Lactate Dehydrogenase In Rats Research Articles

Anaerobic glycolysis and oxidative stress interrelation in erythrocytes under administration of Cornus mas L. fruit extracts to rats with streptozotocin-induced diabetes mellitus

Background. In diabetes mellitus (DM), analysis of changes in the biochemical profile of erythrocytes is the important stage of complex scientific research to clarify the mechanism of action of medicinal products based on plant raw materials. The fruits of Cornus mas L. are widely known. The biologically active compounds of these fruits show multiple biological effects. However, the effect of the fruit extracts of cornelian cherry on the functional state of erythrocytes in diabetes has not been sufficiently studied. The high glucose concentration in erythrocytes induces various structural and functional changes, which lead to numerous disturbances in their metabolism. Glucose transported into erythrocytes by facilitated diffusion via GLUT2 undergoes catabolic breakdown in anaerobic glycolysis (90 % of all glucose) and pentose phosphate pathway (the rest 10 %). ATP and reduced coenzymes of NADH + H+ and NADPH + H+ formed due to metabolism participate in maintaining the structure of hemoglobin. Enzymes of the antioxidant defense system, which prevent hemoglobin oxidation into methemoglobin, are especially important. Hyperglycemia and the development of oxidative stress in diabetes are the cause of a decrease in the activity of antioxidant enzymes and the accumulation of ligand forms of hemoglobin (HbCO2, MetHb, HbA1c). Therefore, the work aimed to investigate the effect of extracts of red and yellow fruits of Cornus mas L. on the content of end products of the glycolytic breakdown of glucose in erythrocytes and biochemical markers of the antioxidant status of these blood cells in rats with streptozotocin-induced diabetes. Materials and Methods. DM 1 type in animals was induced by intraperitoneal injection of streptozotocin. Experiments were performed on male Wistar rats, who, from the 10th day after diabetes induction, were administered per os extracts of red and yellow fruits of the cornelian cherry and loganic acid obtained from yellow fruits at a dose of 20 mg/kg of body weight for 14 days. On the 24th day of the experiment, the rats were decapitated under ether anesthesia, and blood was taken. The content of pyruvate and lactate (as the end products of anaerobic glycolysis) and L-lactate dehydrogenase activity were determined in plasma and erythrocytes, as well as biochemical markers of the antioxidant status of erythrocytes (activity of superoxide dismutase, catalase and glutathione peroxidase, level of reduced glutathione, TBA-reactive substances, concentration of oxidative modifications of proteins and advanced oxidation protein products). Results. The activity of catalase, superoxide dismutase, glutathione peroxidase and the concentration of reduced glutathione significantly increased against the decrease in the content of oxidative modifications of proteins, advanced oxidation protein products, TBA-reactive substances, pyruvate, L-lactate, and lactate dehydrogenase in rats with DM after administration of the fruit extracts of the cornelian cherry. Noteworthy, these biochemical indicators made it possible to assess the intensity of anaerobic glycolysis and the antioxidant status of blood erythrocytes in streptozotocin diabetes. Conclusions. Extracts of Cornus mas L. fruits might be potential natural drugs for the treatment of metabolic disorders in diabetes, as they have a corrective effect on the catabolic breakdown of glucose and the antioxidant defense system of erythrocytes, preventing the development of oxidative stress. It should be pointed out that the extract of red fruits of cornelian cherry showed the best effect among the studied extracts in normalizing these indicators.

α-Bisabolol abrogates isoproterenol-induced myocardial infarction by inhibiting mitochondrial dysfunction and intrinsic pathway of apoptosis in rats.

