LDH Isozymes Research Articles

Kinetic properties of lactate dehydrogenase from trout muscle

The kinetics of pyruvate reduction by LDH isozymes in the presence of NADH is said to be an ordered bisubstrate reaction characterized by Michaelian concentration dependence (Borgmann et al., 1975). If the reaction is ordered, no cooperativity can occur between the two substrates (NADH and pyruvate) (Neet, 1995). Experiments with fish LDH showed, however, that this enzyme might display two types of saturation profile, hyperbolic and sigmoidal, depending on the pH in the reaction medium. At pH 9, for example, the initial velocity of pyruvate reduction by epaxial muscle LDH from goldfish showed a sigmoidal dependence upon substrate concentration, whereas at pH 8 the kinetics were of the hyperbolic type (Hochachka, 1965). Our experiments showed, in addition, that LDH from trout muscle (Onchorhynchus mykiss) could display both types of saturation profile at one and the same pH, but in the presence of different concentrations of the LDH preparation. Rainbow trout (Onchorhyncus mykiss) were obtained in October from the Goc Fishpond of the School of Forestry, University of Belgrade, Serbia. White skeletal muscle was immediately excised and kept in liquid nitrogen until use. After thawing, muscle samples were deprived of skin and spine, cut into small pieces, and homogenized in 5 volumes (w/v) of Tris-EDTA buffer. The homogenate was centrifuged for 10 min at 10 000× g. Partial purification with ammonium sulfate was performed according to Place and Powers, 1984. After centrifugation, solid ammonium sulfate was added to the supernatant to give 30% saturation at 0oC. The suspension was stirred for 1 hour and centrifuged for 30 min at 10 000× g. The pellet was discarded, and the supernatant brought to 70% saturation. The suspension was then stirred again for 1 h at 0oC, and centrifuged as already described. This time, the supernatant was discarded and the pellet redissolved in Tris-EDTA buffer and dialyzed overnight at 4oC against four 1liter replacements of the same buffer. The concentration of protein, determined by the method of Lowry (Lowry et al., 1951), amounted to 5.3 mg/ml of the final enzyme suspension.

Molecular characterization and expression pattern of the equine lactate dehydrogenase A and B genes

The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226, and eLDHB lost negatively charged Glu 14, as compared to the highly conserved residues at these positions in the corresponding amino acid sequences of other mammals. These alterations were identified in six equine species by genomic DNA analysis. A comparison of the equine and human 3D structures revealed that the substituted His 88 and 226 of the eLDHA monomer and the deleted Glu 14 of the eLDHB monomer altered the surface charge of equine LDH tetramers and that these three residues were located in important regions affecting the catalytic kinetics. Also, RT-PCR amplification of the three myosin heavy chain isoforms corroborated that the cervical muscle as postural muscle of the thoroughbred horse was composed of more oxidative myofibers than the dynamic muscle. Based on this property, the mRNA expression patterns of eLDHA, eLDHB, and eGAPDH in various tissues were analyzed by using real-time PCR. The expression levels of these three genes in the cervical muscle were not always relatively higher than in the brain or heart.

Lactate dehydrogenase activity of the rat testis treated with cyproterone acetate during fetal life.

Histological changes, total LDH activity and LDH isozyme patterns of testicular tissues of rats, exposed to cyproterone acetate during fetal life, were studied at different days after birth. While such treated testes revealed all stages of spermatogenesis and normal patterns of LDH isozymes including LDH-X bands, complete loss of isozymes was discernible in the Leydig cell fraction. It is probable that this abnormality in the Leydig cell is a consequence of impairment in the hypothalamo-hypophyseal axis or due to improper scrotal development following CA exposure.

쏘가리(Siniperca scherzeri) 젖산탈수소효소 A4동위효소의 정제 및 특성

The lactate dehydrogenase (EC 1.1.1.27, LDH) isozyme in skeletal muscle of mandrin fish (Siniperca scherzeri) was successfully purified by affinity chromatography and ultrafiltration. The molecular weight of the purified LDH isozyme was 140.4 kDa and its isoelectric point (pI) was 7.0. Optimal pH for enzymatic reaction was 7.5. and value of the purified LDH isozyme were M and 13.31 mM/min using pyruvate as a substrate, respectively. These kinetic properties of the purified LDH isozyme supported the fact that the mandrin fish was a warm-adapted species. The antibody against the purified LDH isozyme may be used in the metabolic physiological studies of ectothermic vertebrates and in the diagnosis of several human diseases.

