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가물치(Channa argus) 젖산탈수소효소 동위효소들의 정제 및 특성

Purification and Characterization of Lactate Dehydrogenase Isozymes in Channa argus

  • 박은미 (청주대학교 생명유전통계학부 생명과학) ;
  • 염정주 (청주대학교 생명유전통계학부 생명과학)
  • Park, Eun-Mi (Department of Life Science, Cheongju University) ;
  • Yum, Jung-Joo (Department of Life Science, Cheongju University)
  • 투고 : 2010.01.04
  • 심사 : 2010.01.22
  • 발행 : 2010.02.28

초록

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 mg/g이며, 특이활성(units/mg)은 골격근 25.88, 심장 0.79, 눈 0.31, 뇌 1.38 units/mg이었으므로 골격근은 혐기적이고, 심장은 호기적이었다. LDH $A_4$, $B_4$, eye-specific $C_4$에 대한 항혈청을 사용한 Western blot, 면역침강반응 및 native-polyacrylamide gel electrophoresis에 의해 $A_4$, $A_3B$, $A_2B_2$, $AB_3$$B_4$가 모든 조직에서 확인되었고, 눈 조직에서 $C_4$$AC_3$, $A_2C_2$, $AC_3$, 뇌 조직에서 $A_3C$도 확인되었다. LDH $A_4$, $A_3B$, $A_2B_2$, $AB_3$, $B_4$, eye-specific $C_4$ 동위효소는 affinity chromatography와 Preparative PAGE Cell에 의해 정제되었다. LDH $A_4$ 동위효소는 $NAD^+$ 유입 후 정제되었고, eye-specific $C_4$$A_4$에 이어 용출되기 시작하였으며 $B_4$는 buffer 유입 후 용출되었다. 정제한 결과 $A_4$$B_4$ 및 eye-specific $C_4$와 분자구조의 일부가 유사하였지만 $B_4$$C_4$는 서로 다른 것으로 나타났으므로, 하부단위체 A는 보존적이고, 하부단위체 B는 A보다 더 빠르게 진화된 것으로 보인다. 피루브산 10 mM에서 $A_4$ 동위효소 39.98%, $A_2B_2$ 21.28%, $B_4$ 19.67% 및 eye-specific $C_4$ 16.87%의 활성이 남아있었고, 피루브산에 대한 $Km^{PYR}$$A_4$ 0.17 mM, $B_4$ 0.27 mM, eye-specific $C_4$ 0.133 mM였다. $A_4$, $B_4$, eye-specific $C_4$, $A_2B_2$, $A_3B$$AB_3$의 최적 pH는 각각 pH 6.50, pH 8.5, pH 5.5, pH 6.0-6.5, 5.0 및 pH 7.5였고, 동질사량체 $A_4$와 이질사량체 동위효소들은 넓은 pH 영역에서 안정하였다. 특히 골격근은 LDH 활성이 크므로 활동성이 크며, 눈조직에서 피루브산 친화력이 강한 eye-specific $C_4$에 의해 피루브산 대사가 빠르게 일어나고, 이어서 $A_4$에 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 mg/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/mg, 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 $A_4$, $B_4$, and eye-specific $C_4$. LDH $A_4$, $A_3B$, $A_2B_2$. $AB_3$ and $B_4$ isozymes were detected in every tissue, $C_4$, $AC_3$, $A_2C_2$ and $A_3C$ were detected in eye tissue, and $A_3C$ was found in brain tissue. LDH $A_4$, $A_3B$, $A_2B_2$, $AB_3$, $B_4$, eye-specific $C_4$ isozymes were purified by affinity chromatography and Preparative PAGE Cells. The LDH $A_4$ isozyme was purified in the fraction from elution with $NAD^+$ containing buffer of affinity chromatography. Eye-specific $C_4$ isozyme was eluted right after $A_4$, after which $B_4$ isozyme was eluted with plain buffer. As a result, one part of molecular structures in $A_4$, $B_4$ and eye-specific $C_4$ were similar, but were different from each other in $B_4$ and $C_4$. 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 $A_4$, $A_2B_2$, $B_4$, and eye-specific $C_4$ 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 $Km^{PYR}$ values were 0.17, 0.27 and 0.133 mM in $A_4$, $B_4$ and eye-specific $C_4$ isozymes, respectively. The optimum pH of LDH $A_4$, $B_4$, eye-specific $C_4$, $A_2B_2$, $A_3B$, and $AB_3$ were pH 6.5, pH 8.5, pH 5.5, pH 6.0-6.5, pH 5.0 and pH 7.5. The $A_4$ 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 $C_4$ isozyme as eye-specific $C_4$ have the highest affinity for pyruvate, and right after the conversion, oxidation of lactate was induced by $A_4$ isozyme. It was found that expression of Ldh-C, affinity to substrate and reaction time of $C_4$ isozyme were different according to the ecological environmental and feeding capturing patterns.

