Subunit Interactions of Vertebrate Lactate Dehydrogenase: II. Molecular Hybridizationa

Yum, Jung-Joo,Kim, Sang-Yeop,Park, Sang-Yoon The Korean Society for Integrative Biology 1981 동물학회지 Vol.24 No.4

In vitro intra- and interspecific molecular hybridizations of lactate dehydrogenase isozymes from six species were performed by freezing and thawing the isozymes in sodium chloride, resulting each of the interspecific hybridization of Fluta alba $H_4 \\times$ Ophicephalus argus $M_4$ and that of O. argus $M_4 \\times$ Parasilurus asotus isozymes showed one hybrid isozyme considered to be an $M_2H_2$ isozyme. The results fit with the suggestion that the isozyme of lower vertebrates is a dimer of homodimer as far as te assemblies of subunit M and subunit H are concerned. 척추동물 6종의 젖산탈수소효소의 초자내 종간 및 종내 분자교잡을 실시한 결과 드렁허리 $H_4$ 동위효소와 가물치 $M_4$ 동위효소의 교잡 및 가물치 $M_4$동위효소와 메기 동위효소의 교잡에서 $H_2M_2$ 동위효소라고 생각되는 교잡형 동위효소를 얻었다. 하등 척추동물 젖산탈수소효소의 하부단위체 M과 H의 상호간 조합에 관한 한 3차 구조의 하부단위체는 동일한 구조를 가진 하부단위체와 이량체를 형성하고 이러한 동질이량체가 사량체를 형성한다고 사료된다.

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.

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.

Affinity Chromatographic and Immunochemical Analysis of the Single Zone Lactate Dehydrogenases

Yum, Jung Joo,Kim, Sang Yeop 청주대학교 산업과학연구소 1984 産業科學硏究 Vol.2 No.2

초산 셀룰로우즈 전기동영상에서 단일대를 나타내는 Gallus 및 Parasilurus asotus의 젖산수소이탈효소(LDH)에 대하여 affinity chromatography 및 면역확산 실험을 실시하였다. CNBr에 의해 활성화된 Sepharose-4 B에 이 단일대 LDH를 크로마트그라피한 결과 5가지 대를 갖는 LDH들에서와 마찬가지로 NAD분획과 NADH분획으로 분리되었다. 순수정제한 3가지 동위효소에 대한 항혈청과 몇가지 효소 추출액사이의 항원-항체 반응에 참여하지 못한 LDH를 전기영동한 결과 단일대 LDH의 형성에 두 가지 동위효소가 모두 관여하고 있음이 확인되었다. 이러한 결과들로부터 단일대의 LDH들은 상이한 동위효소들의 혼합체라고 사료된다. 본 affinity chromatography에서도 NAD가 이질사량체의 H 하부단위체에 결합할 경우 사량체의 conformation이 변화됨이 재확인되었다. Lactate dehydrogenase (LDH) isozymes of Gallus gallus and Parasilurus asotus, which demonstrate single zone in cellulose acetate electrophoresis, were subjected to affinity chromatography and immunodiffusion tests. Affinity chromatography of the single zone LDHs on Sepharose-4B activated by CNBr resulted in the separation of the enzymes into the NAD and NADH fractions just as that of the five zone LDHs. The electrophoresis of LDH isozymes not participated in the antigen-antibody complex formation between several enzyme preparations and the antisera against three purified proteins revealed that both subunits take part in the formation of LDH which shows one zone in the electrophoresis. These results fit with the suggestion that the single zone LDH might be a mixture of different isozymes. It was reaffirmed in this affinity chromatography that the binding of NAD to the H subunits in a heteropolymer alters the conformation of the tetramers.

Increment and Simplification of Zone Number of Vertebrate LDH Isozymes

Yum, Jung Joo,Kim, Sang Yeop 圓光大學校 基礎自然科學硏究所 1984 基礎科學硏究誌 Vol.3 No.1

젖산수소이탈효소(LDH)의 하부단위체의 구조변화를 유발시킬 수 있는 요소들을 찾기 위해 9가지의 상이한 완충용액하에서 척추동물 6종의 LDH 동위효소들의 전기영동을 실시하였다. Gallus gallus 및 Rana nigromaculata 골격근의 단일 LDH는 Na-citrate 완충용액하에서는 분리되지 않으나 Na-acetate 완충용액하에서는 5개의 동위효소로 분리되었고, Parasilurus asotus LDH의 경우 조직에 따라 2개의 zone이 확인되었으며, 두가지 하부단위체를 모두 갖고 있는 다른 조직들의 LDH는 사용된 완충용액의 조성에 따라 1∼5개의 분리양상을 나타냈다. 이러한 동위효소의 증가 및 단순화는 완충용액내의 음이온 또는 해리되지 않은 산이 하부단위체의 표면에 결합되기 때문이라고 사료된다. In an attempt to find factors expected to have effect on the change of tertiary structure of vertebrate LDH, the isozymes from six species were electrophoresed in nine buffers of different components and pHs with emphasis on the so-called one isozyme type LDH. No separation of the skeletal muscle isozymes from Gallus gallus and Rana nigromaculata were revealed in sodium citrate buffer, while five isozymes were exhibited in sodium acetate buffer. The other tissues characterized by both M and H genes have made various separation patterns depending on the buffers except that zymograms of Parasilurus asotus LDH demonstrated only two zones in buffers used, suggesting that the increment and simplification of zone number might be brought about by either the buffer anion or the buffer acid which binds to the surface of the subunit polypeptide.

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.

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.

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.

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.

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.