Mitochondrial dysfunction plays crucial role in the pathologenesis of myocardial infarction (MI). The present study evaluated the protective effect of α-bisabolol against isoproterenol (ISO)-induced mitochondrial dysfunction and apoptosis in rats. Male albino Wistar rats were pre- and co-treated with intraperitoneal injection of α-bisabolol (25mg/kg body weight) daily for 10days. To induce experimental MI, ISO (85mg/kg body weight) was injected subcutaneously to the rats at an interval of 24h for 2days (9th and 10thday). ISO-induced MI was indicated by the decreased activities of heart creatine kinase and lactate dehydrogenase in rats. ISO administration also enhanced the concentrations of heart mitochondrial lipid peroxidation products and decreased the activities/concentrations of mitochondrial antioxidants, Kreb's cycle dehydrogenases and mitochondrial electron transport chain complexes I, II + III and IV in rats. Furthermore, ISO triggers calcium overload and ATP depletion in the rat's heart mitochondria followed by the mitochondrial cytochrome-C release and the activation of intrinsic pathway of apoptosis by upregulating the myocardial pro-apoptotic Bax, P53, APAF-1, active caspase-3, active caspase-9 and down regulating the expressions of anti-apoptotic Bcl-2. α-Bisabolol pre and co-treatment showed considerable protective effects on all the biochemical and molecular parameters studied. Transmission electron microscopic study and mitochondrial swelling assay confirmed our biochemical and molecular findings. The in vitro study on hydroxyl radical also revealed the potent free radical scavenging activity of α-bisabolol. Thus, α-bisabolol attenuates mitochondrial dysfunction and intrinsic pathway of apoptosis in ISO-induced myocardial infarcted rats.

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

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.

Identification of mono-ubiquitinated LDH-A in skeletal muscle cells exposed to oxidative stress

We previously reported that oxidative stress is associated with unloading-mediated ubiquitination of muscle proteins. To further elucidate the involvement of oxidative stress in ubiquitination, we examined the ubiquitination profile in rat myoblastic L6 cells after treatment with hydrogen peroxide. Hydrogen peroxide induced many ubiquitinated proteins with low molecular masses (less than 60 kDa) as well as high molecular masses (more than 160 kDa). Among them, a 42-kDa-ubiquitinated protein was abundantly accumulated and immediately disappeared after the treatment. Microsequencing revealed that the 42-kDa-protein was identical to the mono-ubiquitinated form of rat lactate dehydrogenase A (LDH-A), and we confirmed that hydrogen peroxide induced the mono-ubiquitination of LDH-A in COS7 cells overexpressing LDH-A and ubiquitin. Under unloading conditions, such as tail-suspension and spaceflight, mono-ubiquitinated LDH was accumulated in gastrocnemius muscle. Interestingly, E-64-d plus pepstatin, lysosomal protease inhibitors, further accumulated mono-ubiquitinated LDH-A in the cells after treatment with hydrogen peroxide, while they did not affect the amount of poly-ubiquitinated LDH. In contrast, epoxomicin, a potent proteasome inhibitor, did not change the amount of mono-ubiquitinated LDH-A in L6 cells treated with hydrogen peroxide, although it significantly increased the amount of poly-ubiquitinated LDH. Our results suggest that oxidative stress induces not only poly-ubiquitination but also mono-ubiquitination of LDH-A, which may be involved in its lysosomal degradation during unloading.

Isolation of Diabetes-Associated Kidney Genes Using Differential Display

Differential Display was used to isolate genes that show transcriptional changes in the kidney during the development of diabetes in the GK rat. Eight candidate diabetes-associated cDNA fragments, CDK1-8, were isolated and characterised. cDNA sequencing and subsequent database analysis revealed that CDK2, 4, 5 and 6 showed no significant sequence similarity to previously reported genes, suggesting that they represent novel genes, whereas CDK 1,3,7 and 8 showed significant similarity with rat lactate dehydrogenase, rat amiloride sensitive sodium channel, EST109013 and mouse ubiquitin-like protein respectively. The differential mRNA expression of CDK1-8 was confirmed using differential screening of slot blots. CDK1, 2, 4 and 8 mRNAs appeared to increase whereas CDK3, 5, 6 and 7 mRNAs decreased in the kidneys of GK rats with increasing hyperglycaemia. The altered renal mRNA expression of these genes in association with increased hyperglycemia in the GK rat suggest that they are candidates for a role in the development of diabetic nephropathy.

