LDH Isozymes Research Articles

P176 - LDH isozyme as a prognostic factor for patients with metastatic clear cell renal cell carcinoma (mCRCC)

P176 - LDH isozyme as a prognostic factor for patients with metastatic clear cell renal cell carcinoma (mCRCC)

251P LDH isozyme as a prognostic factor for patients with metastatic clear cell renal cell carcinoma (mCRCC)

251P LDH isozyme as a prognostic factor for patients with metastatic clear cell renal cell carcinoma (mCRCC)

Triple isozyme LDH Inhibition in breast cancer cells does not alter energy production but negatively regulates cell proliferation independent of the absence of lactic acid

Several aggressive human malignant tumors are characterized by an intense glycolytic rate, over‐expression of lactic acid dehydrogenase A (LDHA), and subsequent lactate accumulation, all of which contribute toward an acidic peri‐cellular immunosuppressive tumor microenvironment (TME). While recent focus has been directed at how to inhibit LDHA, it is now becoming clear that the multiple isozymes of LDH must simultaneously be inhibited to suppress lactic acid and halt glycolysis. In this study, we explore the biochemical and genomic consequences of the administration of triple LDH isozyme inhibitor (A, B &C) (GNE‐140) in MDA‐MB‐231 triple‐negative breast cancer cells (TNBC) cells. The findings confirm that GNE‐140 does, in fact, fully block the production of lactic acid, which also results in a block of glucose utilization and severe impedance of the glycolytic pathway. Without a fully functional glycolytic pathway, breast cancer cells continue to thrive, sustain viability, produce ample energy, and maintain mitochondrial potential (ΔΨM). The only observable negative consequence of GNE‐140 in this study was the attenuation of cell division, evident in both 2D and 3D cultures and occurring in fully viable cells. Of important note, the cytostatic effects were not reversible by the addition of exogenous lactic acid. The effects of GNE‐140 on the whole transcriptome were mild (12 up‐regulated differential expressed genes (DEGs) / 77 down‐regulated DEGs) out of the 48,226 evaluated, and downregulated DEGS collectively centered around a loss of genes related to mitosis, cell cycle, G0/G1; G1/S transition, and DNA replication. These data were observed by digital fluorescence cytometry and flow cytometry, both corroborating a G0/G1 phase blockage. In conclusion, the findings in this work suggest that there is an unknown element linking LDH enzyme activity to the cell cycle and that this factor is completely independent of lactic acid. The data also establish that complete inhibition of LDH in cancer cells does not affect cell viability or energy production.

Positive effect of Periostin on repair of Isopreternol induced ischemic damaged cardiomyocyte: an in vitro model.

IntroductionOne of the diseases in developed counties is myocardial infarction that causes the death of many people. A Problem of many new drugs usage concerns their assessment for applying therapeutically for heart disease. Previous studies used animal models but today many researchers tend to apply cellular models for feasibility of cellular model production and application.MethodsFor this purpose, we differentiated human bone marrow mesenchymal stem cells (hBM-MDCs) into cardiomyocyte, then induced damage into cells by Isoproterenol, and finally we assayed repair of cardiomyocyte by Periostin. Damage induction and repair were confirmed by measurement of selected markers for cardiac damage and repair at mRNA level. In addition, we measured LDH activity in culture medium during damage and repair processes.ResultsOur results showed LDHa and b mRNA levels increased and also cardiac markers decreased during damage of cardiomyocytes significantly. Reciprocally LDH isozymes decreased and cardiac markers increased during repair of cardiomyocytes.Discussion/conclusionThese alterations in cardiac markers after Periostin treatment demonstrate that Periostin is an effective factor on repair of cardiomyocytes. LDH activity in culture medium decreases after damage induction and increases during repair process. According to our data, Isoproterenol and Periostin are good inducer to produce damaged and repaired differentiated cardiomyocytes respectively.

Ldh isozymes- It's relation with infertility

Ldh isozymes- It's relation with infertility

Triple Isozyme Lactic Acid Dehydrogenase Inhibition in Fully Viable MDA-MB-231 Cells Induces Cytostatic Effects That Are Not Reversed by Exogenous Lactic Acid.

