• Journal of Life Science
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• v.14 no.4
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• pp.708-715
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• 2004

The lactate dehydrogenase (EC 1.1.1.27, LDH) in liver of Lempetra japonica was purified in buffer of affinity chromatography. The liver-specific $C_4$ isozyme of Gadus macrocephalus was purified by heat treatment, affinity chromatography, and DEAE-Sephacel chromatography. The liver-specific $C_4$ isozyme was eluted in a buffer containing NAD+ and was coeluted with $B_4$isozyme in plain buffer of affinity chromagraphy. Liver-specific $C_4$ isozyme in G. macrocephalus was the most thermostable, and$B_4$isozyme was more stable than $A_4$. 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$C_4$ isozyme was 8.5. The LDH in liver of L. japonica made complexes more with antibody against Coreoperca herzi$A_4$ and liver-specific $C_4$ than with that against eye-specific $C_4$. Therefore, the structure of the LDH in liver of L. japonica might be similarly evolved to that of subunit A and liver-specific $C_4$ 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.

• Journal of Life Science
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• v.15 no.1 s.68
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• pp.71-79
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• 2005

The metabolism of lactate dehydrogenase (EC 1.1.1.27, LDH) and $C_4$ 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 $35-39\%$ in brain and liver tissues, and within $20\%$ in other tissues. The $B_4$ isozyme was increased and isozyme containing subunit C was decreased in muscle tissue. The $B_4$ isozyme was increased in heart and kidney. In P. esocinus, the LDH activity in liver tissues was largely increased to $150\%$ for 30 minute and $70\%$ in other tissues. The $A_4$ isozyme was increased in muscle and $B_4$ isozyme was increased in other tissues. Especially, the metabolism of liver tissue in P. esocinus was regulated by increasing liver-specific $C_4$ and decreasing $A_4$ isozyme. But the metabolism of eye tissue in C. herzi was regulated by decreasing LDH activity and eye-specific $C_4$ 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 $C_4$ and liver-specific $C_4$ 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.

• Journal of Life Science
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• v.18 no.2
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• pp.264-272
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• 2008

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 $A_4,\;B_4$ eye-specific $C_4$ and liver-specific $C_4$. LDH $AC_4$ isozymes were detected predominantly in skeletal muscle, brain and eye tissues, and $B_4$ isozyme was detected in heart. Anodal eye-specific $C_4$ and cathodal liver-specific $C_4$ were coexpressed in A. hasta. The eye-specific $C_4$ isozyme showed higher activity in eye tissue, but liver-specific $C_4$ isozyme showed lower activity in liver. As a result, one part of molecular structures in $A_4\;and\;C_4,\;A_4\;and\;B_4$, and eye-specific $C_4$ and liver-specific $C_4$ were similar, but in $B_4\;and\;C_4$ 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 $A_4$ isozyme of skeletal muscle was purified in the fraction from elution with NAD+ containing buffer of affinity chromatography and eye-specific $C_4$ isozyme was eluted right after $A_4$, so the structure of eye-specific $C_4$ isozyme is similar to $A_4$. 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 $B_4$ isozyme was the thermo-stablest and $C_4$ was stabler than $A_4$ isozyme. The optimum pH of LDH was 6.5. The results mentioned above indicate that isozymes in tissues showed the properties between LDH $A_4\;and\;B_4$ 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.

• Journal of Life Science
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• v.26 no.2
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• pp.155-163
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• 2016

Metabolism of lactate dehydrogenase (EC 1.1.1.27, LDH) was studied to identify the function of LDH-C. Tissues of LDH liver-specific Ldh-C expressed Carassius auratus and eye-specific Ldh-C expressed Lepomis macrochirus after starvation were studied. LDH activity in liver tissue from C. auratus was increased after starvation. And LDH specific activity (units/mg) and LDH/CS were increased in tissues. It means the anaerobic metabolism was taking place in C. auratus after starvation. LDH B₄ isozyme was decreased in skeletal muscle and increased in heart tissue. LDH C₄ isozymes those showed in eye and brain tissues were identified as liver-specific C₄ isozymes and disappeared after starvation. And C hybrid in eye, A₄ isozyme in brain, and both C hybrid and C₄ isozyme in liver tissue were increased, respectively. In L. macrochirus, the level of variation of LDH activities was low but greatly increased especially in eye tissue and LDH A₄ and AC hybrid were increased in brain tissue. The LDH activities in tissues from C. auratus and L. macrochirus remained 30.30-18.64% and 25-18.75%, respectively, as a result of the inhibition by 10 mM of pyruvate. The Km^PYR values of LDH in C. auratus were increased. As a result, LDH liver-specific C₄ isozyme was expressed in liver, brain and eye tissues during starvation. It seems metabolism of lactate was predominant in brain tissue. After starvation, the liver-specific LDH-C was affected more than eye-specific LDH-C.

