Lactate Dehydrogenase Synthesis Research Articles

A practical approach for the synthesis of lactate dehydrogenase A inhibitor GNE-140

GNE-140 is the first lactate dehydrogenase A (LDHA) inhibitor that demonstrates both sub-micromolar cellular potency and adequate pharmacokinetic properties, making it suitable for in vivo applications as a tool compound. However, the synthetic route reported by Genentech is complex, relying on transition metal catalysis and yielding low overall amounts. This study presents a practical, scalable method for constructing the core six-membered ring and quaternary carbon center of GNE-140 without transition metal catalysis, achieving a 48.5% racemic yield in a seven-step sequence, paving the way for future LDHA inhibitor development.

Population Pharmacokinetic/Pharmacodynamic Modelling from the Phase I/II COMPOSER Trial to Predict Lowering of Lactate Dehydrogenase in Crovalimab-Treated Patients with Paroxysmal Nocturnal Hemoglobinuria

Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder characterized by intravascular hemolytic anemia and hemoglobinuria. Lactate dehydrogenase (LDH) is a cytoplasmic enzyme that is abundant in red blood cells. Serum LDH level is used as a biomarker for intravascular hemolysis to monitor response to PNH treatment. Crovalimab is a novel antihuman C5 antibody engineered with Sequential Monoclonal Antibody Recycling Technology (SMART-Ig; Fukuzawa et al, Sci Rep. 2017) that is being evaluated as a therapy for PNH in the 4-part, dose-optimization Phase I/II COMPOSER trial (NCT03157635; Röth et al, Blood. 2020), which includes healthy volunteers and patients with PNH who received or did not receive prior C5 inhibition therapy with eculizumab. In this study, COMPOSER data were used to obtain a dose-concentration effect relationship between crovalimab and LDH through development of a pharmacokinetic (PK)-LDH model. Objectives To describe the LDH time course in patients with PNH and to confirm the adequate LDH-lowering effect of the optimized dose and regimen of crovalimab. Study Design and Methods At the clinical data cutoff date of September 4, 2019, data from 15 treatment-naive patients with PNH were used to develop the population PK-LDH model. Observations collected in patients switching treatment from eculizumab to crovalimab (n = 26) were not used to build the PK-LDH model but were used as an external validation tool to evaluate the predictive performance of the model at steady state. A sequential approach was followed (Zhang et al, J Pharmacokinet Pharmacodynam. 2003;30:387-404; Zhang et al, J Pharmacokinet Pharmacodynam. 2003;30:405-416): the individual PK parameters of crovalimab were derived from a population PK model by an empirical Bayesian analysis, and subsequently, the PK-LDH response relationship was modeled with an indirect response model in which LDH synthesis was inhibited by crovalimab concentrations. The predictive performance of the population PK-LDH model was evaluated using a prediction-corrected visual predictive check (pcVPC). The population PK-LDH analysis and all simulations were performed using NONMEM 7.4.0 software. All data sets were analyzed using SAS 9.4. Results For a typical patient, the maximal LDH-lowering effect was estimated to be 80% and the concentration of crovalimab leading to 90% of maximal reduction of LDH synthesis was estimated to be 34.9 μg/mL (90% CI, 15.9-67.5 μg/mL). The half-life for LDH was estimated to be 3 days and steady state for the LDH-lowering effect was predicted to be reached 15 days after first drug intake. The pcVPC in treatment-naive patients showed that the PK-LDH model had good predictive qualities (Figure 1). An external pcVPC was performed in patients who had switched from eculizumab to crovalimab with good predictive qualities, supporting the use of the model for simulation. Using the optimized crovalimab dose and regimen from Part 4, model-based simulations of the LDH time course showed that > 95% of patients were predicted to have LDH levels of ≤ 1.5 × upper limit of normal (ULN) (Figure 2). A sensitivity analysis, to assess the impact of uncertainty around the estimated concentration of crovalimab leading to 90% of maximal reduction of LDH synthesis, found a moderate impact on the predicted LDH-lowering effect of crovalimab, with a small decrease in the percentage of patients predicted to reach an LDH level of < 1.5 × ULN. Conclusions Simulations show a rapid and sustained decrease in LDH levels to < 1.5 × ULN in over 95% of treatment-naive patients with PNH who receive the optimized dose and regimen of crovalimab. The confirmation of the LDH-lowering effect of crovalimab supports further clinical study of the optimized regimen, which is being evaluated in the Phase III studies COMMODORE 1 (NCT04432584) and COMMODORE 2 (NCT04434092). Disclosures Buatois: F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Sostelly:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Soubret:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third-party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Jaminion:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. Cosson:F. Hoffmann-La Roche Ltd: Current Employment, Other: All authors received support for third party writing assistance, furnished by Scott Battle, PhD, provided by F. Hoffmann-La Roche, Basel, Switzerland.. OffLabel Disclosure: Crovalimab is an anti-C5 monoclonal antibody being evaluated as a therapy for paroxysmal nocturnal hemoglobinuria