Regression of Dalton’s lymphoma in vivo via decline in lactate dehydrogenase and induction of apoptosis by a ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide as ligand

A novel ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide (Ru(II)-CNEB) was found to interact with and inhibit M4-lactate dehydrogenase (M4-LDH), a tumor growth supportive enzyme, at the tissue level. The present article describes modulation of M4-LDH by this compound in a T-cell lymphoma (Dalton's Lymphoma: DL) vis a vis regression of the tumor in vivo. The compound showed a dose dependent cytotoxicity to DL cells in vitro. When a non toxic dose (10 mg/kg bw i.p.) of Ru(II)-CNEB was administered to DL bearing mice, it also produced a significant decline in DL cell viability in vivo. The DL cells from Ru(II)-CNEB treated DL mice showed a significant decline in the level of M4-LDH with a concomitant release of this protein in the cell free ascitic fluid. A significant increase of nuclear DNA fragmentation in DL cells from Ru(II)-CNEB treated DL mice also coincided with the release of mitochondrial cytochrome c in those DL cells. Importantly, neither blood based biochemical markers of liver damage nor the normal patterns of LDH isozymes in other tissues were affected due to the treatment of DL mice with the compound. These results were also comparable with the effects of cisplatin (an anticancer drug) observed simultaneously on DL mice. The findings suggest that Ru(II)-CNEB is able to regress Dalton's lymphoma in vivo via declining M4-LDH and inducing mitochondrial dysfunction-apoptosis pathway without producing any toxicity to the normal tissues.

小児白血病における血清乳酸脱水素酵素アイソザイム(LDH Isozyme)の臨床的意義について

小児白血病における血清乳酸脱水素酵素アイソザイム(LDH Isozyme)の臨床的意義について

Evolutionary Implications of Lactate Dehydrogenases (LDHs) of Hagfishes Compared to Lampreys: LDH cDNA Sequences from Eptatretus burgeri, Paramyxine atami and Eptatretus okinoseanus

Heart muscles of hagfishes Paramyxine atami and Eptatretus okinoseanus express the B4 isozyme of lactate dehydrogenase [L-LDH: NAD oxidoreductase, EC 1.1.1.27] (LDH-B4) whereas their skeletal muscles express LDH-A4. To examine the relationship of hagfish LDHs to lamprey and other vertebrate LDHs, we determined the cDNA sequences of LDH-A from three hagfishes and compared them with previously published sequences. A phylogenic tree shows that hagfishes diverged just after lampreys. The deduced amino acid sequences showed ten regions common to all vertebrate LDHs examined, i.e., the active site, the pocket recognizing the substrate-coenzyme complex, part of a loop at the surface, and the substrate binding site. The cyclostomate-specific regions (S1, S2) were located in the neighborhood of the active site loop. Three regions, IGS1, IGS2 and IGS3, seem to have altered their structures during the differentiation of LDH isozymes, and the regions remain in LDH-B of vertebrates hitherto examined. IGS2 and IGS3, which are in the neighborhood of the active site, may regulate catalytic activity. There were differences in six amino acid residues (6, 10, 20, 156, 269, and 341) in LDHs of hagfishes. These differences might reflect the tolerance to high pressure and low temperature of LDHs from hagfishes at different habitat depths.

풀망둑(Acanthogobius hasta) 젖산탈수소효소의 특성

The lactate dehydrogenase (EC 1.1.1.27, LDH) isozymes in tissues from Acanthogobius hasta were characterized by biochemical, immunochemical and kinetic methods. The activities of LDH in skeletal muscle and eye tissues were 65.30 and 53.25 units, but LDH activities in heart and liver tissues were very low. LDH/CS (EC 4.1.3.7, citrate synthase) in skeletal muscle was the highest as 22.29. Specific activities of LDH in brain, eye and skeletal muscle were 56.45, 38.04 and 11.0 units/mg, respectively. The LDH isozymes in tissues were separated by polyacrylamide gel electrophoresis after immunoprecipitation with antiserum against eye-specific and liver-specific . LDH isozymes were detected predominantly in skeletal muscle, brain and eye tissues, and isozyme was detected in heart. Anodal eye-specific and cathodal liver-specific were coexpressed in A. hasta. The eye-specific isozyme showed higher activity in eye tissue, but liver-specific isozyme showed lower activity in liver. As a result, one part of molecular structures in , and eye-specific and liver-specific were similar, but in were different with each other. 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 LDH isozyme of skeletal muscle was purified in the fraction from elution with NAD+ containing buffer of affinity chromatography and eye-specific isozyme was eluted right after , so the structure of eye-specific isozyme is similar to . And LDH activity remained 35.22-43.47% as a result of the inhibition by pyruvate, the Michaelis-Menten constant values for pyruvate was 0.080-0.098 mM, and Vmax were 153.85 units, 35.09 units in skeletal muscle and eye, respectively. Also the isozyme was the thermo-stablest and was stabler than isozyme. The optimum pH of LDH was 6.5. The results mentioned above indicate that isozymes in tissues showed the properties between LDH isozyme as A. hasta was adapted to hypoxic conditions. Also LDH seems to function more effectively under anaerobic condition because LDH in skeletal muscle and eye tissues have high affinity for pyruvate.