키워드

참고문헌

  1. Ahmad, R. 2009. Functional and adaptive significance of differentially expressed lactate dehydrogenase isoenzymes in tissues of four obligatory air-breathing Channa species. Section Cellular and Molecular Biology 64, 192-196.
  2. Ahmad, R. and A. U. Hasnain. 2005. Ontogenetic changes and developmental adjustments in lactate dehydrogenase isozymes of an obligate air-breathing fish Channa punctatus during deprivation of air access. Comp. Biochem. Physiol. B 140, 271-278. https://doi.org/10.1016/j.cbpc.2004.10.012
  3. Almeida-Val, V. M. F. and A. L. Val. 1993. Evolutionary trends of LDH isozymes in fishes. Comp. Biochem. Physiol. B 105, 21-28. https://doi.org/10.1016/0305-0491(93)90164-Z
  4. Almeida-Val, V. M. F. and P. W. Hochachka. 1995. Air-breathing fishes: metabolic bio-chemistry of the first diving vertebrates. pp. 45-55, In Hochachka, P. W. and T. Mommsen (eds.), Biochemistry and molecular biology of fishes. Elsevier Science, Amsterdam.
  5. Almeida-Val V. M. F., M. N. Paula-Silva, W. P. Duncan, N. P. Lopes, A. L. Val, and S. Land. 1999. Increase of anaerobic potential during growth of an Amazonian cichlid, Astronotus ocellatus. Survivorship and LDH regulation after hypoxia exposure. pp. 437-448, In Val A. L. and Almeida-Val V. M. F. (eds.), Biology of Tropical Fishes. INPA, Manaus.
  6. Almeida-Val, V. M. F., A. L. Val, W. P. Duncan, F. C. A. Souza, M. N. Paula-Silva, and S. Land. 2000. Scaling effects on hypoxia tolerance in the Amazon fish Astronotus ocellatus (Perciformes: Cichlidae): contribution of tissue enzyme levels. Comp. Biochem. Physiol. B 125, 219-226. https://doi.org/10.1016/S0305-0491(99)00172-8
  7. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  8. Chippari-Gomes, A. R., M. A. B. Leitao, M. N. Paula-Silva, L. S. B. Mesquita-Saad, and V. M. F. Almeida-Val. 2003. Metabolic adjustments in Satanoperca aff. jurupari (Perciformes: Cichlidae). Genet. Mol. Evol. 26, 27-32.
  9. Cho, S. K. 2000. Mitochondrial lactate dehydrogenase in tissues of vertebrate. Ph. D. Thesis Cheongju Univ., Korea.
  10. Cho, S. K., B. Ku, H. An, E. M. Park, S. Y. Park, J. B. Kim, and J. J. Yum. 2009. Purification and characterization of lactate dehydrogenase A4 isozyme in mandrin fish (Siniperca scherzeri). J. Life Sci. 19, 256-263. https://doi.org/10.5352/JLS.2009.19.2.256
  11. Cho, S. K., S. Y. Park, and J. J. Yum. 1993. Purification and immunochemistry of lactate dehydrogenase in Lampetra japonica. Korean J. Zool. 36, 505-513.
  12. Cho, S. K. and J. J. Yum. 2004. Lactate dehydrogenase isozyme of hypoxia tropical catfish (Pangasius polyuranodon, Hypostomus plecostomus). J. Life Sci. 14, 702-707.
  13. Dalla Via, J., G. V. den Thillart, O. Cattani, and A. de Zwaan. 1994. Influence of long-term hypoxia exposure on the energy metabolism of Solea solea: II. Intermediary metabolism in blood, liver and muscle. Mar. Ecol. Prog. Ser. 111, 17-27. https://doi.org/10.3354/meps111017
  14. Davis, B. J. 1964. Disc electrophoresis-II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427. https://doi.org/10.1111/j.1749-6632.1964.tb14213.x
  15. Einarsson, J. M., P. Joyce, and Y. W. Kunz. 1995. Kinetic and immunological differences between the retinal specific $C_4$-and $B_4$ lactate dehydrogenase of the cichlid fish Oreochromis mossambicus. Comp. Biochem. Physiol. B 112, 589-598. https://doi.org/10.1016/0305-0491(95)00114-X
  16. Fantin, V. R., J. St-Pierre, and P. Leder. 2006. Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell 9, 425-434. https://doi.org/10.1016/j.ccr.2006.04.023
  17. Goldberg, E. C., E. R. Gagliano, and M. C. G. Passeggi. 1985. Correlation effects in dynamical charge-transfer processes. Phys. Rev. B 32, 4375-4381. https://doi.org/10.1103/PhysRevB.32.4375
  18. Kim, I. S. and E. J. Kang. 1993. Coloured Fishes of Korea. pp. 406, Academy. Korea.
  19. Kim, J. B., S. K. Kim, and J. J. Yum. 2003. Changes of activities and isozymes of lactate dehydrogenase in Pseudogobio esocinus acclimated to acute change of temperature. J. Ind. Sci, Cheongju Univ. 21, 37-44.