A study into the safety of novel bioresorbable matrices for repairing bone tissue defects

The work was aimed at conducting a comparative study into the toxicological safety of the obtained highly purified composite material comprising demineralized bone matrix for repairing bone defects. Â To investigate the biological properties of the demineralized bone matrix and the developed highly purified bone matrix, a culture of immortalized fetal bovine lung (LEK) cells was used. The effect of highly purified bone matrix on cells was studied by the method of cell culture in the presence of the preparation. To accomplish the task, many cell culture techniques were used, including the determination of the percentage of cell death, the level of lactate dehydrogenase synthesis, and the level of glucose uptake by cells exposed to different doses of the highly purified demineralized bone matrix. The results of the study indicate that exposure of a cell culture to the demineralized bone matrix at a dose of 2000 mg/l decreased the level of glucose uptake by cells by 18.2% as compared to control. Exposure to the highly purified bone matrix at the same doses yielded 35% glucose uptake by cells. Â Exposure of the LEK cell line to the demineralized bone matrix at a dose of 2000 mg /l led to the cell death rate exceeding that of control by 97%. When exposed to the highly purified demineralized bone matrix, cell death increased by 10.9% compared to control. Exposure of the cell culture to demineralized bone matrix at a dose of 2000 mg /l indicated that the level of enzyme lactate dehydrogenase (LDH) synthesis by cells was only 11% higher than that in control, whereas the level of LDH synthesis induced by exposure to highly purified bone matrix at the same doses constituted 36.9%. Therefore, the highly purified bone matrix has low toxicity on mammalian cells, which suggests a potential use of the product in clinical practice for repairing bone tissue defects.

Renal Cortical Lactate Dehydrogenase: A Useful, Accurate, Quantitative Marker of In Vivo Tubular Injury and Acute Renal Failure

Studies of experimental acute kidney injury (AKI) are critically dependent on having precise methods for assessing the extent of tubular cell death. However, the most widely used techniques either provide indirect assessments (e.g., BUN, creatinine), suffer from the need for semi-quantitative grading (renal histology), or reflect the status of residual viable, not the number of lost, renal tubular cells (e.g., NGAL content). Lactate dehydrogenase (LDH) release is a highly reliable test for assessing degrees of in vitro cell death. However, its utility as an in vivo AKI marker has not been defined. Towards this end, CD-1 mice were subjected to graded renal ischemia (0, 15, 22, 30, 40, or 60 min) or to nephrotoxic (glycerol; maleate) AKI. Sham operated mice, or mice with AKI in the absence of acute tubular necrosis (ureteral obstruction; endotoxemia), served as negative controls. Renal cortical LDH or NGAL levels were assayed 2 or 24 hrs later. Ischemic, glycerol, and maleate-induced AKI were each associated with striking, steep, inverse correlations (r, −0.89) between renal injury severity and renal LDH content. With severe AKI, >65% LDH declines were observed. Corresponding prompt plasma and urinary LDH increases were observed. These observations, coupled with the maintenance of normal cortical LDH mRNA levels, indicated the renal LDH efflux, not decreased LDH synthesis, caused the falling cortical LDH levels. Renal LDH content was well maintained with sham surgery, ureteral obstruction or endotoxemic AKI. In contrast to LDH, renal cortical NGAL levels did not correlate with AKI severity. In sum, the above results indicate that renal cortical LDH assay is a highly accurate quantitative technique for gauging the extent of experimental acute ischemic and toxic renal injury. That it avoids the limitations of more traditional AKI markers implies great potential utility in experimental studies that require precise quantitation of tubule cell death.

