2주간 기아 후 붕어와 모래무지 조직 내 젖산탈수소효소의 대사

염정주 청주대학교 산업과학연구소 2019 産業科學硏究 Vol.36 No.2

Carassius auratus and Pseudogobio esocinus, which express liver-specific C, were collected in August and maintained in starvation state for two weeks to confirm the function of lactate dehydrogenase (EC 1.1.1.27, LDH) metabolism in the tissues of the starvation group. In C. auratus, LDH activity and specific activity (units/mg) in liver tissue were increased and LDH activity of skeletal muscle tissues was most decreased. LDH liver-specific C4 isozyme was expressed in liver and brain tissue, and A4 isozyme was increased in other tissues. LDH-C was increased in the liver tissue and LDH A4 was increased in the brain tissue from P. esocinus. The activity of monoamine oxidase was decreased and the catalase activity was increased in liver tissue from C. auratus. Therefore, the metabolism of peroxisome was increased. The Km PYR value was increased in the brain tissues of C. auratus, and was inhibited by 10 mM pyruvic acid in the order of subunit B, C, A. The Km PYR values were decreased in the brain and eye tissues of P, esocinus and the affinity for pyruvate was increased. As a result, C4 or C hybrids were expressed in the liver and brain tissues after two weeks of starvation. However, when the period of hunger was prolonged, it was metabolized by C4 isozyme in liver tissue, but lactate was metabolized by A4 isozyme in brain tissue.

풀망둑(Acanthogobius hasta) liver-specific 동위효소와 eye-specific 젖산탈수소효소 동위효소의 동시 발현

염정주 청주대학교 산업과학연구소 2003 産業科學硏究 Vol.21 No.1

A cathodal liver-specific and anodal eye-specific lactate dehydrogenase(EC 1.1.1.27, LDH) isozymes are coexpressed in tissues of Acanthogobius hasta. The activities of LDH were higher in tissues taken from July than from October and it was especially higher in breast muscle. The A₄ isozyme was expressed in skeletal muscle and breast muscle. And the B₄ isozyme was expressed in heart. The liver-specific C₄ isozyme showed lower activity in liver and eye-specific C₄ isozyme showed higher activity in eye tissue. Relative activity(%) of A₄ isozyme was increased and that of C₄ isozyme was decreased in liver taken from July. Therefore C₄ isozyme was played role as lactate oxidase. As a result, Acanthogobius hasta acclimated to hypoxic environment by anaerobic metabolism of LDH isozymes.

Lamprey(Petromyzon marinus) 조직내 3가지 젖산수소이탈효소의 구조적 유사성

염정주,김상엽 圓光大學校 基礎自然科學硏究所 1990 基礎科學硏究誌 Vol.9 No.1

Lamprey(Petromyzon marinus)의 골격근, 심, 간 및 신장조직에 3개의 젖산수소이탈효소 동위효소가 존재함이 전분 젤 전기영동방법으로 확인되었다. Oxamate gel상에서 이 동위효소들을 affinity chromatography하면 동일 분획내에서 3가지 동위효소 들이 모두 확인되므로, 이 동위효소들의 구조는 상당한 부분이 동일함을 암시하고 있다. 이러한 구조적 유사성은 CM-Sepharose 및 DEAE-Sephadex gel상에서의 chromatography에서도 나타났다. 이 동위효소들 중 CI와 AI를 순수정제하였으며 분자량은 140,000정도이었다. CI와 AI 모두 비슷한 정도로 pyruvate에 의해 저해를 받고 있으며 CI는 LDH A_4일 가능성이 높다. 정제된 CI에 대한 항체를 흰쥐로 부터 얻었으며 이 항체는 경골어류 largemouth bass는 물론 mouse의 LDH A_4들과 복합체를 형성하였다. 상기의 결과로부터 lamprey 조직내에는 Ldh-A 및 Ldh-B가 발현되고 있으며 하부단위체 A 및 B의 구조는 서로 상당히 유사하다고 사료된다. Lamprey(Petnmyzon marinus) was demonstrated, by starch gel electrophoresis, to have three lactate dehydrogenase isozymes in the tissues of skeletal muscle, heart, liver and kidney. The three isozymes were found in same fraction after affinity chromatography on oxamate gel, suggesting that the isozymes share common parts to a great extent This structural similarity also could be confirmed by the chromatographies of the isozymes on the CM-Sepharose and DEAE-Sephadex gels. The cathodal(CI) and anodal(AI) isozymes were further purified by extracting the activity zones after the starch gel electrophoresis. Sephadex G-200 gel filtration gave a molecular weight estimate of 140,000 for these isozymes. The CI and Al isozymes were inhibited by pyruvate to a same degree and the CI might be LDH A_4 isozyme. The antibodies against the purified CI made complexes with LDH A_4 isozymes of the largemouth bass and mouse as well as the purified CL It might be suggested, on the basis of these observations, that both Ldh-A and Ldh-B are expressed in lamprey tissues and the structure of subunit A is similar to that of subunit B.

