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.

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

Jung Joo Yum(염정주),Jun Hee Yeon(연준희) 한국생명과학회 2012 생명과학회지 Vol.22 No.1

큰입우럭(Micropterus salmoides) 조직의 젖산탈수소효소(EC 1.1.1.27, LDH)의 특성 및 골격근과 심장조직의 monocarboxylate 수송체 1, 2, 4의 발현을 연구하였다. Native-PAGE 결과골격근에서 LDH A₄, 심장, 간, 눈 및 뇌조직에서 A₄, A₂B₂, B₄, 눈조직에서 eye-specific C₄ 동위효소가 발현되었다. 9월에 심장조직에서 LDH B₄ 동위효소의 활성이 강하고 다른 조직에서는 A₄의 활성이 강했으나, 11월에 심장조직에서 A₄ 동위효소의 활성이 강하게 확인되었다. 골격근과 심장조직에서 LDH/CS로 조직의 혐기적 대사 비율이 높게 확인되었으며, 면역 침강 후 native-PAGE에 의해 LDH eye-specific C₄ 동위효소가 B4보다 A₄ 동위효소에 더 유사한 것으로 확인되었다. LDH A₄ 동위효소가 affinity chromatography에 의해 정제되었고, 하부단위체 A의 분자량은 37.200이었다. 피루브산 10 mM에서 조직 LDH의 활성이 11.05-28.32% 남아 저해 정도가 컸고, 눈조직 LDH의 Km <SUP>PYR</SUP>이 낮았으며, 조직의 최적 pH는 7.5~8.0이였다. 골격근 미토콘드리아에서 LDH A₄ 동위효소, 심장조직의 미토콘드리아에서 B₄와 A₂B₂ 동위효소가 확인되었고, 골격근과 심장조직의 원형질막과 미토콘드리아에서 MCT 1, 2, 4가 확인되었다. 골격근 MCT 1, 2, 4 골격근 MCT 1 60 kDa, MCT 2 54~38 kDa, MCT 4 63 kDa, 심장조직 MCT 1 57 kDa, MCT 2 54~38kDa 및 MCT 4 55.5 kDa이었다. 실험 결과, 큰입우럭이 저산소 조건에 적응되어져 혐기적 대사가 우세하고, 활성이 큰 골격근과 심장조직에서 원형질막과 미토콘드리아 MCT 1, 2, 4를 통해 젖산과 피루브산이 유입되고 유출되며 LDH에 의해 에너지 생성을 효율적으로 조정하는 것으로 사료된다. 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 A₄ isozyme was predominantly located in skeletal muscle. The LDH A₄, A₂B₂, and B₄ isozymes were detected in heart, liver, eye, and brain tissues, while eye-specific C₄ isozyme was detected in eye tissue. In September, strong LDH B₄ isozyme activity was detected in heart tissue. High A₄ isozyme activity was noted in all other tissues except heart tissue. However, in November, strong A₄ 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 C₄ isozyme was more similar to the A₄ than the B₄ isozyme. The LDH A₄ 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 Km <SUP>PYR</SUP> of LDH in eye tissue was very low. The optimum pH for LDH in tissues was pH 7.5~8.0. The LDH A₄ isozyme was detected in mitochondria of skeletal muscle, whereas the B₄ and A₂B₂ 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.