  20. Kim, M. O. and J. J. Yum. 1989. Purification, kinetics and immunochemistry of two homotetrametric lactate dehydrogenase isozyme in Pseudogobio esocinus (Cypriniformes). Korean J. Zool. 32, 420-428.
  21. Koslowski, M., O. Tureci, C. Bell, P. Krause, H. A. Lehr, J. Brunner, G. Seitz, F. O. Nestle, C. Huber, and U. Sahin. 2002. Multiple splice variants of lactate dehydrogenase C selectively expressed in human cancer. Cancer Res. 62, 6750-6755.
  22. Moyes, C. D., O. A. Mathieu-Costello, R. W. Brill, and P. W. Hochachka. 1992. Mitochondrial metabolism of cardiac and skeletal muscle from a fast (Katsuwonus pelamis) and a slow (Cyprinus carpio) fish. Can. J. Zool. 70, 1246-1253. https://doi.org/10.1139/z92-172
  23. O'Brien, J., K. M. Kla, I. B. Hopkins, E. A. Malecki, and M. C. Mckenna. 2007. Kinetic parameters and lactate dehydrogenase isozyme activities support possible lactate uti lization by neurons. Neurochem Res. 32, 597-607.
  24. O'Carra, P. and S. Barry. 1972. Affinity chromatography of lactate dehydrogenase: model studies demonstrating the potential of the technique in the mechanistic investigation as well as in the purification of multi-substrate enzymes. FEBS Letters 21, 281-295. https://doi.org/10.1016/0014-5793(72)80183-2
  25. O'Carra, P., S. Barry, and E. Corcoran. 1974. Affinity chromatographic differentiation of lactate dehydrogenase isozymes on the basis of differential abortive complex formation. FEBS Letters 43, 163-168. https://doi.org/10.1016/0014-5793(74)80992-0
  26. Park, S. Y. and J. J. Yum. 1997. Purification and characterization of lactate dehydrogenase eye-and testis-specific $C_4$ isozyme. J. Ind. Sci, Cheongju Univ. Korea. 15, 263-268.
  27. Philp, A., A. L. Macdonald, and P. W. Watt. 2005. Lactate-a signal coordination cell and system function. J. Exp. Biol. 208, 4561-4575. https://doi.org/10.1242/jeb.01961
  28. Selch, T. M. and S. R. Chipps. 2007. The cost of capturing prey: measuring largemouth bass (Micropterus salmoides) foraging activity using glycolytic enzymes (lactate dehydrogenase). Can. J. Fish Aquat. Sci. 64, 1761-1769. https://doi.org/10.1139/F07-133
  29. Shaklee, J. B. and G. S. Whitt. 1981. Lactate dehydrogenase isozymes of Gadiform fishes: divergent patterns of gene expression indicate a heterogeneous taxon. Copeia 3, 567-578.
  30. Somero, G. N. 2004. Adaptation of enzymes to temperature: searching for basic "strategies". Comp. Biochem. Physiol B 139, 321-33. https://doi.org/10.1016/j.cbpc.2004.05.003
  31. Srere, P. A., H. Brazil, and L. Gonen. 1963. Citrate condensing enzyme of pigeon breast muscle and moth flight muscle. Acta Chem. Scand. 17, S129-134. https://doi.org/10.3891/acta.chem.scand.17s-0129
  32. Tylicki, A., D. Masztaleruk, and S. Strumilo. 2006. Differences in some properties of lactate dehydrogenase from muscles of the carp Cyprinus carpio and trout Salmo gairdneri. Comp Onto. Biochem. 42, 143-147.
  33. Whitt, G. S. 1970. Developmental genetics of the lactate dehydrogenase isozymes of fish. J. Exp. Zool. 175, 1-36. https://doi.org/10.1002/jez.1401750102
  34. Whitt, G. S. 1987. Species differences in isozyme tissue patterns: their utility for systematic and evolutionary analysis. pp. 1-26, In Current topics in biological and medical research. Alan Liss Inc. N.Y.
  35. Yum, J. J. 2008. Characterization of lactate dehydrogenase in Acanthogobius hasta. J. Life Sci. 18, 264-272. https://doi.org/10.5352/JLS.2008.18.2.264
  36. Yum, J. J. and M. O. Kim. 1989. Biochemical properties of lactate dehydrogenase isozymes in Pseudogobio esocinus. J. Ind. Sci., Cheongju Univ. Korea 7, 151-162.
  37. Zietara, M., J. Grouczewska, and E. F. Skorkowski. 1997. Purification and properties of the Threespine Stickleback (Gasterosteus aculeatus) lactate dehydrogenase LDH-$B_4$ and LDH-$C_4$ isoenzymes-Properties and evolutionary relationship. Comp. Boichem. Physiol. B 117, 571-577. https://doi.org/10.1016/S0305-0491(97)00003-5

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