풀망둑(Acanthogobius hasta) 신경조직 젖산탈수소효소의 역학적 특성

염정주 청주대학교 2013 産業科學硏究 Vol.30 No.2

In order to evaluate the effectiveness of lactate dehydrogenase(EC .1.1.27, LDH) in eye and brain tissues from Acanthogobius hasta as a catalyst, the kinetics were studied and the suitable condition for measuring the activity of LDH in fishes was proposed. The LDH inhibition by pyruvate was observed to reduce activity at concentrations greater than 0.2 mM in eye tissue, and 0.3 mM in brain and skeletal muscle, while substrate inhibition by lactate was significant above 60 mM in eye and brain tissues. And the activities of LDH in eye and brain remained at 48.89% and, 63.25% as a result of the inhibition by 1.5 mM of pyruvate. The corresponding value of KmNADH for eye was 0.147 mM, and those of KmPYR and KmLAC for eye, brain and skeletal muscle were 0.028, 0.067, 0.069 mM, and 2.787, 11.986, 8.786 mM, respectively. Therefore, it was found that the activity of LDH in fish had to be measured with 0.5 mM pyruvate and 0.14 mM NADH, or 40 mM lactate and 0.91 mM NAD+ in Tris-HCl buffer of pH 7.5. Conclusively, the LDH in eye tissue had a high affinity for pyruvate. And LDH in eye and brain tissues exhibited higher activity than that of skeletal muscle at lower concentration of lactate. So it was found that the energy produced rapidly by LDH eye-specific C4 isozyme in neural tissues was used for the predatory and defense behavior and lactate played an important role in metabolism of brain.

대구(Gadus macrocephalus) 간조직 미토콘드리아 젖산수소이탈효소의 특성

염정주,조성규,이상학 청주대학교 산업과학연구소 1998 産業科學硏究 Vol.16 No.-

Inhibition rate of mitochondrial LDH activity by Pyruvate was similar to LDH B4 in the fraction washing with 5mM Nacl, and it was intermediated value of B4 and C4 isozyme in the fraction washing with 175mM NaCl and punfied iltochondrial LDH C_(4) isozyme. The result of immunoelecttophoresis and Western blotting, mitochondrial LDH cross-reacted in follows of C_(4)>A_(4)>B_(4) with antibodies against cytosolic A_(4), B_(4) and C_(4) isozyme Cytosolic B_(4) Isozyme for antibody production used in this study was purified from kidney of mouse(Mus musculus) by PreP Cell. Therefore, mltochondrial LDH C4 isozyme was similar to cytosolic C4 isozyme and it had similar elvolutionary Pattern with A_(4) isozyme. Also a part of antigenic determinant in A_(4), B_(4) and C_(4) isozyme had similarity. Isoelectric point(pI) of mitochondrial LDH A_(4), B_(4) and C_(4) isozyme were 5.10, 5.23 and 7.55 by polyacrylamlde gel isoelectric focusing, respectively. And they bound to mitochondrial membrane. Therefore, mitochondrial C_(4) isozyme was different function with cytosolic C_(4) isozyme, but specific control mechanism of cytoplasmic LDH isozymes may be depending on the metabolic condition.