A number of aggressive human malignant tumors are characterized by an intensified glycolytic rate, over-expression of lactic acid dehydrogenase A (LDHA), and subsequent lactate accumulation, all of which contribute toward an acidic peri-cellular immunosuppressive tumor microenvironment (TME). While recent focus has been directed at how to inhibit LDHA, it is now becoming clear that multiple isozymes of LDH must be simultaneously inhibited in order to fully suppress lactic acid and halt glycolysis. In this work we explore the biochemical and genomic consequences of an applied triple LDH isozyme inhibitor (A, B, and C) (GNE-140) in MDA-MB-231 triple-negative breast cancer cells (TNBC) cells. The findings confirm that GNE-140 does in fact, fully block the production of lactic acid, which also results in a block of glucose utilization and severe impedance of the glycolytic pathway. Without a fully functional glycolytic pathway, breast cancer cells continue to thrive, sustain viability, produce ample energy, and maintain mitochondrial potential (ΔΨM). The only observable negative consequence of GNE-140 in this work, was the attenuation of cell division, evident in both 2D and 3D cultures and occurring in fully viable cells. Of important note, the cytostatic effects were not reversed by the addition of exogenous (+) lactic acid. While the effects of GNE-140 on the whole transcriptome were mild (12 up-regulated differential expressed genes (DEGs); 77 down-regulated DEGs) out of the 48,226 evaluated, the down-regulated DEGS collectively centered around a loss of genes related to mitosis, cell cycle, GO/G1–G1/S transition, and DNA replication. These data were also observed with digital florescence cytometry and flow cytometry, both corroborating a G0/G1 phase blockage. In conclusion, the findings in this work suggest there is an unknown element linking LDH enzyme activity to cell cycle progression, and this factor is completely independent of lactic acid. The data also establish that complete inhibition of LDH in cancer cells is not a detriment to cell viability or basic production of energy.

Lactate Dehydrogenase Inhibition: Biochemical Relevance and Therapeutical Potential.

Lactate dehydrogenase (LHD) is a key enzyme of anaerobic metabolism in almost all living organisms and it is also a functional checkpoint for glucose restoration during gluconeogenesis and single-stranded DNA metabolism. This enzyme has a well preserved structure during evolution and among the species, with little, but sometimes very useful, changes in the amino acid sequence, which makes it an attractive target for the design and construction of functional molecules able to modulate its catalytic potential and expression. Research has focused mainly on the selection of modulator especially as far as LDH isozymes (especially LDH-5) and lactate dehydrogenases of Plasmodium falciparum (pfLDH) are concerned. This review summarizes the recent advances in the design and development of inhibitors, pointing out their specificity and therapeutic potentials.

조류 및 포유류 내 미토콘드리아 젖산탈수소효소 동위효소들의 분포와 역할

조류 및 포유류에서 미토콘드리아를 분리하고 모노아민산화효소(EC 1.4.3.4, monoamine oxidase)의 활성을 측정하여 미토콘드리아의 분리도를 확인하였다. 미토콘드리아 각 분획에서 젖산탈수소효소(EC 1.1.1.27, lactate dehydrogenase, LDH)의 발현도를 생화학적 및 면역화학적 방법으로 분석하였다. 조류에 비하여 포유류에서는 미토콘드리아 LDH의 활성이 낮은 것으로 나타났다. 따라서 미토콘드리아 LDH의 역할은 포유류보다 조류에서 중요시된다고 생각된다. 조류 및 포유류 모든 조직의 단백질양은 세포기질보다 미토콘드리아에서 적게 나타났다. 생쥐와 골든햄스터의 세포기질에서는 LDH <TEX>$A_4$</TEX>, <TEX>$A_3B$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$AB_3$</TEX> 및 <TEX>$B_4$</TEX> 다섯 가지 동위효소 이외에 testis-specific LDH <TEX>$C_4$</TEX> 동위효소가 정소 조직에서 나타났다. 닭의 미토콘드리아 내에서는 AB hybrid형 동위효소 한 종류가 나타났다. 포유류에서 미토콘드리아 LDH 동위효소들은 조직 별로 다르게 나타났다. 생쥐의 미토콘드리아 내에서는 LDH <TEX>$A_4$</TEX> 및 testis-specific <TEX>$C_4$</TEX> 동위효소가 나타났다. 미토콘드리아 testis-specific LDH <TEX>$C_4$</TEX> 동위효소는 생쥐에서만 나타났다. 골든햄스터 미토콘드리아 내에서는 LDH <TEX>$B_4$</TEX> 동위효소가 lactate oxidase로서 작용하였다. 조류 및 포유류의 대사조건 및 서식환경에 따라 미토콘드리아 LDH 동위효소의 역할도 구분되는 것으로 생각된다. Mitochondria were isolated from bird and mammals. The activity of monoamine oxidase (EC 1.4.3.4) was then measured to identify mitochondrial isolation. Lactate dehydrogenase (EC 1.1.1.27, lactate dehydrogenase, LDH) isozymes in mitochondrial fractions were analyzed by biochemical and immunochemical methods. The activity of mitochondrial LDH was lower in mammals than in bird. Therefore, the role of mitochondrial LDH seems to be more important in bird than in mammals. The concentration of protein in all tissues of bird and mammals was less in the mitochondria than in the cytosol. In the cytosol of mice and golden hamsters, testis-specific LDH <TEX>$C_4$</TEX> isozyme was expressed in testis in addition to the LDH <TEX>$A_4$</TEX>, <TEX>$A_3B$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$AB_3$</TEX>, and <TEX>$B_4$</TEX> isozymes. A single LDH AB hybrid isozyme was expressed in the chicken mitochondria. In mammals, mitochondrial LDH isozymes were differed according to tissues. LDH <TEX>$A_4$</TEX> and testis-specific LDH <TEX>$C_4$</TEX> isozymes were expressed in the mitochondria of mice. The mitochondrial testis-specific LDH <TEX>$C_4$</TEX> isozyme was expressed only in the mice. In the golden hamster mitochondria, the LDH <TEX>$B_4$</TEX> isozyme functioned as a lactate oxidase. As our results show, the mitochondrial LDH seemed to be playing the different role in the bird and mammals in relation with their metabolic conditions and habitats.