황소개구리(Rana catesbeiana) 조직의 젖산탈수소효소의 역학적 특성

염정주(Jung Joo Yum),하은성(Eun Sung Ha) 한국생명과학회 2014 생명과학회지 Vol.24 No.2

황소개구리(Rana catesbeiana) I (2월)과 II (8월) 조직의 젖산탈수소효소(EC. 1.1.1.27, lactate dehydrogenase, LDH) 동위효소의 발현 및 역학적 특성을 확인하였다. I의 골격근에서 LDH 활성, A4 동위효소 및 LDH/CS (EC 4.1.3.7, citrate synthase)가 높게 측정되었고, II의 여러 조직에서 LDH B4 동위효소의 활성이 증가되었으며, 특히 심장과 뇌조직에서 LDH 활성이 크게 확인되었다. 면역침강반응 후 native-PAGE에 의해 눈조직에서 LDH eye-specific C 동위효소가 확인되었고, LDH C가 LDH B에 유사한 것으로 확인되었다. LDH A4 동위효소를 oxamate-linked affinity chromatography로 정제하였고, 하부단위체 A의 분자량은 32.0 kDa이었다. II의 LDH는 KmPYU이 높았고, 심장과 뇌조직의 VmaxPYU이 높았으며, 조직들의 VmaxLAC도 높고, 젖산에 대한 내성이 큰 것으로 확인되었다. 그리고 정제한 A4 동위효소와 눈조직 LDH가 젖산에 대한 내성이 가장 크게 확인되었다. KmLAC가 KmPYU보다 컸다. 피루브산 10 mM에 의해 I, II 조직 LDH의 하부단위체 B의 비가 증가함에 따라 LDH 활성의 억제정도가 높았다. 실험 결과, 황소개구리에서 LDH eye-specific C가 확인된 것이 특징적이었고, 황소개구리 I의 골격근 LDH의 활성이 높아 혐기적 대사가 우세하였으며, 황소개구리 II의 경우 LDH B가 증가되고 젖산에 대한 내성이 커져 잘 적응되어진 것으로 사료된다. The kinetic properties and isozyme expression of lactate dehydrogenase (EC 1.1.1.27; LDH) in tissues from Rana catesbeiana I and II collected from February (I) and August (II) were studied. LDH activities, A4 isozyme, and LDH/citrate synthase (EC 4.1.3.7; CS) were high in skeletal muscle from R. catesbeiana I, and LDH B4 isozyme increased in several tissues of R. catesbeiana II. In particular, LDH activities were high in heart and brain tissues from R. catesbeiana II. LDH eye-specific C isozyme, detected by native polyacrylamide gel electrophoresis after immunoprecipitation, was expressed in eye tissue and was more similar to the B4 than A4 isozyme. LDH A4 isozyme was purified by oxamate-linked affinity chromatography, and the molecular weight of subunit A was 32.0 kDa. In R. catesbeiana II, levels of KmPYU, VmaxLAC, and tolerance to lactate of LDH were high in all tissues, and VmaxPYU of LDH in heart and brain tissue was highly detected. Purified A4 isozyme and LDH in eye tissue were highly tolerate compared to others. The KmLAC value was highly measured compared to KmPYU. The degree of inhibition by 10 mM of pyruvate on LDH activities in tissues from R. catesbeiana I and II was more pronounced as the ratio of subunit B increased. As a result, characteristic expression of LDH eye-specific C was found in R. catesbeiana. Anaerobic metabolism seemed to predominate as the LDH of skeletal muscle from I showed higher activity. It also appeared that R. catesbeiana II adapted well to incremental increases in LDH B, becoming tolerant to the lactate of LDH in tissues.

기아상태에서 Ldh-C가 발현된 생쥐(Mus musculus) 조직의 젖산탈수소효소의 대사

염정주(Jung Joo Yum),김규동(Gyu Dong Kim) 한국생명과학회 2018 생명과학회지 Vol.28 No.1

젖산탈수소효소(Lactate dehydrogenase, EC 1.1.1.27, LDH)의 기능을 확인하기 위해서 Ldh testis-specific C가 발현된 생쥐(Mus musculus)를 48 hr과 96 hr 기아상태로 유지시킨 후 조직들의 LDH 대사를 LDH 활성, 역학 및 동위효소를 분석하여 연구하였다. 골격근, 간 및 눈조직에서 LDH와 LDH A₄활성이 증가되어 혐기적 대사가 우세하였고, 심장과 신장조직의 LDH 활성은 감소되지만 LDH B₄ 활성이 증가되어 피루브산을 생성하는 호기적 대사가 우세하였다. 하지만 정소조직에서는 LDH C₄가 감소되었고, 뇌조직의 LDH 활성은 조직 중에서 가장 많이 증가되었지만 동위효소의 변화가 작고 피루브산의 양이 감소되었다. 신장조직을 제외한 조직들에서 Km<SUP>PYR</SUP>이 증가되어 피루브산에 대한 친화력이 감소된 것으로 확인되었다. 실험결과 Ldh-A, B가 발현된 조직에서는 상대 농도가 큰 동위효소의 활성이 증가되었으나 Ldh-A, B, C가 발현된 정소조직은 LDH C₄가 감소되어 기능이 저하되었으며 특히 뇌조직에서 LDH는 피루브산 환원효소로서 역할을 하는 것으로 확인되었다. 따라서 이 과정은 기아상태에서 에너지를 생성하는 기작이 될 수 있는 것으로 사료된다. 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 A₄ activity increased and anaerobic metabolism predominated. While LDH activity in the heart and kidney tissues decreased, LDH B4 activity increased and aerobic metabolism predominated, producing pyruvic acid. In the testis tissue, LDH C₄ activity decreased. In the brain tissue, LDH activity increased, but the isozyme change was small and the amount of pyruvic acid decreased. Km<SUP>PYR</SUP> 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, C₄ 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.