Serum Total LDH Activity and LDH-2 Isozyme in Nephrotic Syndrome

Background/Aims: Proteinuria, hypoproteinaemia, hypoalbuminaemia and oedema are major characteristics of nephrotic syndrome. Aims of this study were to detect serum total LDH activity and its isozymes in nephrotic syndrome. Methods: In a cross-sectional study, clinical parameters were compared in three cohorts, namely kidney patients with or without nephrotic syndrome and hypoalbuminaemic controls (NEPHR, NON-NEPHR, CONTR, respectively). Results: Serum total LDH activity in the NEPHR group was increased compared with the NON-NEPHR and CONTR groups (p < 0.001) and correlated with serum total protein (r = –0.549, p < 0.001), serum albumin (r = –0.596, p < 0.001), proteinuria (r = 0.456, p < 0.001) and serum total cholesterol (r = 0.523, p < 0.001). LDH isozyme pattern was analysed in three subgroups of the patients. Serum LDH-2 activity was higher in the NEPHR subgroup compared with the NON-NEPHR and CONTR subgroups (p < 0.001). Serum LDH-2 activity correlated with serum total protein (r = –0.665, p < 0.001), serum albumin (r = –0.615, p < 0.001), proteinuria (r = 0.694, p < 0.001), and serum total cholesterol (r = 0.723, p < 0.001). Linear regression analysis revealed that serum total protein proved to be an independent predictor of serum total LDH activity, while serum total protein and proteinuria were predictors of LDH-2. Conclusions: These findings suggest that serum total LDH activity might be a marker of the activity of the nephrotic syndrome.

Elevated level of serum LDH2 and LDH3 in sputum three positive TB patients of Sahariya Tribe: A preliminary study

Objectives: The objective of this investigation was to find out if sputum-positive (AFB test) test, which is performed to assess mycobacterial infection status, is anyway correlated with any of the LDH isoforms. And if so, can it be used, either alone or together with sputum test, as a rapid on-the-spot marker for field diagnosis of tuberculosis. Design and methods: To analyze the relationship between sputum test results and the level of LDH isozyme (isoforms), 157 individuals were randomly selected from a Sahariya tribal population, with a known history of tuberculosis, for sputum and blood collection. The Ziehl–Neelsen’s staining of sputum smear was done as per RNTCP (Revised National Tuberculosis Control Programme) protocol. In all the samples, serum LDH level was estimated spectrophotometrically while the levels of individual isoforms were assessed on native PAGE. Results: The LDH content was significantly higher in blood sera of sputum-positive (three positive) individuals (444 ± 270 IU) as compared to sputum-negative samples (242 ± 125 IU). Analysis on the association of different LDH isoforms (LDH1, 2, 3, 4 and 5) with sputum test revealed significantly higher frequency of LDH2 and LDH3 in sputum-positive samples as compared to sputum-negative samples. In sputum three positive cases, however, the frequency of LDH3 appeared much higher (in 60% cases) than LDH2 (in 33% cases), which was found to be almost same in sputum negative but higher in two positive samples (44.4%). Conclusions: The present data suggests a strong association of LDH3 with sputum three positive or severe cases of mycobacterial infection, indicating a likely possibility of using LDH3 as a supporting diagnostic marker in, at least, cases of chronic tuberculosis.

RCE1 Corneal Epithelial Cell Line: Its Variability on Phenotype Expression and Differential Response to Growth Factors

By serial transfer of rabbit corneal epithelial cells, the spontaneous RCE1 cell line was previously established. These cells mimic the stage-dependent differentiation of the corresponding cell type. RCE1 cells were cultured either on plastic culture dishes or on collagen rafts to compare the epithelial ultrastructure after growth on these substrata. Phenotypic variability was studied after subcloning of cells. The differentiation ability of each subclone was determined by Western blot with antibodies against the differentiation-linked keratin pair K3/K12 and by measuring LDH activity and LDH isozymes in cytosolic extracts. The proliferative response of RCE1 cells to EGF, TGFalpha, amphiregulin, bFGF or IL-6 was determined under serum-free culture conditions. Cells grown on collagen rafts formed 5- to 7-layered epithelia with characteristics closer to those found in normal corneal epithelium than cells cultivated on plastic substrata, which formed 3- to 5-layered epithelia. Subcloning experiments demonstrated that every proliferative cell is able to grow and constitute stratified epithelia expressing K3/K12 keratins. LDH levels in RCE1 epithelia were similar to those of cultured or freshly harvested corneal epithelia; however, they showed a slightly altered LDH isozyme set, with prevalence of LDH-3 isoform. Whereas EGF and TGF-alpha were equipotent, amphiregulin elicited a 4-fold lower proliferative response. Also, bFGF was 10-fold less mitogenic than EGF, and IL-6 had the lowest effect with an ED(50) 20-fold lower than EGF. The results demonstrate that every RCE1 proliferative cell has the ability to generate epithelial sheets. We conclude that EGF and TGF-alpha are the major effectors of RCE1 cell proliferation.