북방산개구리(Rana dybowskii) 조직 젖산탈수소효소 동위효소의 대사 조정

염정주 청주대학교 2017 産業科學硏究 Vol.34 No.2

The metabolic adjustment of LDH was investigated by quantifying lactate dehydrogenase (EC.1.1.1.27, LDH) activity and protein and characterizing purified LDH A4 isozyme in Rana sybowskii tissues collected in August. The LDH activity in skeletal muscle, heart, liver, eye and brain tissue was found to be particularly high at 1196.10, 231.50, 119.61, 50.48 and 76.13 units and the amount of protein was low at 15.21, 10.60, 24.57, 22.40 and 4.32 mg. Therfore the specific activity of LDH was high at 21.79, 4.87, 2.25, and 17.62 units/mg, respectively. Native- polyacrylamide electrophoresis showed stronger identification of LDH A4 isozyme in skeletal muscle and heart tissue. And A4 isozyme was slightly stronger in brain tissue, B4 isozyme weakened and eye-C hybrid appeared on the anode side. In the skeletal muscle, liver, and eye tissues, several bands appeared on the most negative side and are thought to be liver-specific C. The LDH A4 isozyme was purified by affinity chromatography at 65.6-72 ml after NAD+ entry, and all LDH isozymes were purified 6158.11 units with a yield of 73.54%. The LDH A4 was confirmed to be purified by SDS-PAGE, and the molecular weight of the subunit A was 29.9 kDa. The purified LDH A4 isozyme showed maximal activity in 0.5 mM pyruvic acid and then decreased in activity to 13.92% in 10 mM pyruvic acid. Therefore, KmPYR and VmaxPYR were 0.11 mM and 97.76 units, respectively. Therefore, although the affinity of LDH A4 to pyruvic acid was lowered slightly due to the increase in the temperature of the environment, it was considered that the LDH activity was greatly increased, and the anaerobic metabolism using pyruvic acid for energy generation was greatly increased.

북방산개구리와 참개구리 조직의 젖산탈수소효소의 역학적 특성

염정주,하은성 청주대학교 산업과학연구소 2016 産業科學硏究 Vol.33 No.2

The kinetic properties of lactate dehydrogenase(EC 1.1.1.27,LDH) were studied in tissues from Rana dybowskii and Rana nigromaculata. An expression of LDH-C was detected, furthermore, the mesurement condition for the activity of LDH was proposed in the study. LDH activity and specific activity of skeletal muscle from R. nigromaculata were the highest at 290.79units and 4.82, respectively, and it was higher than those from R. dybowskii. And higher concentration of LDH A4 isozyme was detected in every tissues except heart. Moreover, the isozymes containing LDH-C were identified by immunoprecipitation and native-polyacrylamide electrophoresis in heart, liver, eye, and brain tissues from R. dybowskii and in eye tissue of R. nigromaculata. The activity of LDH in tissues was reduced at concentration over 0.2mM of pyruvate, Therefore, the activity of LDH have to be measured with 0.2mM of pyruvate. The activity of LDH in tissues from R. dybowskii remained at 8.14-1.95% and those in tissues from R. nigromaculata remained at 8.81-3.62% as a result of the inhibition by 10mM pyruvate. And the degree of inhibition of isozyme B by pyruvate was higher than that of isozyme A. The maximum activity of LDH in skeletal muscle was measured at 105mM of lactate. The KmPYU values were 0.03-0.09mM from R. dybowskii and 0.02-0.17mM from R. nigromaculata, which was considerably low. As a conclusion, the tissues from R. nigromaculata have a relatively more anaerobic metabolism, and the LDH in both species have a high affinity for pyruvate. The Ldh-C was identified characteristically, and LDH-A,B,C are considered to be similar.