젖산탈수소효소 eye-specific C₄ 동위효소의 생화학적 특성

Jung Joo Yum(염정주),Bora Ku(구보라) 한국생명과학회 2012 생명과학회지 Vol.22 No.2

눈조직의 젖산탈수소효소(LDH, EC 1.1.1.27) eye-specific C₄ 동위효소의 특성을 native-PAGE, Western blotting, 면역침강반응 및 효소 역학을 이용하여 연구하였고, LDH eye-specific C₄ 동위효소의 활성을 측정하는 조건을 제시하였다. 파랑볼우럭(Lepomis macrochirus)과 큰입우럭(Micropterus salmoides) 눈조직의 세포기질에서 LDH eye-specific C₄ 동위효소가 확인되었으며 B₄ 동위효소보다 A₄ 동위효소에 유사하였다. 파랑볼우럭 눈조직의 LDH/CS는 9월에 증가하여 혐기적 대사가 높게 이루어졌다. 파랑볼우럭과 큰입우럭 눈조직 미토콘드리아 LDH의 전기영동상은 세포기질 LDH와 유사하였다. 눈조직의 LDH eye-specific C₄ 동위효소는 Preparative native-PAGE에 의해 정제되었다. 파랑볼우럭과 큰입우럭의 LDH eye-specific C₄ 동위효소의 활성은 피루브산 0.2 mM과 0.1 mM 이상의 농도에서 각각 감소되었고, 피루브산 10 mM에서 5.2%, 15.8% 활성이 남아 저해 정도가크게 나타났다. 그리고 눈조직의 LDH 활성도 젖산 22 mM과 24 mM 이상의 농도에서 각각 감소되어졌다. 파랑볼우럭 eye-specific C₄ 동위효소의 KmPYR는 0.088 mM, 큰입우럭 eye-specific C₄ KmPYR는 0.033 mM였으며, 세포기질과 미토콘드리아 eye-specific C₄ 동위효소는 ?-ketobutyric acid에서 활성이 높게 나타났다. 눈조직과 eye-specific C₄ 동위효소의 활성은 피루브산 0.5 mM과 Tris-HCl 완충액, pH 7.5에서 측정하는 것이 적합한 것으로 확인되었다. 실험 결과, 큰입우럭 LDH eye-specific C₄ 동위효소는 파랑볼우럭 LDH eye-specific C₄보다 피루브산에 대한 친화력이 큰 것으로 확인되었고, 더 낮은 피루브산의 농도에서 최대 활성에 이르고, 더 높은 젖산의 농도에서 최대 활성을 나타냈다. 따라서 LDH eye-specific C₄ 동위효소에 의해 생성된 에너지가 포식 행동의 초기 반응에 사용되는 것으로 사료된다. The properties of lactate dehydrogenase (LDH, EC 1.1.1.27) eye-specific C₄ isozyme were studied by polyacrylamide gel electrophoresis, Western blotting, immunoprecipitation, and enzyme kinetics. Furthermore, we proposed the optimal conditions for measuring the activity of LDH eye-specific C₄ isozyme. The isozymes were detected in the cytosol of eye tissues from Lepomis macrochirus and Micropterus salmoides and were more similar to the A₄ than the B₄ isozyme. LDH/CS in the eye tissue of L. macrochirus was increased in September, so the ratio of anaerobic metabolism was high. The electrophoretic patterns of mitochondrial LDH were similar to those of cytosolic LDH in the eye tissues of L. macrochirus and Micropterus salmoides. LDH eye-specific C₄ isozyme from eye tissue was purified by preparative native-PAGE. The activities of LDH eye-specific C₄ isozymes in L. macrochirus and M. salmoides were reduced at concentrations greater than 0.2 mM and 0.1 mM of pyruvate, respectively. These concentrations remained at 5.2% and 15.8% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The LDH activities of eye tissues were reduced at concentrations greater than 22 mM and 24 mM of lactate, respectively, in L. macrochirus and M. salmoides. The Km PYR of eye-specific C₄ was 0.088 mM in L. macrochirus and it was 0.033 mM in M. salmoides. The activities of cytosolic and mitochondrial eye-specific C₄ isozymes were high in α-ketobutyric acid. Furthermore, the activities of eye tissue and eye-specific C₄ isozyme had to be measured with 0.5 mM of pyruvate and a buffer solution of pH 7.5. As a conclusion, the eye-specific C₄ isozyme in M. salmoides has a high affinity for pyruvate and exhibits maximum activity at a lower concentration of pyruvate and at higher concentration of lactate than that in L. macrochirus. Therefore, it seems that the energy produced by the LDH eye-specific C₄ isozyme in M. salmoides was used at the first stage of predatory behavior.