Olmesartan Attenuates the Impairment of Endothelial Cells Induced by Oxidized Low Density Lipoprotein through Downregulating Expression of LOX-1

Oxidized low density lipoprotein (ox-LDL) and its receptor, lectin-Like ox-LDL receptor-1 (LOX-1), play important roles in the development of endothelial injuries. Olmesartan can protect endothelial cells from the impairment caused by various pathological stimulations. In the present study we investigated whether olmesartan decreased the impairment of endothelial cells induced by ox-LDL by exerting its effects on LOX-1 both in vitro and in vivo. Incubation of cultured endothelial cells of neonatal rats with ox-LDL for 24 h or infusion of ox-LDL in mice for 3 weeks led to the remarkable impairment of endothelial cells, including increased lactate dehydrogenase synthesis, phosphorylation of p38 mitogen-activated protein kinases (p38 MAPK) and expression of apoptotic genes such as B-cell leukemia/lymphoma 2 (Bcl-2)-associated X protein (Bax) and caspase-3. Simultaneously, the cell vitality and expression of Bcl-2 gene were greatly reduced. All these effects, however, were significantly suppressed by the treatment with olmesartan. Furthermore, ox-LDL promoted up-regulation of LOX-1 expression either in cultured endothelial cells or in the aortas of mice, which was reversed with the administration of olmesartan. Our data indicated that olmesartan may attenuate the impairment of endothelial cell via down-regulation of the increased LOX-1 expression induced by ox-LDL.

Effects of the overexpression of fructose-1,6-bisphosphate aldolase on fermentation pattern and transcription of the genes encoding lactate dehydrogenase and pyruvate formate-lyase in a ruminal bacterium, Streptococcus bovis

Whether fructose-1,6-bisphosphate (FBP) triggers the transcriptional regulation of the gene expression of lactate dehydrogenase (LDH) and pyruvate formate-lyase (PFL) in Streptococcus bovis was examined by constructing a recombinant strain that overexpresses FBP aldolase (FBA). When the recombinant strain was grown on glucose, intracellular FBP was much lower as compared to the parent strain, whereas dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde-3-phosphate (GAP) were slightly higher. Intracellular ATP and ADP were slightly lower, but the NADH/NAD(+) ratio was not different. When glucose was replaced by lactose, a less readily utilized substrate, there was no great difference in FBP, DHAP, GAP, or adenine nucleotides. Overexpression of FBA decreased the level of LDH-mRNA, and increased the level of PFL-mRNA. Consequently, FBP concentration was positively related to the LDH-mRNA level and inversely related to the PFL-mRNA level. On the contrary, DHAP and GAP concentrations were positively related to the PFL-mRNA level and inversely related to the LDH-mRNA level. The levels of these mRNA were proportional to the amounts of corresponding enzymes in cells. As a result, the ratio of formate to lactate produced was increased by the overexpression of FBA. From these results, it could be presumed that FBP is involved in the transcriptional control of LDH and PFL synthesis in S. bovis.

Regulation of fermentation in a ruminal bacterium, Streptococcus bovis, with special reference to rumen acidosis