ASSESMENT OF BIOACTIVITY OF A FISCHERELLA SPECIES COLONIZING AZADIRACHTA INDICA (NEEM) BARK

In order to investigate whether a. Fischerella sp. colonizing Azadirachta indica (neem) bark is able to show, like neem derived extracts/products, bioactivity with respect to glucose metabolizing potential in animal models, laboratory cultivated neem colonized cyanobacterial biomass was fed to mice and its effects on liver lactate dehydrogenase (LDH: a key enzyme of glucose metabolism) vis a vis blood glucose level was monitored. Neem bark extract and purified azadirachtin (a bioactive neem product) were also tested simultaneously for comparison. As compared to the control, none of the samples could produce much variation in blood glucose levels except a transitory decline, 34% and 26% at 2 nd & 12 th h, in azadirachtin and Fischerella (isolated from neem) fed mice respectively. However, all the test samples showed significant increase in liver LDH activity after 2nd h of treatment (p< 0.01) followed by a decline at 6th h. Thereafter, the enzyme activity increased progressively up to 24 th h in mice fed with azadirachtin and neem colonized Fischerella only (p < 0.001 at 24 th h). Expression pattern of the enzyme was also studied using polyacrylamide gel electrophoresis. Liver specific M4-LDH (homo-tetramer of M sub-unit) was detected in all the cases and time-dependent changes in expression of the enzyme paralleled the alterations observed in its activities. To ascertain whether such a bioactivity is common with Fischerella sp. colonizing other trees also, liver LDH profile was compared between mice fed with Fischerella isolated from neem and jamun (Eugenia jambolana) barks separately. Though Fischerella from jamun did not alter liver LDH profile, Fischerella isolated from neem could produce significant increase in the enzyme activity (p < 0.001) and corresponding changes in the expression of LDH isozymes. The results suggest that Fischerella sp. inhabiting neem bark, like bioactivity of its host plant products, modulates activity and expression of liver LDH in mice.

Effects Of Clenbuterol On MyoD, UCP3, MCT1, CD147, And Myosin-hc Isoforms Of Rat Skeletal Muscles

Clenbuterol is one of the beta-2 adrenergic receptor agonists with powerful muscle anabolic and lipolytic effects and is prohibited to use as doping drug for athletes. Since previously we have reported that clenbuterol induced the muscular hypertrophy, the possible decreased endurance capacity by the decreased expression of lactate transporter (MCT1 and MCT4) in rat skeletal muscles, and the transition of muscle fiber type from slow to fast-type (JAP 91(1), 85–90, 2001). However, the mechanism of these changes with clenbuterol was not clear yet. PURPOSE This study was performed to examine the effects of clenbuterol on the molecular adaptation system of muscle. METHODS In the present study, clenbuterol was subcutaneously administered in 8wks female Sprague-Dawley rats (CLEB: n=8) during 4 weeks. The effects of clenbuterol on slow-twitch soleus and fast-twitch EDL muscles were examined using reverse-transcription polymerase chain reaction (RT-PCR) analysis of the mRNAs of MCT1, MyoD, CD147, and UCP3. Furtermore, the effects of CLEB on proteins were examined using polyacrylamide gel (PAGE) systems of non-denatured PAGE for LDH isozymes and denatured SDS-PAGE for the both of Myosin HCs, MCT1, and CD147. RESULTS The muscle wet weights increased in both soleus and EDL muscles. The MyoD and UCP3 mRNAs increased drastically in SOL. They may explained the accelelated higher lipolytic function in soleus muscle by the accumulated fast-twitch fibers with fiber type transition from slow-to-fast. Furthermore, the increased MyoD mRNA may explain the muscle hypertrophy in soleus, but no changes of MyoD mRNA in EDL may need another explanation for the hypertrophy like as autocrine of IGF-1. The contents of MCT1 and its mRNA decreased in EDL and they tended to decrease in SOL. The contents of CD147 significantly decreased in EDL, but the CD147 mRNA showed no change in both SOL and EDL. In EDL, the decreased MCT1 might be explained by the decreased CD147 which is important to translocate MCT1 to plasma membrane. The difference of these changes between the proteins and mRNAs might be due to the posttranslational modification. These findings suggest that clenbuterol is able to induce lactate accumulation easily and to decrease the endurance capacity. CONCLUSIONS These results suggest that the clenbuterol induced many chages on skeletal muscles via positive feedback mechanism of MyoD in myogenesis as well as via cAMP of beta-2 adrenergic receptor system. It is necessary to carry out more detail experiments to explain the specific adaptation mechanism. Supported by A grant-in-aid (No. 15500437 to T.K.) from the Ministry of Education, Science, Sports, and Culture of Japan.