Identification of a Silencer Module Which Selectively Represses Cyclic AMP-Responsive Element-Dependent Gene Expression

The cyclic AMP (cAMP)-inducible promoter from the rat lactate dehydrogenase A subunit gene (LDH A) is associated with a distal negative regulatory element (LDH-NRE) that represses inherent basal and cAMP-inducible promoter activity. The element is of dyad symmetry, consisting of a palindromic sequence with two half-sites, 5'-TCTTG-3'. It represses the expression of an LDH A/chloramphenicol acetyltransferase (CAT) reporter gene in a dose-dependent, orientation- and position-independent fashion, suggesting that it is a true silencer element. Uniquely, it selectively represses cAMP-responsive element (CRE)-dependent transcription but has no effect on promoters lacking a CRE sequence. The repressing action of LDH-NRE could be overcome by cotransfection with LDH A/CAT vector oligonucleotides containing either the LDH-NRE or CRE sequence. This suggests that the reversal of repression was caused by the removal of functional active, limiting transacting factors which associate with LDH-NRE as well as with CRE. Gel mobility shift, footprinting, and Southwestern blotting assays demonstrated the presence of a 69-kDa protein with specific binding activity for LDH-NRE. Additionally, gel supershift assays with anti-CREB and anti-Fos antibodies indicate the presence of CREB and Fos or antigenically closely related proteins with the LDH-NRE/protein complex. We suggest that the LDH-NRE and CRE modules functionally interact to achieve negative modulation of cAMP-responsive LDH A transcriptional activity.

Analysis of the rat lactate dehydrogenase A subunit gene promoter/regulatory region.

The rat lactate dehydrogenase (LDH) A subunit gene promoter contains a putative AP-1 binding site at -295/-289 bp, two consensus Sp1 binding sites at -141/-136 bp and -103/-98 bp, and a single copy of a consensus cyclic AMP-responsive element (CRE) at -48 to -41 bp upstream of the transcription initiation site. Additionally, an as yet unidentified silencer element is located within the -1173/-830 bp 5'-flanking region. Transient transfection analyses of a -1173/+25 bp LDH A-chLoramphenicol acetyltransferase fusion gene has indicated a complete inability of the promoter fragment to direct basal or forskolin-induced transcription. Deletion of the -1173/-830 bp sequence restored basal and cyclic AMP (cAMP)-inducible activity. Point mutations in the Sp1 binding sites of a -830/+25 bp promoter fragment reduced basal but not the relative degree of cAMP-inducible activity. cAMP-regulated transcriptional activity was dependent upon an 8 bp CRE, -TGACGTCA-, located at the -48/-41 bp upstream region. Mutations in the CRE abolished cAMP-mediated induction and reduced basal activity by about 65%. The CRE binds a 47 kDa protein which has previously been identified as CRE binding protein (CREB)-327, an isoform of the activating transcription factor/CREB transcription factor gene family. Co-transfection of a vector that expresses the catalytic subunit of cAMP-dependent protein kinase stimulates LDH A subunit promoter activity suggesting that cAMP induces LDH A subunit gene expression through phosphorylative modification of CREB-327. This study emphasizes a fundamental role of several modules including Sp1 and CREB binding sites in regulating basal and cAMP-mediated transcriptional activity of the LDH A gene.

Rat lactate dehydrogenase A and B subunit concentrations are not regulated by mRNA abundance in liver and heart

RNA was isolated from rat liver and heart tissues at various times up to 12 weeks after birth, and probed on slot blots with lactate dehydrogenase A and B cDNA probes. Although the relative abundances of LDH A in liver and LDH B in heart increased substantially in the 12 weeks after birth, mRNAs for both isoenzymes remained remarkably stable in both tissues over the same period. The implications of these observations for the regulation of constitutive gene expression are discussed.

Purification and radioimmunoassay of rat lactate dehydrogenase A and B subunits.