모래무지 젖산수소이탈효소의 생화학적 특성

廉貞周,金明玉 청주대학교 산업과학연구소 1989 産業科學硏究 Vol.7 No.-

모래무지(Pseudogobio esocinus) 젖산수소이탈효소 (EC.1.1.1.27, Lactate dehydrogenase, LDH)의 생화학적 특성을 연구하였다. Polyacrylamide gel electrophoresis 후 가장 음극(-)쪽의 LDH 동위효소는 sodium deoxycholate에 의해 선택적으로 불활성화되었으므로 LDH A₄동위효소이다. Urea에 의한 불활성화 정도는 B₄>C₄>A₄의 순서로 낮아졌으며, 잉어목(Cypnnformes) 어류 LDH의 경우와 일치하지 않았다. 골격근, 간 및 신장조직 LDH의 열에 대한 안정성의 정도는 B₄>C₄>A₄의 순서로 낮아졌다. 골격근조직 LDH의 최적 pH는 6.5였으며, 황산암모늄 분별침전 범위는 30~55%였다. Biochemical properties of lactate dehydrogenase isozymes in Pseudogobio esocinus were characterized. The most cathodal LDH isozyme after polyacrylamide gel electrophoresis was selectively inactivated by sodium deoxycholate, indicating that the zone was LDH A_(4)isozyme. The LDH isozymes exhibited a hierachy of susceptibility to inactivation by urea of B_(4)>C_(4)>A_(4) ; inconsistent with findings for cyprinid fishes. Thermostability of the skeletal muscle, liver and kidney LDH isozymes agreed with data obtained for LDH isozymes from other vertebrate species, B_(4) isozyme was most thermostable and C_(4) isozyme was more stable than A_(4) isozyme. The optimum pH of LDH isozyrmes in skeletal muscle was 6.5. The ammonium sulfate precipitation range of LDH isozymes in skeletal muscle was 30-55%.

기아상태에서 Ldh-C가 발현된 어류 조직의 젖산탈수소효소의 대사

염정주(Jung Joo Yum),김규동(Gyu Dong Kim) 한국생명과학회 2016 생명과학회지 Vol.26 No.2

젖산탈수소효소(Lactate dehydrogenase, EC 1.1.1.27, LDH) LDH-C의 기능을 확인하기 위해 liver-specific Ldh-C가 발현된 붕어(Carassius auratus)와 eye-specific Ldh-C가 발현된 파랑볼우럭(Lepomis macrochirus)을 기아 상태로 유지시킨 후(S) 조직들의 LDH 대사를 연구하였다. 기아 후 붕어 간조직의 LDH 활성이 크게 증가되었으며 LDH 비활성(units/mg)과 LDH/CS는 조직들에서 증가되어 혐기적 대사가 이루어짐을 확인하였다. 기아 후 LDH B4 동위효소가 골격근조직에서 감소되었고 심장조직에서 증가되었다. 눈과 뇌조직에 나타났던 LDH C4 동위효소는 liver-specific C4로 확인되었으며 기아 후에 없어지고, 눈조직은 C hybrid, 뇌조직은 A4, 간조직은 C hybrid와 C4 동위효소가 각각 증가되었다. 그러나 파랑볼우럭 조직에서 LDH 활성의 변화는 작았으나 눈조직에서 가장 크게 증가되었으며, 뇌조직은 LDH A4와 AC hybrid가 증가되었다. 피루브산 10 mM에 의해 기아 후 붕어 조직의 LDH 활성은 30.30-18.64%, 파랑볼우럭 조직의 LDH는 25-18.75% 남았으며, 붕어는 Km<SUP>PYR</SUP> 값이 증가되었다. 실험결과 LDH liver-specific C 동위효소가 기아 중에 간, 뇌 및 눈조직에서 발현되었고, 기아 후 뇌조직에서 젖산의 대사가 우세하고, 붕어 LDH liver-specific C가 파랑볼우럭 LDH eye-specific C보다 영향을 더 받는 것으로 사료된다. 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<SUP>PYR</SUP> 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.