ABSTRACTWhen high‐starch diets are fed to ruminants, Streptococcus bovis often predominates in the rumen and produces lactate as the major fermentation product. Lactate production by S. bovis is regulated by the activity ratio of lactate dehydrogenase (LDH) to pyruvate formate‐lyase (PFL) in response to an energy supply or the intracellular pH. The activities of LDH and PFL are affected not only by the amounts of enzyme proteins, but also by the concentrations of allosteric effectors, such as fructose‐1, 6‐bisphosphate (FBP) and triose phosphates. Synthesis of LDH and PFL is regulated at the transcriptional level in opposing directions. The inverse relationship between LDH and PFL synthesis amplifies a change in the proportion of lactate in the total fermentation products. A high capacity to regulate lactate production in response to low pH allows S. bovis to grow at low pH and as one of the most acid‐tolerant bacteria among the prevailing ruminal bacteria, S. bovis also has a high capacity to extrude intracellular H+ by an F1F0 H+‐ATPase, which is important for acid tolerance. In addition, S. bovis is able to augment H+‐ATPase synthesis at the transcriptional level in response to low pH. The activities of H+‐ATPase and pyruvate kinase are enhanced by FBP and strongly inhibited by inorganic phosphate (Pi). Intracellular FBP and Pi concentrations affect sugar metabolism in S. bovis through regulation of the expression of catabolic genes and controlling the activity of sugar transport systems. Synthesis of the enzymes involved in sugar transport and fermentation appears to be regulated by the catabolite control protein A (CcpA) –phosphotransferase system (PTS) system.

Regulation of lactate dehydrogenase synthesis in a ruminal bacterium, Streptococcus bovis.

Lactate dehydrogenase (LDH) was purified from three strains of Streptococcus bovis, and the gene ldh was cloned and sequenced. The ldh of S. bovis from a goat (TH1) was different from the ldhs of two other strains from cattle (TH2, JB1) in that Asp(220) was substituted for Glu. Northern blot analysis revealed that the LDH-mRNA of S. bovis was approximately 1.0 kbp, which was transcribed in a monocistronic fashion. When cells were grown at pH 6.9 in a batch culture, the level of ldh transcript decreased as the growth phase changed; from exponential growth to the cessation of growth. The level of ldh transcript was higher in cells grown at pH 4.5 than at pH 6.9. This observation was consistent with the amounts of LDH in cells and the percentages of lactate produced. These results support the hypothesis that S. bovis regulates LDH synthesis at the transcriptional level probably in response to intracellular pH.

The predominant role of surfactants in the modulation of toxicity of detergent products: An in vitro analysis of shampoos

20 shampoos and their component surfactants were studied in vitro using a murine epidermal cell line (HEL/30). Lactate dehydrogenase (LDH) leakage into the cell medium and the inhibition of protein neosynthesis were evaluated as markers of cytotoxicity after 2 hr of exposure. The coefficients of correlation were −0.83 and −0.69 for comparisons between total surfactant content and inhibition of protein synthesis and leakage of LDH, respectively. There was an inverse relationship between the inhibition of protein synthesis and the amphoteric surfactant content of the shampoo. An excellent correlation ( r = 0.94) was found between LDH leakage values and scores from eye irritation tests in vivo on seven of the shampoos. The results obtained suggest that this model allows quantitative evaluation of the cytotoxicity of detergent products (shampoos) and of the contribution of their major components (surfactants) to that toxicity.

Insulin and other stimulants have nonparallel translational effects on protein synthesis.

Isolated pancreatic acini from streptozocin-induced diabetic rats were used to study the role of insulin on the synthesis of specific cellular proteins. When acini were incubated with 0-100 nM insulin for 2 h and then pulsed with [35S]methionine, a dose-dependent increase in [35S]methionine incorporation into total cellular proteins was observed. When acinar cell lysates were subjected to gel electrophoresis, 12 major newly synthesized protein bands were resolved. Insulin (100 nM) increased the incorporation of [35S]methionine into all bands but with significantly different rates, varying from 84 to 216% of control. Next, specific antibodies to amylase, trypsin, ribonuclease, myosin, and lactate dehydrogenase (LDH) were used to evaluate the biosynthesis of known proteins. Insulin stimulated labeled amino acid incorporation into amylase by 148% over control. Insulin stimulated the synthesis of trypsinogen to a similar degree, but ribonuclease synthesis showed a significantly smaller increase of 53% over control. Insulin stimulated myosin and LDH synthesis by 169 and 184%, respectively. A differential pattern of protein synthesis was also observed when acini were treated with two other stimulators of protein synthesis, cholecystokinin and hemin. Both of these stimulators had a reduced effect on ribonuclease synthesis compared with amylase and trypsinogen synthesis but failed to increase myosin synthesis. When the RNAs extracted from control acini and acini treated with 100 nM insulin were translated in vitro, the proteins synthesized were quantitatively similar. This study therefore indicates that insulin has translational effects on acinar protein synthesis, and these effects are nonparallel for various specific acinar cell proteins.