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.

환경변화에 따른 꺽지 ( Coreoperca herzi ) 젖산수소이탈효소 동위효소의 적응상

박선영,염정주 ( Seon Young Park,Jung Joo Yum ) 한국환경생물학회 1995 환경생물 : 환경생물학회지 Vol.13 No.2

Lactate dehydrogenase(EC 1.1.1.27. Lactate dehydrogenase: LDH) in the tissues of Coreoperca herzi was studied on the expression of Ldh-A, B, and C by environmental factors according to the seasonal changes. The water temperature varied from 1 to 24 1, the pH range was 5.6 to 9.0. and the level of dissolved oxygen was 8.4 to 17.5㎎/ℓ. The activity of LDH had the differences in tissues. It showed higher activity in skeletal muscle and heart and lower activity in kidney, eye. brain, and liver. The relative activity of subunit A and B was affected by the pH range and the level of dissolved oxygen. It was showed that the kind of subunits had the difference on the tissue specificity. The relative activity of homotetrameric isozymes was higher than that of heterotetramers in each tissue. The relative activity of heterotetrameric isozymes was followed A_2B_2, A_3B, and AB_3 isozymes. The LDH AB_3 isozyme was absent in heart, liver, and brain tissues in spite of changes in environmental factors. The activity of LDH C_4 isozyme was lower than subunit A and B and it was especially lower level in a spawning season. The degree of inhibition of LDH by 10mM pyruvate was measured high level. The LDH isozymes were changed with the environmental changes, but if they were adapted to environmental situation they may maintain the metabolic states.

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

조성규,염정주,Cho Sung Kyu,Yum Jung Joo 한국생명과학회 2005 생명과학회지 Vol.15 No.1

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 $C_4$ 동위효소를 확인하였다. DO 18 ppm에 갑작스럽게 순응시 꺽지 LDH활성은 뇌와 간조직에서 각각 $35-39\%$ 변화되었고, 다른 조직에서는 $20\%$이내로 변화가 적었다. 골격근조직은 LDH $A_4$ 동위효소가 증가되고 하부단위체 C를 포함하는 동위효소는 감소되었다. 심장조직은 $B_4$ 동위효소가 조금 증가되었다. 신장조직도 $B_4$가 증가하고 눈 조직은 eye-specific $C_4$와 C hybrid가 감소되어 조절되었다. DO 증가에 갑작스럽게 순응한 모래무지 간조직에서 LDH 활성은 30분에서 급격하게 $150\%$이상 크게 증가되었고, 다른 조직에서는 $70\%$ 이상 변화되었다. 그리고 골격근조직은 $A_4$ 동위효소가 증가하고 다른 조직들에서는 $B_4$가 증가되었으며 특히 간 조직의 대사는 liver-specific $C_4$가 증가하고 $A_4$ 는 감소되어 조절되었다. 그러나 꺽지 눈 조직의 대사는 LDH 활성이 감소하고 eye-specific $C_4$ 동위효소가 감소되어 조절되었다. 따라서 DO증가에 순응시 모래무지는 꺽지에 비해 LDH 활성이 크게 증가되었고 동위효소의 변화정도도 크게 나타났으며, eye-spe-cific $C_4$와 liver-specific $C_4$ 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 $C_4$ 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.

햄스터와 소의 젖산탈수소효소에 대한 미토콘드리아 inhibitor의 영향

조성규,이상학,염정주,Cho Sung Kyu,Lee Sang Hak,Yum Jung Joo 한국생명과학회 2005 생명과학회지 Vol.15 No.1

젖산탈수소효소(EC 1.1.1.27, lactate dehydrogenase, LDH) inhibitor는 햄스터와 소 골격근 미토콘드리아에서 분리하였다. 햄스터 골격근조직의 LDH inhibitor는 175 mM NaCl과 초음파로 분리하였다. 소 골격근조직의 미토콘드리아에서 분리된 inhibitor는 열에 강한 특성을 보였고, $A_4$ 동위효소에 대한 저해정도가 높았으며, 분자량은 22,000 kDa으로 나타났다. Inhibitor는 심장조직을 제외한 골격근, 신장 및 간 조직의 미토콘드리아에서 LDH 결합 시 중요하게 관여하고 있었다. The lactate dehydrogenase (EC 1.1.1.27, LDH) inhibitors were isolated from the LDH-free crude mitochondrial fraction of skeletal muscle in Syrian hamster (Mesocricetus auratus) and Korean native cattle (Bos taurus coreanae). The LDH inhibitor in skeletal muscle of M. auratus was successfully isolated by the treatment with 175 mM NaCl and ultrasonic. The LDH inhibitor in skeletal muscle of B. taurus coreanae was highly stable to heat and LDH fu isozyme was largely inhibited by the LDH inhibitor. The molecular weight of inhibitor was 22 kDa. Inhibitor played an important role in the binding of LDH with the mitochondria in tissues of skeletal muscle, kidney and liver except heart.