Activities of cytochrome c oxidase and mitochondrial lactate dehydrogenase isozymes and Cox1, Cox2, Cox4, and Cox6 gene subunit expression in cold adaptation of Salmo trutta L.

Characteristic changes in some parameters of white muscle mitochondria (mitochondrial volume, activity of cytochrome c oxidase (COX, EC 1.9.3.1) and the level of Cox1, Cox2, Cox4, and Cox6 subunit gene expression and activity and kinetic characteristics of mitochondrial lactate dehydrogenase isozymes (mtLDH, EC 1.1.1.27)) in adaptation to seasonal decrease in temperature from 16 to 6°C of one-year juvenile brown trout Salmo trutta L. from rivers of Lake Onega basin were investigated. A 1.5-fold increase in the activity of COX, and the increase in the levels of gene expression of both Cox4 and Cox6 nuclear subunits and increased activity of mitochondrial LDH isozymes, which have a low affinity towards lactate, has been shown. The possible role of nuclear and mitochondrial subunits of cytochrome c oxidase in improving the efficiency of the enzyme in molecule biogenesis and further modulation of its activity, as well as regulation of pyruvate formation to maintain the required rate of oxidative phosphorylation in mitochondria under low temperature were discussed.

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

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 can adapt to the hypoxia environment.

Abstract 87: Urinary LDH activities in patients with Kawasaki disease

Background: Kawasaki disease (KD) is a systemic vasculitis with unknown origin. KD patients often have leukocytes in urine as well as urinary tract infection (UTI). The aim of this study is to evaluate urinary LDH and its isozyme, which is useful in diagnosis with UTI, in patients with KD. Patients and methods: 158 patients with KD (including 4 with incomplete KD) and 52 patients with febrile sickness were enrolled as control group in this study. Urine samples were obtained at least one point. Results: Total level of urinary LDH in patients with KD was significantly higher than that in control group. Urinary LDH level in the acute phase was significantly higher than that with convalescent phase in patients with KD. Urinary LDH isozyme showed predominant elevations of LDH1 and LDH2. Some patients showed the elevation of urinary LDH level before the clinical criteria of KD were fulfilled. There was no correlation between urinary LDH concentration and pyuria. Conclusion: The levels of LDH isozyme (LDH4 and LDH5) in urine is known to increase in patients with acute upper urinary tract infection, while the level of its isozyme (LDH1 and LDH2) increased in acute phase of KD. The elevated levels of total urinary LDH and its isozyme (LDH1 and LDH2) may be useful for early diagnosis of KD and also for diagnosis of incomplete KD.