Human tear enzyme changes as indicators of the corneal response to anterior hypoxia.

A non-invasive biochemical technique for quantifying the effects of anterior corneal hypoxia on the in vivo corneal epithelium of the human eye is described. Following short-term exposure of the cornea to low atmospheric oxygen pressures, lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in tears are altered so that the tear LDH/MDH ratio is elevated. The degree of elevation of the ratio and its timing are related to the severity of hypoxia. Possible explanations for the elevation of the tear LDH/MDH ratio include unbinding of intracellular LDH and increased cell membrane permeability. For severe hypoxia, de novo LDH synthesis may also contribute. These changes suggest that control mechanisms within the corneal epithelium may be responding to optimize the efficiency of anaerobic metabolism during conditions of environmental stress, and offer the prospect of a non-invasive technique for monitoring epithelial metabolic stress.

Diurnal variation in human tear enzymes

A fluorometric technique for measuring the activities of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) in human tear fluid is described. Measurement of separate LDH and MDH activities during the day resulted in fluctuating values with no definite pattern. However, a consistent diurnal pattern emerged when the tear LDH MDH ratio was measured for individual tear sample. The tear LDH MDH ratio was highest upon waking, declining soon after to a stable level for the remainder of the day. Evidence for a corneal epithelial origin of tear LDH and MDH is discussed, as well as the ability of the technique to detect differences in the metabolic status of the epithelium between the open-eye and closed-eye environments. Possible causes of the elevation of the tear LDH MDH ratio following lid closure are unbinding of intracellular M-type LDH within epithelial cells and increased cell membrane permeability, both processes occurring in response to reduced cellular energy content under the more hypoxic conditions of lid closure. Although the nature of the present experiment did not allow any conclusive statement, de novo LDH synthesis may also be contributing to the increased tear LDH MDH ratio. Deliberate corneal epithelial surface disruption was found to have no quantifiable effect on the tear LDH MDH ratio.

Drosophila lactate dehydrogenase: developmental aspects.

Partially purified lactate dehydrogenase (LDH) from third-instar larvae displays two bands (one major and one minor) on polyacrylamide gels. Analogous preparations from pupae and adults exhibit three LDH-staining bands (one major and two minor) in a similar pattern. The migration of the major band is similar for larvae, pupae, and adults, while the two minor LDH bands of pupae and adults migrate more slowly than the minor larval band. It has been shown that larval LDH incubated with beta-nicotinamide adenine dinucleotide exhibits two additional minor bands with an electrophoretic mobility similar to that of the minor bands of both pupae and adults. The intensity of the minor larval LDH band (exhibited also by untreated preparations) is drastically reduced. This fact indicates that the life-cycle stage-dependent LDH isozymic distribution is possibly due to a posttranslational effect(s). Highly purified LDH from larvae, pupae, or adults, obtained by an affinity chromatography procedure, displays just one dispersed band, located in the area between the band 5 and the band 6 exhibited by crude extract preparations. These data, in combination with the lack of difference in catalytic properties among enzymes from larvae, pupae, and adults, suggest that LDH synthesis is controlled by the same single structural gene at all developmental stages.

Program of early development in the mammal: Post-transcriptional control of a class of proteins synthesized by mouse oocytes and early embryos