We have developed procedures for purifying lactate dehydrogenase isoenzymes from rat tissues that involve two affinity-chromatography steps and that facilitate the isolation of milligram quantities of highly purified proteins within 2--3 days. Antibodies raised against pure A and B subunits in rabbits and hens were used in radioimmunoassays and showed negligible cross-reactivity with heterologous subunits. The radioimmunoassays provide a sensitive method for measuring nanogram amounts of A-subunit and B-subunit polypeptides in tissue homogenates and were employed to characterize the enzyme purification procedures.

Separation of rat lactate dehydrogenase isoenzyme C4 from other isoenzymes by affinity and ion-exchange chromatography

Lactate dehydrogenase C, an isoenzyme composed of C polypeptide subunits and found only in mature testes and spermatozoa, differs kinetically, chemically and immunologically from the five common isoenzymes of lactate dehydrogenase, each of which is a tetramer of A and/or B subunits. In the rat lactate dehydrogenase C exists in two molecular forms, isoenzymes C4 and A1C3. In addition to these two forms of lactate dehydrogenase C, rat testicular homogenate contains all the five isoenzymes of A and B type. Purification of isoenzyme C4 requires its separation from the other six isoenzymes, of which isoenzymes A1C3 and A3B1 are the most difficult ones to separate. In the present study isoenzyme A3B1, along with other enzymes, was separated from isoenzyme C4 by AMP-Sepharose chromatography by using a gradient of increasing concentration of NAD+-pyruvate adduct. In the next step, isoenzyme A1C3 was separated from isoenzyme C4 by DEAD-cellulose chromatography, resulting in a pure lactate dehydrogenase isoenzyme C4 preparation.

Effects of lead, copper, and zinc on the rat's lactate dehydrogenase in vivo and in vitro.

Following subacute intoxication of rats with Pb-, Cu-, and Zn-salts (separately or in mixture) for 5 weeks, the chelating agent D-penicillamine was administered for 3 weeks. In the course of the 3-month experiment, lactate dehydrogenase (LDH) was estimated in serum and in cytoplasmic fraction of the kidney. Pb2+ treatment resulted in an increase of LDH activity, Cu2+ in a slight decrease, whereas Zn2+ had no effect, respectively. Mixture of these metals caused a significant rise in the enzymatic activity. Seven weeks after the stoppage of the administration of toxic substances, altered LDH activity, both in serum and in kidney returned to normal. D-penicillamine treatment was found to accelerate a restoration of the enzyme activity. In the experiments in vitro, Cu2+ inhibited significantly the kidney LDH activity, Pb2+ and Zn2+ being 100- and 400-times less efficient, respectively. Cu2+ inhibition was reversed by D-penicillamine, whereas inhibition of LDH by Zn2+ or Pb2+ was irreversible.

Evidence for the lack of feedback regulation of gene activity and for the absence of subunit exchange between lactate dehydrogenase tetramers in vivo.

Frog and rat lactate dehydrogenase (LDH, L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) isozymes, highly purfied by affinity chromatography, were injected into newly fertilized from (Rana pipiens) eggs to study the effect of the injected isozymes on the expression of the LDH genes of the developing frog embryo. From these experiments three conclusions can be drawn: (i) homologous and heterologous LDH, even when present in more than twice the normal amount of LDH, does not play any role in the switching on or off of the frog LDH genes or in regulating the level of gene activity. No evidence was found in support of any feedback regulation of LDH synthesis by the LDH molecule itself. (ii) Injected rat LDH is very stable in the frog embryo, and the contrary to what can be demonstrated in vitro, all the isozymes show the same stability. No evidence was found for selective degradation of any of the injected five isozymes. (iii) The fact that the injected isozymes disappear 12 days after the injection without forming hybrid molecules between themselves or with the stored or newly synthesized frog LDH is evidence in favor of the hypothesis that the tetramer is the degradative unit of LDH, not the monomer as others have postulated. The LDH tetramers do not dissociate the recombine under the physiological conditions present in frog cells.