가물치(Channa argus) 젖산탈수소효소 동위효소들의 정제 및 특성

Eun Mi Park(박은미),Jung Joo Yum(염정주) 한국생명과학회 2010 생명과학회지 Vol.20 No.2

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 ㎎/g이며, 특이활성(units/mg)은 골격근 25.88, 심장 0.79, 눈 0.31, 뇌 1.38 units/㎎이었으므로 골격근은 혐기적이고, 심장은 호기적이었다. LDH A₄, B₄, eye-specific C₄에 대한 항혈청을 사용한 Western blot, 면역침강반응 및 native- polyacrylamide gel electrophoresis에 의해 A₄, A₃B, A₂B₂, AB₃ 및 B₄가 모든 조직에서 확인되었고, 눈 조직에서 C₄와 AC₃, A₂C₂, AC₃, 뇌 조직에서 A₃C도 확인되었다. LDH A₄, A₃B, A₂B₂, AB₃, B₄, eye-specific C₄ 동위효소는 affinity chromatography와 Preparative PAGE Cell에 의해 정제되었다. LDH A₄ 동위효소는 NAD+ 유입 후 정제되었고, eye-specific C₄는 A₄에 이어 용출되기 시작하였으며 B₄는 buffer 유입 후 용출되었다. 정제한 결과 A₄는 B₄ 및 eye-specific C₄와 분자구조의 일부가 유사하였지만 B₄와 C₄는 서로 다른 것으로 나타났으므로, 하부단위체 A는 보존적이고, 하부단위체 B는 A보다 더 빠르게 진화된 것으로 보인다. 피루브산 10 mM에서 A₄ 동위효소 39.98%, A₂B₂ 21.28%, B4 19.67% 및 eye-specific C₄16.87%의 활성이 남아있었고, 피루브산에 대한 Km<SUP>PYR</SUP>은 A₄ 0.17 mM, B4 0.27 mM, eye-specific C₄ 0.133 mM였다. A₄, B₄, eye-specific C₄, A₂B₂, A₃B 및 AB₃의 최적 pH는 각각 pH 6.50, pH 8.5, pH 5.5, pH 6.0-6.5, 5.0 및 pH 7.5였고, 동질사량체 A₄와 이질사량체 동위효소들은 넓은 pH 영역에서 안정하였다. 특히 골격근은 LDH 활성이 크므로 활동성이 크며, 눈조직에서 피루브산 친화력이 강한 eye-specific C₄에 의해 피루브산 대사가 빠르게 일어나고, 이어서 A₄에 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 ㎎/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/㎎, 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 A₄, B₄, and eye-specific C₄. LDH A₄, A₃B, A₂B₂. AB₃ and B₄ isozymes were detected in every tissue, C4, AC₃, A₂C₂ and A₃C were detected in eye tissue, and A₃C was found in brain tissue. LDH A₄, A₃B, A₂B₂, AB₃, B₄, eye-specific C₄ isozymes were purified by affinity chromatography and Preparative PAGE Cells. The LDH A₄ isozyme was purified in the fraction from elution with NAD+containing buffer of affinity chromatography. Eye-specific C₄ isozyme was eluted right after A₄, after which B₄ isozyme was eluted with plain buffer. As a result, one part of molecular structures in A₄, B₄ and eye-specific C₄ were similar, but were different from each other in B₄ and C₄. 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 A₄, A₂B₂, B₄, and eye-specific C₄ 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 Km<SUP>PYR</SUP> values were 0.17, 0.27 and 0.133 mM in A₄, B₄ and eye-specific C₄ isozymes, respectively. The optimum pH of LDH A₄, B₄, eye-specific C₄, A₂B₂, A₃B, and AB₃ were pH 6.5, pH 8.5, pH 5.5, pH 6.0-6.5, pH 5.0 and pH 7.5. The A₄ 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 C₄ isozyme as eye-specific C₄ have the highest affinity for pyruvate, and right after the conversion, oxidation of lactate was induced by A₄ isozyme. It was found that expression of Ldh-C, affinity to substrate and reaction time of C₄ isozyme were different according to the ecological environmental and feeding capturing patterns.