• Journal of Life Science
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• v.20 no.3
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• pp.416-423
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• 2010

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 ($27{\pm}0.5{\rightarrow}18{\pm}0.5^{\circ}C$) and dissolved oxygen (DO) change ($6{\pm}1{\rightarrow}18\;ppm$) 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 $A_4$ isozyme was shown to have a more cathodal mobility relative to the $B_4$ isozyme. In the liver tissue, the LDH $A_4$ isozyme increased with temperature drop while the $B_4$ isozyme decreased. The LDH $A_4$ and $C_4$ isozymes increased with DO increment, while the $B_4$ isozyme decreased. In the eye tissue, the LDH $A_4$ and B4 isozymse increased with temperature drop while the $B_4$ isozyme decreased. The LDH $A_4$ and $B_4$ isozymes increased with DO increment, but the $C_4$ isozyme and isozymes including the subunit C decreased. In the heart tissue, LDH activity increased with DO increment, as well as the LDH $B_4$ isozyme. In the brain tissue, the LDH $A_4$ and $B_4$ isozymes increased with temperature drop. The LDH $B_4$ isozyme increased with DO increment. Accordingly, since the liver- and eye-specific Ldh-C are influenced by changes in DO and the LDH $B_4$ and $C_4$ isozymes are relatively controlled in the liver and eye tissues, the $C_4$ isozyme can be considered to have a lactate oxidase function.

• Journal of Life Science
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• v.22 no.1
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• pp.98-109
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• 2012

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 $A_4$ isozyme was predominantly located in skeletal muscle. The LDH $A_4$, $A_2B_2$, and $B_4$ isozymes were detected in heart, liver, eye, and brain tissues, while eye-specific $C_4$ isozyme was detected in eye tissue. In September, strong LDH $B_4$ isozyme activity was detected in heart tissue. High $A_4$ isozyme activity was noted in all other tissues except heart tissue. However, in November, strong $A_4$ 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 $C_4$ isozyme was more similar to the $A_4$ than the $B_4$ isozyme. The LDH $A_4$ 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 $K_m^{PYR}$ of LDH in eye tissue was very low. The optimum pH for LDH in tissues was pH 7.5~8.0. The LDH $A_4$ isozyme was detected in mitochondria of skeletal muscle, whereas the $B_4$ and $A_2B_2$ 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.

• Journal of Life Science
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• v.28 no.1
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• pp.1-8
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• 2018

To confirm the function of lactate dehydrogenase (LDH) (EC 1.1.1.27, LDH), its metabolism was studied by activity, kinetics, and isozyme analysis in tissues of Ldh testis-specific C expressing mice (Mus musculus) maintained in a state of starvation for 48 hr and 96 hr. In skeletal muscle, liver, and eye tissues, LDH and LDH $A_4$ activity increased and anaerobic metabolism predominated. While LDH activity in the heart and kidney tissues decreased, LDH $B_4$ activity increased and aerobic metabolism predominated, producing pyruvic acid. In the testis tissue, LDH $C_4$ activity decreased. In the brain tissue, LDH activity increased, but the isozyme change was small and the amount of pyruvic acid decreased. $K{_m}^{PYR}$ increased in tissues other than kidney tissue, and the affinity for pyruvic acid decreased. Consequently, in Ldh-A and B-expressing tissues, the activities of isozymes with higher concentrations increased. However, in Ldh-A, B, and C-expressing tissue, $C_4$ decreased and the function of the tissue also decreased. In particular, LDH in brain tissue played a role as a pyruvate reductase. Therefore, this process might be the mechanism for producing energy in the state of starvation.

