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생명과학회지 = Journal of life science v.18 no.2 = no.94, 2008년, pp.264 - 272   피인용횟수: 8

풀망둑(Acanthogobius hasta) 젖산탈수소효소의 특성
Characterization of Lactate Dehydrogenase in Acanthogobius hasta

염정주   (청주대학교 생명유전통계학부 생명과학UU0001267  );
  • 초록

    풀망둑(Acanthogobius hasta) 조직의 젖산탈수소효소(EC 1.1.1.27 Lactate dehydrogenase, LDH) 동위 효소의 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/mg였다. 각 조직에 대해 $A_4,\;B_4$ , eye-specific $C_4$ , 및 liver-specific $C_4$ 에 대한 항혈청으로 면역 침강 반응시킨 후 Polyacrylamide gel 전기영동 하였다. 골격근, 뇌 및 눈 조직에서 $A_4$ 동위효소가 우세하게 확인되었고, 심장에서는 $A_4$ 동위효소가 확인되었다. 또한 양극의 eye-specific t와 음극의 liver-specific $C_4$ 가 한 종에서 함께 발현되었으며, 눈 조직의 eye-specific $C_4$ 는 활성이 크고 간 조직의 liver-Specific $C_4$ 의 활성은 낮게 나타났다. 결과 $A_4$ 와 $C_4,\;A_4$ 와 $B_4$ 및 eye-specific $C_4$ 와 liver-specific $C_4$ 의 분자구조의 일부가 서로 유사하지만 $B_4$ 와 $C_4$ 의 구조는 서로 다른 것으로 나타났으므로 하부단위체 A는 보존적이고 하부단위체 B는 하부단위체 A보다 빠르게 진화된 것으로 사료된다. 골격근 조직의 LDH $A_4$ 동위효소는 affinity chromatography에서 $NAD^+$ 를 함유한 buffer를 유입한 후 용출된 분획에서 정제되었고, eye-specific $C_4$ 동위효소는 $A_4$ 분획에 이어 용출되었으므로 eye-specific $C_4$ 가 $A_4$ 의 분자 구조와 유사한 것으로 보인다. 그리고 LDH에 대한 피루브산의 저해 실험 결과 35.22-43.47% 의 활성이 남았고, $Km_{pyr}$ 은 0.080-0.098 mM이고 골격근과 눈조직의 Vmax은 153.85와 35.09 units였다. 또한 $B_4$ 동위효소가 열에 대해 가장 안정하였고 $C_4$ 는 $A_4$ 보다 안정하였으며, 최적 pH는 6.5로 나타났다. 본 실험 결과 풀망둑은 저 산소 환경조건에 적응되어져 조직들의 동위효소들이 $A_4$ 와 $B_4$ 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/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.


  • 주제어

    Acanthogobius hasta .   lactate dehydrogenase .   eye-specific $C_4$ isozyme .   Km .   Vmax.  

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  • 이 논문을 인용한 문헌 (8)

    1. Cho, Sung-Kyu ; Ku, Bo-Ra ; An, Hyo-Jung ; Park, Eun-Mi ; Park, Seon-Young ; Kim, Jae-Bum ; Yum, Jung-Joo 2009. "Purification and Characterization of Lactate Dehydrogenase A4 Isozyme in Mandrin Fish (Siniperca scherzeri)" 생명과학회지 = Journal of life science, 19(2): 256~263     
    2. Park, Eun-Mi ; Yum, Jung-Joo 2010. "Purification and Characterization of Lactate Dehydrogenase Isozymes in Channa argus" 생명과학회지 = Journal of life science, 20(2): 260~268     
    3. Cho, Sung-Kyu ; Yum, Jung-Joo 2011. "Purification and Characterization of Eye-Specific Lactate Dehydrogenase C4 Isozyme in Greenling (Hexagrammos otakii)" 생명과학회지 = Journal of life science, 21(11): 1565~1572     
    4. Yum, Jung-Joo ; Yeon, Jun-Hee 2012. "Lactate Dehydrogenase and Monocarboxylate Transporters 1, 2, and 4 in Tissues of Micropterus salmoides" 생명과학회지 = Journal of life science, 22(1): 98~109     
    5. Yum, Jung-Joo ; Ku, Bo-Ra 2012. "Biochemical Properties of Lactate Dehydrogenase Eye-Specific C4 Isozyme: Lepomis macrochirus and Micropterus salmoides" 생명과학회지 = Journal of life science, 22(2): 209~219     
    6. Yum, Jung Joo ; Ha, Eun Sung 2014. "Kinetic Properties of Lactate Dehydrogenase in Tissues from Rana catesbeiana" 생명과학회지 = Journal of life science, 24(2): 118~127     
    7. Yum, Jung Joo ; Kim, Gyu Dong 2016. "Metabolism of Lactate Dehydrogenase in Tissues from Ldh-C Expressed Fish at Starved State" 생명과학회지 = Journal of life science, 26(2): 155~163     
    8. Ku, Bora ; Cho, Sung Kyu ; Yum, Jung Joo 2016. "Metabolic Adjustments of Lactate Dehydrogenase Isozymes to the Environmental Temperature in Bluegill (Lepomis macrochirus)" 생명과학회지 = Journal of life science, 26(10): 1105~1112     

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    1. 1989 "Pseudogobio esocinus (Cypriniformes) 젖산수소이탈효소 동질사량체들의 정제, 역학 및 면역화학" 동물학회지 = The Korean journal of zoology 32 (4): 420~428    
    2. 1993 "칠성장어(Lampetra japonica) 조직내 젖산수소이탈효소 동위효소들의 이질성" 동물학회지 = The Korean journal of zoology 36 (3): 319~328    
    3. 1995 "대구 간조직 미토콘드리아 젖산수소이탈효소의 생화학적 특성" 한국동물학회 뉴스레터 = The Zoological Society Korea : Newsletter 12 (2): 110.1~110.1    
    4. 1999 "방사선 조사 후 생쥐(Mus musculus)조직의 형태와 젖산탈수소효소 동위효소의 재분포" 환경생물 = Korean journal of environmental biology 17 (3): 263~270    
    5. 2004 "저산소 환경에 서식하는 열대성 catfish (Pangasius Polyuranodon, Hypostomus Ple-Costomus)의 젖산탈수소효소 동위효소" 생명과학회지 = Journal of life science 14 (4): 702~707    
    6. 2004 "칠성장어(Lampetra japnica) 간조직 젖산탈수소효소와 대구(Gadus macrocephalus) liver-Specific C4동위효소의 특성 및 진화적 관계" 생명과학회지 = Journal of life science 14 (4): 708~715    
    7. 2005 "급격한 용존산소량 증가에 순응한 꺽지(Coreoperca herzi)와 모래무지(Pseudogobioesocinus) 젖산탈수소효소 활성과 동위효소의 변화" 생명과학회지 = Journal of life science 15 (1): 71~79    
    8. 2005 "햄스터와 소의 젖산탈수소효소에 대한 미토콘드리아 inhibitor의 영향" 생명과학회지 = Journal of life science 15 (1): 100~105    
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