Sources of variation analysis and derivation of reference intervals for ALP, LDH, and amylase isozymes using sera from the Asian multicenter study on reference values

BackgroundSources of variation (SV) of ALP, LDH, and amylase isozymes were explored. MethodsWe analyzed 3511 sera from well-defined healthy individuals recruited during the 2009 Asian project for derivation of common reference intervals (RIs). Up-to-date electrophoresis auto-analyzer and reagents were employed for high resolution and reproducibility. SVs including sex, age, body mass index (BMI), ABO blood groups, and levels of drinking, smoking, and exercise were analyzed by multiple regression analysis. RIs were determined by parametric methods after refining healthy individuals by use of latent reference values exclusion method. ResultsAge-related changes in ALP2–3 were different in females: ALP2, linear increase from 20–64y; ALP3, lowering until 45 y and rising steeply thereafter. ALP2 increased with BMI especially in females. ALP5 was barely detectable except in blood-types O and B. Age-related increases in LDH1–LDH3 were noted in females, whereas BMI-related increases were found only for LDH2–LDH5 in both sexes. Pancreatic amylase showed age-related increase in females and was slightly higher in blood-type O. RIs for absolute and relative activities of each isozyme were derived in consideration of sex and age. ConclusionsInvestigation of these isozymes revealed various age-, BMI-, and blood-type-related changes that are all relevant in clinical interpretation of enzyme test results.

큰입우럭(Micropterus salmoides) 조직의 젖산탈수소효소 및 Monocarboxylate 수송체(MCT) 1, 2, 4

The properties of lactate dehydrogenase (EC 1.1.1.27, LDH) and expression of monocarboxylate transporters (MCTs) 1, 2, and 4 were studied in tissues from Micropterus salmoides. Native-PAGE revealed that the LDH isozyme was predominantly located in skeletal muscle. The LDH , , and isozymes were detected in heart, liver, eye, and brain tissues, while eye-specific isozyme was detected in eye tissue. In September, strong LDH isozyme activity was detected in heart tissue. High isozyme activity was noted in all other tissues except heart tissue. However, in November, strong isozyme activity was detected in heart tissue. The LDH/CS (Citrate synthase, EC 4.1.3.7) ratio in skeletal muscle and heart tissues indicated that anaerobic metabolism was high in those tissues. Native-PAGE after immunoprecipitation showed that eye-specific isozyme was more similar to the than the isozyme. The LDH isozyme was purified by affinity chromatography. The molecular weight of subunit A was 37,200. The LDH activity in tissues was consistently 11.05~28.32% due to inhibition by 10 mM pyruvate. The of LDH in eye tissue was very low. The optimum pH for LDH in tissues was pH 7.5~8.0. The LDH isozyme was detected in mitochondria of skeletal muscle, whereas the and isozymes were detected in heart tissue mitochondria. Western blot analysis indicated that MCTs 1, 2, and 4 were located in the plasma membrane and mitochondria of skeletal muscle and heart tissues. The sizes of MCTs 1, 2, and 4 in skeletal muscle were 60, 54~38, and 63 kDa, while those in heart tissue were 57, 54~38, and 55.5 kDa, respectively. In conclusion, M. salmoides appears to use anaerobic metabolism predominantly when adapted to a hypoxic environment. In highly activated skeletal muscle and heart tissue, energy production is controlled by inward and outward flows of pyruvate and lactate through MCTs 1, 2, and 4 in the plasma membrane and mitochondria, with effective adjustment by LDH isozymes.

저항성훈련이 흰쥐 골격근의 GLUT-4 단백질 및 LDH 동위효소 발현에 미치는 영향

The purpose of the present study was to investigate the effect of climbing resistance training on GLUT-4 protein and LDH isozyme activities of the soleus and gastrocnemius muscles in rats. Each experimental group was randomly divided into a control group (n

쥐노래미 eye-specific LDH C4동위효소의 정제 및 특성

Eye-specific lactate dehydrogenase (EC 1.1.1.27, LDH) isozyme in the eyes of greenlings (Hexagrammos otakii) was successfully purified by affinity chromatography and continuous-elution electrophoresis. The molecular weight of the purified eye-specific LDH isozyme was 154.8 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimal pH for enzymatic reaction of the eye-specific LDH isozyme was pH 8.5. value of the purified eye-specific LDH isozyme was M using pyruvate as a substrate. These results indicate that we must consider pH when measuring eye-specific LDH isozyme activity. The eye-specific LDH isozyme had a higher binding affinity for the substrate as a pyruvate than LDH A4 isozyme. Antibodies produced against the purified eye-specific LDH isozyme may be used in the diagnosis of several human diseases and in comparative physiological studies of fishes.

