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쥐노래미 eye-specific LDH C4 동위효소의 정제 및 특성

Purification and Characterization of Eye-Specific Lactate Dehydrogenase C4 Isozyme in Greenling (Hexagrammos otakii)

  • 조성규 (청주대학교 산업과학연구소) ;
  • 염정주 (청주대학교 자연과학부 생명과학)
  • Cho, Sung-Kyu (Industrial Science Research Institute, Cheongju University) ;
  • Yum, Jung-Joo (Department of Life Science, Cheongju University)
  • 투고 : 2011.08.31
  • 심사 : 2011.11.08
  • 발행 : 2011.11.30

초록

쥐노래미(Hexagrammos otakii) 눈 조직의 젖산탈수소효소(EC 1.1.1.27, lactate dehydrogenase, LDH) eye-specific $C_4$ 동위효소를 affinity chromatography 및 continuous-elution electrophoresis system으로 정제하였다. 정제된 eye-specific LDH $C_4$ 동위효소의 분자량은 154.8 kDa이었다. 효소반응의 최적 pH는 8.5로 나타났다. 피루브산을 기질로 하였을 때 eye-specific LDH $C_4$ 동위효소의 $K^{PYR}_m$ 값은 $1.88{\times}10^{-5}$ M로 나타났다. 본 실험결과 eye-specific LDH $C_4$ 동위효소의 활성 측정 시 pH를 고려해야만 하고 eye-specific LDH $C_4$ 동위효소는 LDH A4 동위효소보다 피루브산에 대한 기질결합친화성이 큰 것으로나타났다. 생성된 eye-specific LDH $C_4$ 동위효소에 대한 항체는 사람에서의 병증진단 및 어류에서의 비교생리학적 연구에 폭 넓게 사용될 것이라고 생각된다.

Eye-specific lactate dehydrogenase (EC 1.1.1.27, LDH) $C_4$ isozyme in the eyes of greenlings (Hexagrammos otakii) was successfully purified by affinity chromatography and continuous-elution electrophoresis. The molecular weight of the purified eye-specific LDH $C_4$ isozyme was 154.8 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimal pH for enzymatic reaction of the eye-specific LDH $C_4$ isozyme was pH 8.5. $K^{PYR}_m$ value of the purified eye-specific LDH $C_4$ isozyme was $1.88{\times}10^{-5}$ M using pyruvate as a substrate. These results indicate that we must consider pH when measuring eye-specific LDH $C_4$ isozyme activity. The eye-specific LDH $C_4$ isozyme had a higher binding affinity for the substrate as a pyruvate than LDH A4 isozyme. Antibodies produced against the purified eye-specific LDH $C_4$ isozyme may be used in the diagnosis of several human diseases and in comparative physiological studies of fishes.