Effects Of Clenbuterol On MyoD, UCP3, MCT1, CD147, And Myosin-hc Isoforms Of Rat Skeletal Muscles

Clenbuterol is one of the beta-2 adrenergic receptor agonists with powerful muscle anabolic and lipolytic effects and is prohibited to use as doping drug for athletes. Since previously we have reported that clenbuterol induced the muscular hypertrophy, the possible decreased endurance capacity by the decreased expression of lactate transporter (MCT1 and MCT4) in rat skeletal muscles, and the transition of muscle fiber type from slow to fast-type (JAP 91(1), 85–90, 2001). However, the mechanism of these changes with clenbuterol was not clear yet. PURPOSE This study was performed to examine the effects of clenbuterol on the molecular adaptation system of muscle. METHODS In the present study, clenbuterol was subcutaneously administered in 8wks female Sprague-Dawley rats (CLEB: n=8) during 4 weeks. The effects of clenbuterol on slow-twitch soleus and fast-twitch EDL muscles were examined using reverse-transcription polymerase chain reaction (RT-PCR) analysis of the mRNAs of MCT1, MyoD, CD147, and UCP3. Furtermore, the effects of CLEB on proteins were examined using polyacrylamide gel (PAGE) systems of non-denatured PAGE for LDH isozymes and denatured SDS-PAGE for the both of Myosin HCs, MCT1, and CD147. RESULTS The muscle wet weights increased in both soleus and EDL muscles. The MyoD and UCP3 mRNAs increased drastically in SOL. They may explained the accelelated higher lipolytic function in soleus muscle by the accumulated fast-twitch fibers with fiber type transition from slow-to-fast. Furthermore, the increased MyoD mRNA may explain the muscle hypertrophy in soleus, but no changes of MyoD mRNA in EDL may need another explanation for the hypertrophy like as autocrine of IGF-1. The contents of MCT1 and its mRNA decreased in EDL and they tended to decrease in SOL. The contents of CD147 significantly decreased in EDL, but the CD147 mRNA showed no change in both SOL and EDL. In EDL, the decreased MCT1 might be explained by the decreased CD147 which is important to translocate MCT1 to plasma membrane. The difference of these changes between the proteins and mRNAs might be due to the posttranslational modification. These findings suggest that clenbuterol is able to induce lactate accumulation easily and to decrease the endurance capacity. CONCLUSIONS These results suggest that the clenbuterol induced many chages on skeletal muscles via positive feedback mechanism of MyoD in myogenesis as well as via cAMP of beta-2 adrenergic receptor system. It is necessary to carry out more detail experiments to explain the specific adaptation mechanism. Supported by A grant-in-aid (No. 15500437 to T.K.) from the Ministry of Education, Science, Sports, and Culture of Japan.

급격한 용존산소량 증가에 순응한 꺽지(Coreoperca herzi)와 모래무지(Pseudogobioesocinus) 젖산탈수소효소 활성과 동위효소의 변화