Molecular Characterization and Expression of LDHA and LDHB mRNA in Testes of Japanese Quail (Coturnix japonica)

The LDH isozymes are key catalysts in the glycolytic pathway of energy metabolism. It is well known that the distribution of the LDH isozymes vary in accordance with the metabolic requirements of different tissues. The substrates required for energy production change noticeably at successive stages of testes development suggesting a significant flexibility in the expression of glycolytic enzymes. Therefore, expression of LHDA and LDHB mRNAs was examined in adult and prepubertal quail testis. The mRNA of both LDHA and LDHB were expressed and no significant difference was observed in prepubertal testes. The mRNA levels of LDHB significantly increased during testicular development. In the adult testis, LDHA mRNA was not expressed. Expression studies revealed the presence of different LDH isozymes during testicular development. In contrast, electrophoresis of both testicular samples revealed only single band at a position indicative of an extreme type of LDH isozyme in quail testes. Furthermore, nucleotide and amino acid sequence analysis revealed significant similarity to chicken, duck and rock pigeon. These sequence results confirmed the similarity of LDHA and LDHB subunit protein in different avian species.

Oxamic acid analogues as LDH-C4-specific competitive inhibitors

We performed kinetic studies to determine whether oxamate analogues are selective inhibitors of LDH-C4, owing to their potential usefulness in fertility control and treatment of some cancers. These substances were shown to be competitive inhibitors of LDH isozymes and are able to discriminate among subtle differences that differentiate the active sites of LDH-A4, LDH-B4 and LDH-C4. N-Ethyl oxamate was the most potent inhibitor showing the highest affinity for LDH-C4. However, N-propyl oxamate was the most selective inhibitor showing a high degree of selectivity towards LDH-C4. Non-polar four carbon atoms chains, linear or branched, dramatically diminished the affinity and selectivity towards LDH-C4. N-Propyl oxamate significantly reduced ATP levels, capacitation and mouse sperm motility, in line with results shown by others, suggesting that LDH-C4 plays an essential role in mouse fertility.

Molecular cloning, characterization, and immunolocalization of two lactate dehydrogenase homologous genes from Taenia solium

Two novel genes encoding lactate dehydrogenase A (LDHA) and B (LDHB) homologues, respectively, were identified from the cDNA libraries of adult Taenia solium (T. solium). The two deduced amino acid sequences both show more than 50% identity to the homologues for Danio rerio, Xenopus laevis, Schistosoma japonicum, Sus scrofa, Homo sapiens, et al. The identity of the amino acid sequence between TsLDHA and TsLDHB is 57.4%, and that of the nucleotide sequence is 61.5%. Recombinant TsLDHA homologue (rTsLDHA) and TsLDHB homologue (rTsLDHB) were expressed in Escherichia coli BL21/DE3 and purified. Though there were some differences in the sequence, the two LDH isozyme homologues show similarity in the conserved LDH domain, topological structure, primary immunological traits, localization on the tegument of T. solium adult, and partial physicochemical properties. The linear B-cell epitope analysis of TsLDHA and TsLDHB discovered a TsLDHA specific epitope. The purified rTsLDHA and rTsLDHB could be recognized by rat immuno-sera, serum from swine, or a patient infected with T. solium, respectively, but Western blot analysis showed cross-reactions, not only between these two LDH members but also with other common human tapeworms or helminths. The results suggested that the two LDH homologues are similar in the characteristics of LDH family, and they are not specific antigens for immunodiagnosis.