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참고문헌

  1. 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
  2. An, C. S., S. K. Cho, and J. J. Yum. 2010. Variation of lactate dehydrogenase isozymes in angelfish (Pterophyllum scalare) according to acute environmental change. J. Life Sci. 20, 416-423. https://doi.org/10.5352/JLS.2010.20.3.416
  3. Andriutsa, K. A. and A. K. Andriutsa. 1987. Diagnostic and pathogenetic significance of determining lactate dehydrogenase isoenzyme activity in various biological media in patients with viral hepatitis. Ter. Arkh. 59, 89-94.
  4. Baldwin, J. and P. S. Lake. 1987. Lactate dehydrogenase homopolymer of hagfish heart and the single lactate dehydrogenase of lampreys display greater immunochemical similarity to LDH $C_{4}$ than to LDH $B_{4}$ of teleost fish. J. Exp. Zool. 242, 99-102. https://doi.org/10.1002/jez.1402420114
  5. Baldwin, J., K. Mortimer, and A. Patak. 1988. Do ascidians possess the ancestral subunit type of vertebrate lactate dehydrogenase. J. Exp. Zool. 246, 109-114. https://doi.org/10.1002/jez.1402460202
  6. 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
  7. Cho, S. K. 2000. Mitochondrial lactate dehydrogenase in tissues of vertebrate. Ph. D. Thesis, Cheongju University, Cheongju, Korea.
  8. 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 $A_{4}$ isozyme in mandrin fish (Siniperca scherzeri). J. Life Sci. 19, 256-263. https://doi.org/10.5352/JLS.2009.19.2.256
  9. Cho, S. K., S. Y. Park, and J. J. Yum. 1993. Purification and immunochemistry of lactate dehydrogenase isozyme in Lampetra japonica. Korean J. Zool. 36, 505-513.
  10. Cho, S. K. and J. J. Yum. 1993. Heterogeneity of lactate dehydrogenase isozymes in tissues of Lampetra japonica. Korean J. Zool. 36, 319-328.
  11. 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. https://doi.org/10.5352/JLS.2004.14.4.702
  12. Cho, S. K. and J. J. Yum. 2005. Changes of activities and isozymes of lactate dehydrogenase in Coreoperca herzi and Pseudogobio esocinus acclimated to rapid increase of dissolved oxygen. J. Life Sci. 15, 71-79. https://doi.org/10.5352/JLS.2005.15.1.071
  13. Coonrod, S., A. Vitale, C. Duan, S. Bristol-Gould, J. Herr, and E. Goldberg. 2006. Testis-specific lactate dehydrogenase (LDH-$C_{4}$; Ldh3) in murine oocytes and preimplantation embryos. J. Androl. 27, 502-509. https://doi.org/10.2164/jandrol.05185
  14. Coppes, Z. 1992. Lactate dehydrogenase in teleosts: the role of LDH-$C_{4}$ isozyme. Comp. Biochem. Physiol. B 102, 673-677. https://doi.org/10.1016/0305-0491(92)90063-W
  15. Coronel, C. E., C. Burgos, N. M. Gerez de Burgos, L. E. Rovai, and A. Blanco. 1983. Catalytic properties of the sperm-specific lactate dehydrogenase (LDH X or $C_{4}$) from different species. J. Exp. Zool. 225, 379-385. https://doi.org/10.1002/jez.1402250305
  16. 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
  17. Donovan, J. and P. Brown. 1991. Cardiac Puncture of Rabbit, Unit 1.7.6., In Coligan, J. E. et al. (eds.), Current Protocols in Immunology, Vol. 1, John Wiley & Sons Inc., New York.
  18. Fisher, S. E., J. B. Shaklee, S. D. Ferris, and G. S. Whitt. 1980. Evolution of five multilocus isozyme systems in the chordates. Genetica 52/53, 73-85. https://doi.org/10.1007/BF00121817
  19. Goldberg, E. 1972. Amino acid composition and properties of crystalline lactate dehydrogenase X from mouse testes. J. Biol. Chem. 247, 2044-2048.
  20. Goldberg, E., E. M. Eddy, C. Duan, and F. Odet. 2010. LDHC: the ultimate testis-specific gene. J. Androl. 31, 86-94. https://doi.org/10.2164/jandrol.109.008367
  21. Higuti, I. H., S. W. Grande, R. Sacco, and A. J. Nascimento. 2003. Isolation of alkalophilic CGTase-producing bacteria and characterization of cyclodextrin-glycosyltransferase. Braz. Arch. Biol. Technol. 46, 183-186. https://doi.org/10.1590/S1516-89132003000200007
  22. Holbrook, J. J., A. Liljas, S. J. Steindel, and M. G. Rossmann. 1975. Lactate dehydrogenase, pp. 191-192, In Boyer, P. D. (ed.), The Enzymes., 3rd eds., Vol. XI. Academic Press Inc., New York.