Following fertilization of mouse eggs there is a singular change in the pattern of newly synthesized proteins, as analyzed by high-resolution two-dimensional gel electrophoresis. The synthesis of six polypeptides, designated here as “fertilization proteins” FP1-FP6, is greatly enhanced in one-cell embryos as compared to ovulated eggs. Their synthesis continues for about 36 hr, during which the first cleavage division takes place, and is virtually terminated by the eight-cell stage of development. These six proteins all have apparent molecular weights of approximately 35,000 daltons and isoelectric points ranging from about 6.1 (FP1) to 6.9 (FP6). Peptide mapping of [ 35S]methionine-labeled FP1-FP4 on SDS-polyacrylamide gels revealed very similar electrophoretic patterns of radiolabeled peptides, suggesting that the four polypeptides are structurally related to one another. Although FP1-FP4 are synthesized by mouse oocytes, they represent minor products, with only about 0.2% of the [ 35S]methionine incorporated into oocyte proteins present in FP1-FP4 in growing and fully grown oocytes. This is to be compared with the synthesis of lactate dehydrogenase (LDH) and tubulin (α and β) which are major products, representing as much as 1.8 and 2.0%, respectively, of total protein synthesis in growing and fully grown oocytes. On the other hand, in an mRNA-dependent, cell-free translation system (rabbit reticulocyte lysate) to which RNA prepared from mouse oocytes is added, the incorporation of [ 35S]methionine into FP1, FP2, FP3, or FP4 is comparable to incorporation into either LDH or tubulin. Along with LDH, tubulin, and actin, FP1-FP4 are the major cell-free translation products observed on fluorograms of two-dimensional gels; such is the case using RNA prepared from oocytes at all stages of growth (20 to 85 μm in diameter). Comparisons of [ 35S]methionine incorporation into FP1-FP4, LDH, and tubulin in cultured oocytes and in a cell-free translation system indicate that the synthesis of FP1-FP4 in mouse oocytes is very low relative to levels of mRNA coding for these proteins that are present throughout oocyte growth. The results suggest that expression of FP1-FP4 during oogenesis is under post-transcriptional control, and that following fertilization of mouse eggs there is a marked stimulation of translation of mRNA coding for FP1-FP4. It is possible that the mRNA may have been transcribed from days to weeks earlier during oocyte growth.

Effect of environmental pH on fermentation balance of Lactobacillus bulgaricus.

When Lactobacillus bulgaricus NLS-4 was grown anaerobically in continuous culture with limiting glucose, a shift in the pH of the medium from the acidic to the alkaline range caused this normally homofermentative bacterium to catabolize glucose in a heterofermentative fashion. The change in the nature of the fermentation was accompanied by a decrease in lactate dehydrogenase biosynthesis in alkaline conditions. The lactate dehydrogenase from this organism did not require fructose 1,6-diphosphate or manganese ions (Mn2+) for catalytic activity. Involvement of the phosphoroclastic split in the pyruvate conversion in an alkaline environment was also confirmed. The high lactate dehydrogenase synthesis in acidic medium together with the participation of the phosphoroclastic split under alkaline conditions may explain the shift from homolactic to heterolactic fermentation of L. bulgaricus NLS-4 with the change of environmental pH.

Patterns of synthesis and degradation of lactate dehydrogenase during the cell cycle of burkitts lymphoma cells

1. 1. The pattern of lactate dehydrogenase synthesis during all phases of a synchronous cell cycle was determined by labelling the enzyme with [ 3H]leucine and titrating the amount of LDH present in individual cultures by means of affinity chromatography. 2. 2. Two different labelling techniques were used, both of which supported the hypothesis of a periodic pattern for total LDH synthesis during the cell cycle, with two peaks of synthesis occurring during S phase, one early in S phase and one late, and two other peaks—one occurring during the G 2/M phase and one during the G 1 phase. 3. 3. When the percentage contribution of each of the individual isoenzymes towards total LDH activity during the cell cycle was taken into account, it became evident that the relative specific incorporation of tritium into both LDH-1 and LDH-5 greatly exceeded that of tritium into LDH-2, 3 or 4. 4. 4. Studies on the relative degradation rates of the total soluble protein, total LDH and the individual LDH isoenzymes gave indications that the isoenzymes were degraded extremely rapidly compared to total soluble proteins.