급격한 환경변화에 대한 angelfish (Pterophyllum scalare) 젖산탈수소효소 동위효소의 변화

열대 저산소 환경에 적응되어 있는 angelfish (Pterophyllum scalare)를 급격한 온도변화(<TEX>$27{\pm}0.5{\rightarrow}18{\pm}0.5^{\circ}C$</TEX>) 및 DO 변화(<TEX>$6{\pm}1{\rightarrow}18\;ppm$</TEX>)에 2시간 동안 적응시킨 후 젖산탈수소효소(EC 1.1.1.27, lactate dehydrogenase, LDH) 동위효소의 특성 및 유전자발현을 연구하였다. LDH 동위효소의 특성은 native-polyacrylamide gel 전기영동, Western blot 분석 및 효소활성 측정으로 확인하였다. 전기영동 결과 liver- 및 eye-specific Ldh-C 유전자는 간, 눈 및 뇌 조직에서 발현되었다. Western blot 분석 결과 LDH <TEX>$A_4$</TEX> 동위효소는 <TEX>$B_4$</TEX> 동위효소보다 음극 쪽에 나타났다. 간 조직에서 온도 저하 시 LDH <TEX>$A_4$</TEX> 동위효소가 증가하고 <TEX>$B_4$</TEX> 동위효소는 감소하였으며, DO 증가 시 LDH <TEX>$A_4$</TEX> 및 <TEX>$C_4$</TEX> 동위효소가 증가하고 <TEX>$B_4$</TEX> 동위효소는 감소하였다. 눈 조직에서는 온도 저하 시 LDH <TEX>$A_4$</TEX> 및 <TEX>$B_4$</TEX> 동위효소가 증가하고 <TEX>$C_4$</TEX> 동위효소는 감소하였으며, DO 증가 시 LDH <TEX>$A_4$</TEX> 및 <TEX>$B_4$</TEX> 동위효소는 증가하지만 <TEX>$C_4$</TEX> 동위효소 및 하부단위체 C를 포함하는 동위효소는 감소하였다. 심장 조직에서는 DO 증가 시 LDH 활성이 증가하였고, LDH <TEX>$B_4$</TEX> 동위효소가 증가하였다. 뇌 조직에서는 온도 저하 시 LDH <TEX>$A_4$</TEX> 및 <TEX>$B_4$</TEX> 동위효소가 증가하였고, DO 증가 시 LDH <TEX>$B_4$</TEX> 동위효소는 증가하였다. 따라서 liver- 및 eye-specific Ldh-C는 DO 변화에 의해 영향을 받으며 간 및 눈 조직에서 LDH <TEX>$B_4$</TEX> 및 <TEX>$C_4$</TEX> 동위효소는 서로 상대적으로 조절되므로 <TEX>$C_4$</TEX> 동위효소는 lactate oxidase로서 기능을 나타내는 것으로 사료된다. In this study, the properties and gene expression of the lactate dehydrogenase (EC 1.1.1.27, LDH) isozyme were studied in angelfish (Pterophyllum scalare) - known for their adaptation to the low oxygen environment of the tropics - which were acclimated to acute temperature change (<TEX>$27{\pm}0.5{\rightarrow}18{\pm}0.5^{\circ}C$</TEX>) and dissolved oxygen (DO) change (<TEX>$6{\pm}1{\rightarrow}18\;ppm$</TEX>) for 2 hours. The properties of the LDH isozymes were confirmed in the native-polyacrylamide gel electrophoresis, Western blot analysis and enzyme activity measurement. Liver- and eye-specific Ldh-C gene were expressed in liver, eye and brain tissues. Through Western blot analysis, the LDH <TEX>$A_4$</TEX> isozyme was shown to have a more cathodal mobility relative to the <TEX>$B_4$</TEX> isozyme. In the liver tissue, the LDH <TEX>$A_4$</TEX> isozyme increased with temperature drop while the <TEX>$B_4$</TEX> isozyme decreased. The LDH <TEX>$A_4$</TEX> and <TEX>$C_4$</TEX> isozymes increased with DO increment, while the <TEX>$B_4$</TEX> isozyme decreased. In the eye tissue, the LDH <TEX>$A_4$</TEX> and B4 isozymse increased with temperature drop while the <TEX>$B_4$</TEX> isozyme decreased. The LDH <TEX>$A_4$</TEX> and <TEX>$B_4$</TEX> isozymes increased with DO increment, but the <TEX>$C_4$</TEX> isozyme and isozymes including the subunit C decreased. In the heart tissue, LDH activity increased with DO increment, as well as the LDH <TEX>$B_4$</TEX> isozyme. In the brain tissue, the LDH <TEX>$A_4$</TEX> and <TEX>$B_4$</TEX> isozymes increased with temperature drop. The LDH <TEX>$B_4$</TEX> isozyme increased with DO increment. Accordingly, since the liver- and eye-specific Ldh-C are influenced by changes in DO and the LDH <TEX>$B_4$</TEX> and <TEX>$C_4$</TEX> isozymes are relatively controlled in the liver and eye tissues, the <TEX>$C_4$</TEX> isozyme can be considered to have a lactate oxidase function.