풀망둑( Acanthogobius hasta) 젖산탈수소효소의 특성

염정주(Jung Joo Yum) 한국생명과학회 2008 생명과학회지 Vol.18 No.2

풀망둑(Acanthogobius hasta) 조직의 젖산탈수소효소(EC 1.1.1.27. Lactate dehydrogenase, LDH) 동위효소의 특성을 생화학적, 면역화학적 및 역학적 방법에 의해 연구하였다. 풀망둑 골격근과 눈 조직의 젖산탈수소효소 활성이 65.30과 53.25 units였고, 심장과 간 조직에서는 낮게 나타났다. 골격근 조직의 LDH/CS는 22.29로 가장 높고, LDH 특이활성도는 뇌 56.45, 눈 38.04 및 골격근 11.04 units/㎎였다. 각 조직에 대해 A₄, B₄, eye-specific C₄, 및 liver-specific C₄에 대한 항혈청으로 면역 침강 반응시킨 후 polyacrylamide gel 전기영동 하였다. 골격근, 뇌 및 눈 조직에서 A₄ 동위효소가 우세하게 확인되었고, 심장에서는 B₄ 동위효소가 확인되었다. 또한 양극의 eye-specific C₄와 음극의 liver-specific C₄가 한 종에서 함께 발현되었으며, 눈 조직의 eye-specific C₄는 활성이 크고 간 조직의 liver-specific C₄의 활성은 낮게 나타났다. 결과 A₄와 C₄, A₄와 B₄ 및 eye-specific C₄와 liver-specific C₄의 분자구조의 일부가 서로 유사하지만 B₄와 C₄의 구조는 서로 다른 것으로 나타났으므로 하부단위체 A는 보존적이고 하부단위체 B는 하부단위체 A보다 빠르게 진화된 것으로 사료된다. 골격근 조직의 LDH A₄ 동위효소는 affinity chromatography에서 NAD?를 함유한 buffer를 유입한 후 용출된 분획에서 정제되었고, eye-specific C₄ 동위효소는 A₄ 분획에 이어 용출되었으므로 eye-specific C₄가 A₄의 분자 구조와 유사한 것으로 보인다. 그리고 LDH에 대한 피루브산의 저해 실험 결과 35.22-43.47%의 활성이 남았고, Kmpyr은 0.080-0.098 mM 이고 골격근과 눈 조직의 Vmax은 153.85와 35.09 units였다. 또한 B₄ 동위효소가 열에 대해 가장 안정하였고 C₄는 A₄보다 안정하였으며, 최적 pH는 6.5로 나타났다. 본 실험 결과 풀망둑은 저 산소 환경조건에 적응되어져 조직들의 동위효소들이 A₄와 B₄ 동위효소 사이의 특성을 나타냈고, 골격근과 눈 조직에서 피루브산에 대한 LDH의 친화력이 상당히 크므로 LDH가 혐기적 조건에서 효율적으로 기능을 하는 것으로 사료된다. 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/㎎, respectively. The LDH isozymes in tissues were separated by polyacrylamide gel electrophoresis after immunoprecipitation with antiserum against A₄, B₄, eye-specific C₄ and liver-specific C₄. LDH A₄ isozymes were detected predominantly in skeletal muscle, brain and eye tissues, and B₄ isozyme was detected in heart. Anodal eye-specific C₄ and cathodal liver-specific C₄ were coexpressed in A. hasta. The eye-specific C₄ isozyme showed higher activity in eye tissue, but liver-specific C₄ isozyme showed lower activity in liver. As a result, one part of molecular structures in A₄ and C4, A₄ and B4, and eye-specific C₄ and liver-specific C₄ were similar, but in B₄ and C₄ 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 A₄ isozyme of skeletal muscle was purified in the fraction from elution with NAD? containing buffer of affinity chromatography and eye-specific C₄ isozyme was eluted right after A4, so the structure of eye-specific C₄ isozyme is similar to A₄. 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 B₄ isozyme was the thermo-stablest and C₄ was stabler than A₄ isozyme. The optimum pH of LDH was 6.5. The results mentioned above indicate that isozymes in tissues showed the properties between LDH A₄ and B₄ 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.