review is to determine the particularities in the energy metabolism of cancer cells, focusing on the main nutritional substrates, such as glucose and glutamine, evaluating lactate dehydrogenase as a potential marker of malignancy and estimating activators and inhibitors in cancer treatment.
Topics: Animals; Carcinogenesis; Citric Acid Cycle; Disease Progression; Energy Metabolism; Glucose; Glutamine; Glycolysis; Humans; L-Lactate Dehydrogenase; Neoplasms; Oxygen Consumption
PubMed: 33649975
DOI: 10.1007/s00204-021-02974-9 -
Chemphyschem : a European Journal of... Mar 2021Parkinson's disease is a neurodegenerative disorder involving a functional protein, α-synuclein, whose primary function is related to vesicle trafficking. However,...
Parkinson's disease is a neurodegenerative disorder involving a functional protein, α-synuclein, whose primary function is related to vesicle trafficking. However, α-synuclein is prone to form aggregates, and these inclusions, known as Lewy bodies, are the hallmark of Parkinson's disease. α-synuclein can alter its conformation and acquire aggregating capacity, forming aggregates containing β-sheets. This protein's pathogenic importance is based on its ability to form oligomers that impair synaptic transmission and neuronal function by increasing membrane permeability and altering homeostasis, generating a deleterious effect over cells. First, we establish that oligomers interfere with the mechanical properties of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane, as demonstrated by nanoindentation curves. In contrast, nanoindentation revealed that the α-synuclein monomer's presence leads to a much more resistant lipid bilayer. Moreover, the oligomers' interaction with cell membranes can promote lactate dehydrogenase (LDH) release, suggesting the activation of cytotoxic events.
Topics: Animals; Cell Line, Tumor; Cell Membrane; Hydrophobic and Hydrophilic Interactions; L-Lactate Dehydrogenase; Lipid Bilayers; Mice; Phosphatidylcholines; Protein Aggregates; Protein Multimerization; alpha-Synuclein
PubMed: 33482036
DOI: 10.1002/cphc.202000831