Regulation of lactate hydrogen synthesis in Bacillus subtilis

The regulation of lactate dehydrogenase in Bacillus subtilis was determined under a variety of growth conditions and in mutants blocked in the citric acid cyle. The synthesis of lactate dehydrogenase increased sharply concomitantly upon the exhaustion of glucose from the medium and the onset of the stationay phase. The synthesis of lactate dehydrogenase may be under catabolite repression control. Studies with mutants bloced in the citric acid cylce showed that lactate dehydrogenase is regulated independently of either the oxidative or reductase branches of the cycle. Certain citric acid cycle mutants, e.g., aconitase or succinate dehydrogenase, exhibited very low levels of lactate dehydrogenase while others, e.g., malate dehydrogenase or isocitrate dehydrogenase, showed normal levels. A stage 0 sporulation mutant expressed levels of lactate dehydrogenase more than one-thousand-fold higher than the low group of citric acid cycle mutants. The induction of lactate dehydroganase was shown to be independent of the accumulation of its substrate, pyruvate.

Protein and lactate dehydrogenase levels in Aedes aegypti

Lactate dehydrogenase (LDH) and total protein concentrations of adult female Aedes aegypti were determined following emergence from the pupal cuticle through a period of 20 days, during which time 2 blood meals were taken. Preceding the blood meal, LDH exhibited distinct peaks 5 and 10 days following emergence. LDH activity remained constant for 2 hr following a blood meal, then rose rapidly to a value 10 times pre-blood meal values 7 hr after feeding. This activity then steadily declined to reach pre-blood meal values within 72 hr. This pattern was repeated in mosquitoes given a second blood meal as well as in virgin mosquitoes taking blood, suggesting that the blood meal stimulates either the synthesis or activation of LDH.

Increased Synthesis of Lactate Dehydrogenase "H" Subunit by a Malignant Tumor

Abstract In a patient with a rapidly growing malignant teratoma in the anterior mediastinum, the only biochemical abnormality detected at first was an abnormally high serum lactate dehydrogenase (EC 1.1.1.27) activity, almost 50% of the activity being present in lactate dehydrogenase isoenzyme-1 (LD1). At the time of death, the total lactate dehydrogenase activity had increased still further, 77% of it being in the LD1 fraction. Both the tumor and its hepatic metastases were shown to have a supranormally active LD1 fraction. These patterns appear to be attributable to an increased synthesis of the "H" subunit by the tumor.

Synthesis and Degradation of Rat Liver Lactate Dehydrogenase M4: HYBRIDIZATION IN THE PURIFICATION OF LACTATE DEHYDROGENASE ISOENZYMES

Abstract For the measurement of lactate dehydrogenase (LDH) isoenzyme biosynthesis a new method is presented which is based on the quantitative hybridization of isoenzyme M4 with isoenzyme H4. After reversible dissociation the formation of active LDH M4 was more rapid and occurred to a greater extent than the formation of LDH H4. The formation of hybrid isoenzymes from the two subunits resulted in an intermediate total activity. In the reaction mixture, 1 mm NADH resulted in a quantitative recovery of all isoenzymes. Rat liver proteins were labeled after a single injection of [3H]leucine or in double labeling experiments after injections of [14C]leucine and [3H]leucine. LDH M4 (LDH5) of the liver homogenate was separated from the other isoenzymes by DEAE-Sephadex chromatography. The LDH M4 fraction was subjected to a freeze-thaw cycle in neutral phosphate buffer containing 1 m NaCl and 1 mm NADH. A second DEAE-Sephadex chromatography was performed and the LDH M4 fraction obtained was hybridized with nonradioactive LDH H4 with the freeze-thaw technique. A third chromatography on DEAE-Sephadex was performed. The hybridized LDH M subunits attached to the column were eluted with a NaCl gradient. The radioactivity eluted was localized to the fractions containing LDH HM3, LDH H2M2, and LDH H3M. The radioactivity per unit of LDH M4 activity in the liver homogenate was calculated. Using this technique, it was demonstrated that cycloheximide, but not actinomycin D, inhibited the incorporation of radioactive leucine into LDH M4. This indicates that the synthesis of lactate dehydrogenase is coded by a relatively stable messenger RNA. The half-life of rat liver LDH M4 was found to be 4.1 days. The degradation constant, kd, was 0.173 day-1. The synthesis rate, ks, was calculated to be 84 molecules·cell-1·s-1.