용존산소(DO)의 급격한 증가에 순응된 꺽지(Coreoperca herzi)와 모래무지(Pseudogobio esocinus) 조직내 젖산탈수소효소(EC 1.1.1.27, lactate dehydrogenase, LDH)의 대사와 <TEX>$C_4$</TEX> 동위효소를 확인하였다. DO 18 ppm에 갑작스럽게 순응시 꺽지 LDH활성은 뇌와 간조직에서 각각 <TEX>$35-39\%$</TEX> 변화되었고, 다른 조직에서는 <TEX>$20\%$</TEX>이내로 변화가 적었다. 골격근조직은 LDH <TEX>$A_4$</TEX> 동위효소가 증가되고 하부단위체 C를 포함하는 동위효소는 감소되었다. 심장조직은 <TEX>$B_4$</TEX> 동위효소가 조금 증가되었다. 신장조직도 <TEX>$B_4$</TEX>가 증가하고 눈 조직은 eye-specific <TEX>$C_4$</TEX>와 C hybrid가 감소되어 조절되었다. DO 증가에 갑작스럽게 순응한 모래무지 간조직에서 LDH 활성은 30분에서 급격하게 <TEX>$150\%$</TEX>이상 크게 증가되었고, 다른 조직에서는 <TEX>$70\%$</TEX> 이상 변화되었다. 그리고 골격근조직은 <TEX>$A_4$</TEX> 동위효소가 증가하고 다른 조직들에서는 <TEX>$B_4$</TEX>가 증가되었으며 특히 간 조직의 대사는 liver-specific <TEX>$C_4$</TEX>가 증가하고 <TEX>$A_4$</TEX> 는 감소되어 조절되었다. 그러나 꺽지 눈 조직의 대사는 LDH 활성이 감소하고 eye-specific <TEX>$C_4$</TEX> 동위효소가 감소되어 조절되었다. 따라서 DO증가에 순응시 모래무지는 꺽지에 비해 LDH 활성이 크게 증가되었고 동위효소의 변화정도도 크게 나타났으며, eye-spe-cific <TEX>$C_4$</TEX>와 liver-specific <TEX>$C_4$</TEX> 동위효소는 lactate oxidase로서 대사를 조절하였다. 그러므로 환경변화에 순응하여 나타나는 대사는 종이 이전에 어떤 서식조건에 적응되었는가에 따라 다른 것으로 사료된다. The metabolism of lactate dehydrogenase (EC 1.1.1.27, LDH) and <TEX>$C_4$</TEX> isozyme were studied in tissues of Coreoperca herzi and Pseudogobio esocinus acclimated to rapid increase of dissolved oxygen (DO). In C. herzi the LDH activity was changed <TEX>$35-39\%$</TEX> in brain and liver tissues, and within <TEX>$20\%$</TEX> in other tissues. The <TEX>$B_4$</TEX> isozyme was increased and isozyme containing subunit C was decreased in muscle tissue. The <TEX>$B_4$</TEX> isozyme was increased in heart and kidney. In P. esocinus, the LDH activity in liver tissues was largely increased to <TEX>$150\%$</TEX> for 30 minute and <TEX>$70\%$</TEX> in other tissues. The <TEX>$A_4$</TEX> isozyme was increased in muscle and <TEX>$B_4$</TEX> isozyme was increased in other tissues. Especially, the metabolism of liver tissue in P. esocinus was regulated by increasing liver-specific <TEX>$C_4$</TEX> and decreasing <TEX>$A_4$</TEX> isozyme. But the metabolism of eye tissue in C. herzi was regulated by decreasing LDH activity and eye-specific <TEX>$C_4$</TEX> isozyme. The LDH activity and LDH isozyme in P. esocinus were largely increased than C. herzi acclimated to rapid increase of DO. And eye-specific <TEX>$C_4$</TEX> and liver-specific <TEX>$C_4$</TEX> isozymes played role as lactate oxidase. Therefore, the response of species acclimated to rapid increase of DO seems to be variable, perhaps due to prior exposure to environmental conditions.

운동강도의 차이가 흰쥐의 심근 세포기질 및 미토콘드리아 LDH 동위효소 변화에 미치는 영향

생후 7주령 흰쥐를 대상으로 저강도집단, 중강도집단, 고강도집단, 통제집단으로 분리하여 8주 동안 트레드밀 운동을 실시한 후 심장을 적출하여 조직의 세포기질을 분리하고 전기영동을 실시하여 세포기질 LDH 동위효소의 분포양상을 확인하였고, 활성을 측정하였다. 또한 미토콘드리아를 순수분리한 뒤 분리된 미토콘드리아를 외막 내막 막간물질, 매트릭스로 각각 분리하여 각 분획을 전기영동을 실시하고 효소활성을 측정하였다. 운동 강도에 따른 장기간의 지구성훈련으로 심장조직의 세포기질 LDH 동위효소 활성은 운동집단과 비 운동집단에서 모든 LDH 동위효소의 활성을 나타냈다. 세포기질 LDH 동위효소의 운동 강도에 따른 활성변화는 모든 동위효소가 운동강도의 증가에 따라 증가하는 결과를 나타냈으며 모든 강도에서 <TEX>$AB_3$</TEX> 동위효소의 활성이 가장 크게 나타났다. 심장조직 미토콘드리아 각 분획에서 운동 강도에 따른 LDH 등위효소의 분포와 활성은 외막의 경우 다른 분획에 비하여 동위효소의 분포 및 활성이 매우 약하게 나타났다. 운동강도가 증가하면서 모든 분획의 동위효소가 <TEX>$B_4$</TEX>쪽으로 활성이 강해지는 현상을 보이며, 외막에서 내막, 매트릭스로 이동하면서 각 분획에 LDH 동위효소의 분포도 많아지고 활성도 또한 증가하는 현상을 나타냈다. To achieve the purpose of this study, forty-eight male Sprague-Dawley rats were assigned to control and three endurance exercise group. 36 rats were forced to exercise according to exercise intensity for 8 weeks and 12 rats were untrained for control group. Cardiac cytosol was extracted from cardiac tissue and cardiac mitochondria was purified from the cytosol. Purified mitochondria were separated into four fraction: inner membrane, outer membrane inter membrane space and matrix. The changes of cytosolic and mitochondrial LDH isozymes activity were measure. Relative activity <TEX>$(\%)$</TEX> of cytosol for low and control group showed the following order of prevalence <TEX>$AB_3>A_2B_2>B_4>A_3B>A_4$</TEX> for moderate and high group : <TEX>$AB_3>B_4>A_2B_2>A_3B>A_4$</TEX>. Outer membrane for low group showed <TEX>$AB_3>B_4>A_2B_2$</TEX>, for moderate group:<TEX>$ B_4>AB_3>A_2B_2$</TEX>, for high and control group: <TEX>$B_4>A_3B$</TEX>. Inter membrane space for low, moderate and high group showed <TEX>$B_4>AB_3>A_2B_2>A_3B>A_4$</TEX>, for control group: <TEX>$B_4>A_3B>AB_3>A_2B_2>A_4$</TEX>. Inner membrane for all group showed <TEX>$B_4>AB_3>A_2B_2>A_3B>A_4$</TEX>. Matrix for control, low, moderate and high group showed <TEX>$B_4>AB_3>A_2B_2>A_3B>A_4$</TEX>. These results suggest that long term exercise intensity effect on cardiac tissue cytosolic and mitochondrial activity and <TEX>$A_4,\;A_3B,\;A_2B_2,\;AB_3\;and\;B_4$</TEX> isozymes were found entirely in mitochondrial fraction.