• The Korean Journal of Nuclear Medicine
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• v.28 no.1
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• pp.112-123
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• 1994

${\gamma}$-Glutamyltransferase (GGT: E.C. 2.3.2.2.) is a glycoprotein enzyme which is involved in glutathione metabolism and amino acid transport through the plasma membrane. It is distributed widely in several organs including liver and kidney. Several isozymes of GGT have been reported and some of the isozymes may be associated with hepatocarcinogenesis. We have produced six monoclnal antibodies (mAbs) against GGT purified from the liver of 2-acetamidofluorene (AAF) treated rats. All of the six mAbs were obtained by immunizing mice with liver GGT Six hybridomas which produced anti-GGT Abs were extensively subcloned and injected into the peritoneal cavity of BALB/c mice to obtain large quantities of Abs. These mAbs were purified from ascites by ammonium sulfate precipitation and protein A sepharose CL-4B column chromatography. Using these mAbs we preformed enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC), and autoradiography (ARG) to study the distribution of GGT isozyme in tissue. The results indicate that GGT-mAb 1 is specific for the AAF treated liver GGT, GGT-mAb 5 for the normal liver GGT, and GGT-mAb 6 for the normal kindey GGT. These mAbs may be used to evaluate the distribution of GGT isozymes in different tissues.

• Journal of Life Science
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• v.14 no.4
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• pp.702-707
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• 2004

In native-polyacrylamide gel electrophoresis of Pangasius polyuranodon, the lactate dehydrogenase (EC 1.1.1.27, LDH) $A_4$, $A_3$B, $A_2$$B_2$,$AB_3$ and $B_4$ isozymes were expressed in various tissues. The LDH $A_4$ and liver-specific $C_4$ isozymes were expressed in the tissues of Hypostomus Plecostomus. The bands of LDH in skeletal muscle, heart and eye tissues were not detected while one band was detected in kidney and liver, and four bands were detected in brain. The detected one band in liver was identified as alcohol dehydrogenase and an anodal band of skeletal muscle was identified as nothing dehydrogenase. The LDH in skeletal muscle, heart and eye might function as pyruvate reductase. The degree of inhibitions of LDH in skeletal muscle and heart of P. polyuranodon by 10 mM pyruvate were measured 57.6% and 73.8%, respectively. However, those of LDH in tissues of H. plecostomus were measured 52.7-61.8% so tissue specificity did not appear. Therefore, H. ple-costomus might be more acclimated to hypoxic environment by anaerobic metabolism of LDH iso-zymes than P. polyuranodon.

• Journal of fish pathology
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• v.18 no.1
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• pp.67-80
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• 2005

Phosphoinositide-specific phospholipase $C\delta$ $PLC\delta$) plays an important role in many cellular responses and is involved in the production of second messenger. The present study was conducted to obtain the biochemical characteristics of the expressed recombinant $PLC\delta$ in E. coli cloned from Misgurnus mizolepis and partially purified $PLC\delta$ enzymes from liver tissues of M. mizolepis (wild ML-$PLC\delta$). The ML $PLC\delta$ gene was cloned and expressed under the previous report (Kim et al., 2004), and purified the recombinant protein by successive chromatography using $Ni^{2+}$-NTA affinity column and gel iltration FPLC column. The wild ML-$PLC\delta$ protein was solublized with 2 M KCI and purified by successive chromatography on open heparin-Sephagel and analytical TSKgel heparin-5PW. Both the recombinant and wild ML-$PLC\delta$ form of protein showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP$_2$) or phosphatidylinositol (PI). Its activity was absolutely $Ca^{2+}$- dependant, which was similar to mammalian $PLC\delta$ isozymes. Maximal PI-hydrolytic activations of recombinant and wild ML- TEX>$PLC\delta$ was at pH 7.0 and pH 7.5, respectively. In addition, the enzymatic activities of recombinant and wild ML-$PLC\delta$ were increased in concentration-dependent manner by detergent, such as sodium deoxycholate SDC), phosphatidylethanolamine (PE) and phosphatidylcholine (PC). The activities decreased in contrast by a polyamine, such as spermine. Western blotting showed that several types of $PLC\delta$ isozymes exist in various organs. Taken together our results, it suggested that the biochemical characteristics of ML-$PLC\delta$ are similar with those of mammalian $PLC\delta1$ and ${\delta}3$ isozymes.