  23. Kim, J. B., S. K. Cho, and J. J. Yum. 2004. Changes of activities and isozymes of lactate dehydrogenase in Coreoperca herzi acclimated to acute increase of temperature for short-term period. J. Ind. Sci., Cheongju Univ., Korea. 22, 43-50.
  24. Kim, M. O. and J. J. Yum. 1989. Purification, kinetics and immunochemistry of two homotetrameric lactate dehydrogenase isozymes in Pseudogobio esocinus (Cypriniformes). Korean J. Zool. 32, 420-428.
  25. Kolev, Y., H. Uetake, Y. Takagi, and K. Sugihara. 2008. Lactate dehydrogenase-5 (LDH-5) expression in human gastric cancer: association with hypoxia-inducible factor (HIF-1$\alpha$) pathway, angiogenic factors production and poor prognosis. Ann. Surg. Oncol. 15, 2336-2344. https://doi.org/10.1245/s10434-008-9955-5
  26. 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.
  27. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage $T_{4}$. Nature 227, 680-685. https://doi.org/10.1038/227680a0
  28. Lam, C. W., M. H. Chan, and C. K. Wong. 2004. Severe acute respiratory syndrome: clinical and laboratory manifestations. Clin. Biochem. Rev. 25, 121-132.
  29. Markert, C. L., J. B. Shaklee, and G. S. Whitt. 1975. Evolution of a gene: multiple genes for LDH isozymes provide a model of the evolution of gene structure, function and regulation. Science 189, 102-114. https://doi.org/10.1126/science.1138367
  30. 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, 281-285. https://doi.org/10.1016/0014-5793(72)80183-2
  31. O'Carra, P., S. Barry, and E. Corcoran. 1974. Affinity chromatographic differentiation of lactate dehydrogenase isoenzymes on the basis of differential abortive complex formation. FEBS Letters 43, 163-168. https://doi.org/10.1016/0014-5793(74)80992-0
  32. Painter, P. C., S. Van Meter, R. L. Dabbs, and G. E. Clement. 1994. Analytical evaluation and comparison of Dupont aca lactate dehydrogenase-1 (LD1) isoenzyme assay diagnostic efficiency for acute myocardial infarction detection with other LD1 methods and aca CK-MB. A two-site study. Angiology 45, 585-595. https://doi.org/10.1177/000331979404500701
  33. Park, S. Y., S. K. Cho, and J. J. Yum. 2004. Characterization and evolutionary relationship of lactate dehydrogenase in liver of Lampetra japonica and liver-specific $C_{4}$ isozyme in Gadus macrocephalus. J. Life Sci. 14, 708-715. https://doi.org/10.5352/JLS.2004.14.4.708
  34. Park, S. Y. and J. J. Yum. 1993. Lactate dehydrogenase isozymes of cypriniform and perciform fishes: expression of the Ldh-C gene. J. Ind. Sci., Cheongju Univ., Korea. 11, 265-277.
  35. 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, 263-270.
  36. Park, E. M. and J. J. Yum. 2010. Purification and characterization of lactate dehydrogenase isozymes in Channa argus. J. Life Sci. 20, 260-268. https://doi.org/10.5352/JLS.2010.20.2.260
  37. Park, E. M. and J. J. Yum. 2011. Activities of lactate dehydrogenase and ratios of lactate dehydrogenase/citrate synthase in tissues of Odontobutis interrupta. J. Ind. Sci., Cheongju Univ., Korea. 28, 15-24.
  38. Rehse, P. H. and W. S. Davidson. 1986. Purification and properties of a C-type isozyme of lactate dehydrogenase from the liver of the Atlantic cod (Gadus morhua). Comp. Biochem. Physiol. B 84, 145-150. https://doi.org/10.1016/0305-0491(86)90196-3
  39. Sensabaugh, G. F. and N. O. Kaplan. 1972. A lactate dehydeogenase specific to the liver of gadoid fish. J. Biol. Chem. 247, 585-593.
  40. Shaklee, J. B., K. L. Kepes, and G. S. Whitt. 1973. Specialized lactate dehydrogenase isozymes: the molecular and genetic basis for the unique eye and liver LDHs of teleost fishs. J. Exp. Zool. 185, 217-240. https://doi.org/10.1002/jez.1401850209
  41. Whitt, G. S. 1970. Developmental genetics of the lactate dehydrogenase isozymes of fish. J. Exp. Zool. 175, 1-35. https://doi.org/10.1002/jez.1401750102
  42. Whitt, G. S. 1981. Evolution of isozyme loci and their differential regulation, pp. 271-289, In Scudder, G. G. E. and J. L. Reveal (eds.), Evolution today: proceedings of second international congress of systematic and evolutionary biology. Hunt Institute for Botanical Documentation, Carnegie-Mellon Univ. Press, Pittsburgh.
  43. 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

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