Ontogenetic changes and developmental adjustments in lactate dehydrogenase isozymes of an obligate air-breathing fish Channa punctatus during deprivation of air access

In air-breathing snakehead Channa punctatus, Ldh-B is expressed at all ontogenetic and developmental stages, while Ldh-A is expressed temporally in pre-hatchlings 12–13 days ahead of bimodal respiration marked by air-breathing. Remarkable differences are observed in the LDH isozyme expression among various ontogenetic and developmental stages upon denying air access. When denied air access, water-breathing larvae show two distinct characteristics: (i) they survive longer than transitory air-breathers due to independence from air-breathing and (ii) there is more transient induction of Ldh-B than Ldh-A. Transition to bimodal breathing, which occurred post-hatching in 15-day old larvae, is coincidental with inducibility of Ldh-A and concomitant down-regulation of Ldh-B. Heart tissue from air-breathing adults denied air access shows a preferential expression of LDH-A subunit and slight down-regulation of LDH-B. Heterotetramers of A and B subunits participate in adjusting LDH levels among those stages which either precede air-breathing switchover, or are subsequent to this transition. The contribution of heterotetramers depends on the stage-specific levels of LDH homotetramers A 4 or B 4. Scaling of muscle mass during growth, tolerance to extended deprivation of air access and induction of Ldh-A are correlated. Response to restoring air contact indicated that advanced air-breathing stages of C. punctatus possess an inherent capacity to sense surface air. In kinetic properties, LDH isozymes of C. punctatus are teleost-like but species specificity is displayed in oxidative potential by cardiac muscle and in L-lactate reduction by skeletal muscle.

칠성장어(Lampetra japnica) 간조직 젖산탈수소효소와 대구(Gadus macrocephalus) liver-Specific C4동위효소의 특성 및 진화적 관계