가물치(Channa argus) 젖산탈수소효소 동위효소들의 정제 및 특성

가물치(Channa argus) 조직의 젖산탈수소효소 동위효소(EC 1.1.1.27, lactate dehydrogenase, LDH)를 정제하고 생화학적, 면역학적 및 역학적 방법으로 특성을 연구하였다. LDH 활성은 골격근이 380.4 units로 가장 높고 심장 13.4, 눈 3.5, 뇌 조직 5.4 units이었으며, 심장의 CS 활성은 20.7 unit로 가장 높고, LDH/CS는 골격근 172.9, 심장 0.6, 눈 0.32, 뇌 0.47이고, 단백질 양은 골격근 14.7 mg/g이며, 특이활성(units/mg)은 골격근 25.88, 심장 0.79, 눈 0.31, 뇌 1.38 units/mg이었으므로 골격근은 혐기적이고, 심장은 호기적이었다. LDH <TEX>$A_4$</TEX>, <TEX>$B_4$</TEX>, eye-specific <TEX>$C_4$</TEX>에 대한 항혈청을 사용한 Western blot, 면역침강반응 및 native-polyacrylamide gel electrophoresis에 의해 <TEX>$A_4$</TEX>, <TEX>$A_3B$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$AB_3$</TEX> 및 <TEX>$B_4$</TEX>가 모든 조직에서 확인되었고, 눈 조직에서 <TEX>$C_4$</TEX>와 <TEX>$AC_3$</TEX>, <TEX>$A_2C_2$</TEX>, <TEX>$AC_3$</TEX>, 뇌 조직에서 <TEX>$A_3C$</TEX>도 확인되었다. LDH <TEX>$A_4$</TEX>, <TEX>$A_3B$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$AB_3$</TEX>, <TEX>$B_4$</TEX>, eye-specific <TEX>$C_4$</TEX> 동위효소는 affinity chromatography와 Preparative PAGE Cell에 의해 정제되었다. LDH <TEX>$A_4$</TEX> 동위효소는 <TEX>$NAD^+$</TEX> 유입 후 정제되었고, eye-specific <TEX>$C_4$</TEX>는 <TEX>$A_4$</TEX>에 이어 용출되기 시작하였으며 <TEX>$B_4$</TEX>는 buffer 유입 후 용출되었다. 정제한 결과 <TEX>$A_4$</TEX>는 <TEX>$B_4$</TEX> 및 eye-specific <TEX>$C_4$</TEX>와 분자구조의 일부가 유사하였지만 <TEX>$B_4$</TEX>와 <TEX>$C_4$</TEX>는 서로 다른 것으로 나타났으므로, 하부단위체 A는 보존적이고, 하부단위체 B는 A보다 더 빠르게 진화된 것으로 보인다. 피루브산 10 mM에서 <TEX>$A_4$</TEX> 동위효소 39.98%, <TEX>$A_2B_2$</TEX> 21.28%, <TEX>$B_4$</TEX> 19.67% 및 eye-specific <TEX>$C_4$</TEX> 16.87%의 활성이 남아있었고, 피루브산에 대한 <TEX>$Km^{PYR}$</TEX>은 <TEX>$A_4$</TEX> 0.17 mM, <TEX>$B_4$</TEX> 0.27 mM, eye-specific <TEX>$C_4$</TEX> 0.133 mM였다. <TEX>$A_4$</TEX>, <TEX>$B_4$</TEX>, eye-specific <TEX>$C_4$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$A_3B$</TEX> 및 <TEX>$AB_3$</TEX>의 최적 pH는 각각 pH 6.50, pH 8.5, pH 5.5, pH 6.0-6.5, 5.0 및 pH 7.5였고, 동질사량체 <TEX>$A_4$</TEX>와 이질사량체 동위효소들은 넓은 pH 영역에서 안정하였다. 특히 골격근은 LDH 활성이 크므로 활동성이 크며, 눈조직에서 피루브산 친화력이 강한 eye-specific <TEX>$C_4$</TEX>에 의해 피루브산 대사가 빠르게 일어나고, 이어서 <TEX>$A_4$</TEX>에 의해 젖산이 산화되어지는 것으로 사료되므로, 종의 생태환경 및 먹이 획득 양식에 따라 LDH-C 발현, 기질에 대한 친화도 및 대사 시간이 다른 것으로 사료된다. The lactate dehydrogenase (EC 1.1.1.27, LDH) isozymes in tissues from Channa argus were purified and characterized by biochemical, immunochemical and kinetic methods. The activity of LDH in skeletal muscle was the highest at 380.4 units and those in heart, eye and brain tissues were 13.4, 3,5 and 5.4 units, respectively. Citrate synthase (EC 4.1.3.7, CS) activity in heart tissue was the highest at 20.7 units. LDH/CS in skeletal muscle, heart, eye and brain tissues were 172.9, 0.6, 0.32 and 0.47. Protein concentration in skeletal muscle tissue was 14.7 mg/g and specific activities of LDH in skeletal muscle, heart, eye and brain tissues were 