칠성장어(Lampetra japonica) 간조직 젖산탈수소효소(EC 1.1.1.27, lactate dehydrogenase, LDH) 동위효소는 affinity chromatography에서 buffer를 유입한 후 용출된 분획에서 정제되었다. 대구(Gadus macrocephalus)의 liver-specific <TEX>$C_4$</TEX>동위효소는 열처리한 후 affinity chromatography하여 NAD+ 를 함유한 buffer에서 용출되기 시작하여 buffer를 유입한 후 <TEX>$B_4$</TEX> 동위효소와 함께 용출되어, DEAE-Sephacel chromatography에 의해 정제되었다. 대구 간조직에서 열에 대한 안정성은<TEX>$C_4$</TEX><TEX>$B_4$</TEX><TEX>$A_4$</TEX> 동위효소의 순서로 나타났다. Chromate-focusing에 의해 정제한 칠성장어 간조직의 pH 7.45 분획의 LDH 동위효소는 정제된 간조직 LDH보다 피루브산에 의한 기질저해도가 컸다. 칠성장어 간조직 LDH의 최적 pH는 7.5, liver-specific <TEX>$C_4$</TEX>동위효소는 pH 8.5였다. 칠성장어 간조직 LDH는 항원-항체반응에서 꺽지 <TEX>$A_4$</TEX> 항체와 liver-specific <TEX>$C_4$</TEX> 항체의 순서로 반응하였고 eye-specific <TEX>$C_4$</TEX> 항체와는 반응 정도가 낮았다. 따라서 칠성장어 간조직 LDH는 하부단위체 A와 liver-specific <TEX>$C_4$</TEX>의 구조와 유사하게 진화되었으며, 하부단위체 C 는 진화속도가 매우 빠른 것으로 확인되었다. 칠성장어 간조직의 LDH는 단일 동위효소가 아니라, 하부단위체 A, B 및 C로 구성된 동위효소들인 것으로 사료된다. The lactate dehydrogenase (EC 1.1.1.27, LDH) in liver of Lempetra japonica was purified in buffer of affinity chromatography. The liver-specific <TEX>$C_4$</TEX> isozyme of Gadus macrocephalus was purified by heat treatment, affinity chromatography, and DEAE-Sephacel chromatography. The liver-specific <TEX>$C_4$</TEX> isozyme was eluted in a buffer containing NAD+ and was coeluted with <TEX>$B_4$</TEX>isozyme in plain buffer of affinity chromagraphy. Liver-specific <TEX>$C_4$</TEX> isozyme in G. macrocephalus was the most thermostable, and<TEX>$B_4$</TEX>isozyme was more stable than <TEX>$A_4$</TEX>. The LDH in the fraction of pH 7.45 purified from the liver of L. iaponica by chromatofocusing was more inhibited by pyruvate than purified LDH. The optimum pH of the LDH isozyme in the liver of L. japonica was 7.5 and that of liver-specific<TEX>$C_4$</TEX> isozyme was 8.5. The LDH in liver of L. japonica made complexes more with antibody against Coreoperca herzi<TEX>$A_4$</TEX> and liver-specific <TEX>$C_4$</TEX> than with that against eye-specific <TEX>$C_4$</TEX>. Therefore, the structure of the LDH in liver of L. japonica might be similarly evolved to that of subunit A and liver-specific <TEX>$C_4$</TEX> isozyme in liver tissue of G. macrocephalus. The evolution rate of subunit C is faster than that of subunit A. LDH in liver of L. japonica has not one isozyme but isozymes and it was also found out to have not only subunit A and B but also subunit C.

Differential expression for tissue-specific isozymes in three species of Hypostomus Lacépède, 1803 (Teleostei: Loricariidae)

The expression patterns of 14 enzymatic systems in skeletal muscle, liver and heart tissues of three species of Hypostomus from the Iguaçu River basin (Brazil) were investigated. Although the patterns were similar for the three species, different tissues showed differential expressions, and the data showed that differential tissue expressions of isoperoxidases may be due to preferential combination or association of polypeptide subunits. The detected patterns for SOD isozymes showed that the quaternary structures of these enzymes were in disagreement with the subunit number reported for the majority of other vertebrate groups. Tissue-specific restriction on heterotetramer formation also were described in LDH and MDHP isozymes. Thus, these tissue-specific gene expression character in the species of Hypostomus have the greatest potential to be recognized and applied in systematic studies among species of Hypostomus.

Differential Expression of Lactate Dehydrogenase Isozymes (LDH) in Human Placenta with High Expression of LDH-A4Isozyme in the Endothelial Cells of Pre-eclampsia Villi

To evaluate the role of LDH isozymes in the human placenta during the third trimester, placentae were obtained from patients with normal pregnancy and pre-eclampsia. LDH-A4isozyme was immunolocalized primarily in the fetal endothelial cells while LDH-B4isozyme was predominantly present in syncytiotrophoblasts. This distinct cellular expression pattern of LDH isozymes was confirmed in HUVE and JEG cells. In addition to demonstrating the presence of five LDH isozymes in the placenta, zymograms showed that there was predominant activity of LDH-A4isozyme in HUVE cells and high activity of LDH-B4in JEG cells. Quantitative studies of LDH by agarose gel electrophoresis and Northern analysis in patients concluded that LDH-A4isozyme was increased in pre-eclampsia. The LDH-A4isozyme activity increased (P< 0.01) approx 1.6-fold in pre-eclampsia but there was no difference in the LDH-B4isozyme activity between placentae from normal compared to pre-eclampsia pregnancy. The level of LDH-A mRNA was increased (P< 0.05) approx twofold in pre-eclampsia. We conclude that the LDH-A gene in the endothelial cells of the placenta within the fetal microvasculature is increased in pre-eclampsia, probably as a result of hypoxia. LDH-A4isozyme activity and gene expression in placental endothelial cells, therefore, is a marker for the endothelial pathology seen in pre-eclampsia.