25.88, 0.79, 0.31 and 1.38 units/mg, respectively. Therefore, skeletal muscle tissue was anaerobic and heart tissue was aerobic. The LDH isozymes in tissues were identified by polyacrylamide gel electrophoresis, immunoprecipitation and Western blot with antiserum against <TEX>$A_4$</TEX>, <TEX>$B_4$</TEX>, and eye-specific <TEX>$C_4$</TEX>. LDH <TEX>$A_4$</TEX>, <TEX>$A_3B$</TEX>, <TEX>$A_2B_2$</TEX>. <TEX>$AB_3$</TEX> and <TEX>$B_4$</TEX> isozymes were detected in every tissue, <TEX>$C_4$</TEX>, <TEX>$AC_3$</TEX>, <TEX>$A_2C_2$</TEX> and <TEX>$A_3C$</TEX> were detected in eye tissue, and <TEX>$A_3C$</TEX> was found in brain tissue. LDH <TEX>$A_4$</TEX>, <TEX>$A_3B$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$AB_3$</TEX>, <TEX>$B_4$</TEX>, eye-specific <TEX>$C_4$</TEX> isozymes were purified by affinity chromatography and Preparative PAGE Cells. The LDH <TEX>$A_4$</TEX> isozyme was purified in the fraction from elution with <TEX>$NAD^+$</TEX> containing buffer of affinity chromatography. Eye-specific <TEX>$C_4$</TEX> isozyme was eluted right after <TEX>$A_4$</TEX>, after which <TEX>$B_4$</TEX> isozyme was eluted with plain buffer. As a result, one part of molecular structures in <TEX>$A_4$</TEX>, <TEX>$B_4$</TEX> and eye-specific <TEX>$C_4$</TEX> were similar, but were different from each other in <TEX>$B_4$</TEX> and <TEX>$C_4$</TEX>. Therefore the subunit A may be conservative in evolution, and the evolution of subunit B seems to be faster than that of subunit A. The activity of LDH <TEX>$A_4$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$B_4$</TEX>, and eye-specific <TEX>$C_4$</TEX> isozymes remained at 39.98, 21.28, 19.67 and 16.87% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The <TEX>$Km^{PYR}$</TEX> values were 0.17, 0.27 and 0.133 mM in <TEX>$A_4$</TEX>, <TEX>$B_4$</TEX> and eye-specific <TEX>$C_4$</TEX> isozymes, respectively. The optimum pH of LDH <TEX>$A_4$</TEX>, <TEX>$B_4$</TEX>, eye-specific <TEX>$C_4$</TEX>, <TEX>$A_2B_2$</TEX>, <TEX>$A_3B$</TEX>, and <TEX>$AB_3$</TEX> were pH 6.5, pH 8.5, pH 5.5, pH 6.0-6.5, pH 5.0 and pH 7.5. The <TEX>$A_4$</TEX> and heterotetramer isozymes stabilized a broad range of pH. Especially, LDH activities in skeletal muscle tissue were high, resulting in a high degree of muscle activity.LDH metabolism in eye tissue seems to be converted faster from pyruvate to lactate by eye-specific <TEX>$C_4$</TEX> isozyme as eye-specific <TEX>$C_4$</TEX> have the highest affinity for pyruvate, and right after the conversion, oxidation of lactate was induced by <TEX>$A_4$</TEX> isozyme. It was found that expression of Ldh-C, affinity to substrate and reaction time of <TEX>$C_4$</TEX> isozyme were different according to the ecological environmental and feeding capturing patterns.