Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Purification and Characterization of Lactate Dehydrogenase A4 Isozyme in Mandrin Fish (Siniperca scherzeri)

쏘가리(Siniperca scherzeri) 젖산탈수소효소 A4 동위효소의 정제 및 특성

  • Cho, Sung-Kyu (Industrial Science Research Institute, Cheongju University) ;
  • Ku, Bo-Ra (Department of Life Science, Cheongju University) ;
  • An, Hyo-Jung (Department of Life Science, Cheongju University) ;
  • Park, Eun-Mi (Department of Life Science, Cheongju University) ;
  • Park, Seon-Young (Department of Life Science, Cheongju University) ;
  • Kim, Jae-Bum (Department of Anatomy, School of Medicine, Sungkyunkwan University) ;
  • Yum, Jung-Joo (Department of Life Science, Cheongju University)
  • 조성규 (청주대학교 산업과학연구소) ;
  • 구보라 (청주대학교 생명유전통계학부 생명과학) ;
  • 안효정 (청주대학교 생명유전통계학부 생명과학) ;
  • 박은미 (청주대학교 생명유전통계학부 생명과학) ;
  • 박선영 (청주대학교 생명유전통계학부 생명과학) ;
  • 김재범 (성균관대학교 의과대학 해부학교실) ;
  • 염정주 (청주대학교 생명유전통계학부 생명과학)
  • Published : 2009.02.28
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Abstract

The lactate dehydrogenase (EC 1.1.1.27, LDH) $A_4$ isozyme in skeletal muscle of mandrin fish (Siniperca scherzeri) was successfully purified by affinity chromatography and ultrafiltration. The molecular weight of the purified LDH $A_4$ isozyme was 140.4 kDa and its isoelectric point (pI) was 7.0. Optimal pH for enzymatic reaction was 7.5. ${K_m}^{PYR}$ and $V_{max}$ value of the purified LDH $A_4$ isozyme were $4.86{\times}10^{-5}$ M and 13.31 mM/min using pyruvate as a substrate, respectively. These kinetic properties of the purified LDH $A_4$ isozyme supported the fact that the mandrin fish was a warm-adapted species. The antibody against the purified LDH $A_4$ isozyme may be used in the metabolic physiological studies of ectothermic vertebrates and in the diagnosis of several human diseases.

쏘가리 젖산탈수소효소(EC 1.1.1.27, lactate dehydrogenase, LDH) $A_4$ 동위효소를 affinity chromatography 및 ultrafiltration으로 정제하였다. 정제된 LDH $A_4$ 동위효소의 분자량은 140.4 kDa이었고 등전점(pI)은 7.0이었다. 효소반응의 최적 pH는 7.5로 나타났다. 피루브산을 기질로 하였을 때 LDH $A_4$ 동위효소의 ${K_m}^{PYR}$값은 $4.86{\times}10^{-5}$ M, $V_{max}$값은 13.31mM/min로 나타났다. LDH $A_4$ 동위효소의 역학실험은 쏘가리가 온수성어류라는 점을 보여주었고 정제된 LDH $A_4$ 동위 효소에 대한 항체는 변온 척추동물의 대사생리학적 특성을 연구하거나 여러 가지 병증의 진단에 유용하게 사용될 수 있을 것이다.

Keywords

References

  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/10.1016/0300-9629(93)90168-4
  2. Andriutsa, K. A. and A. K. Andriutsa. 1987. Diagnostic and pathogenetic significance of determining lactate de hydrogenase isoenzyme activity in various biological media in patients with viral hepatitis. Ter. Arkh. 59, 89-94
  3. Bouafia, F., J. Drai, J. Bienvenu, C. Thieblemont, D. Espinouse, G. Salles, and B. Coiffier. 2004. Profiles and prognostic values of serum LDH isoenzymes in patients with haematopoietic malignancies. Bull. Cancer 91, E229-240
  4. 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
  5. Cho, S. K. 2000. Mitochondrial lactate dehydrogenase in tissues of vertebrate. 88pp., Ph.D. Thesis Cheongju Univ., Korea.
  6. 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
  7. Cho, S. K. and J. J. Yum. 1993. Heterogeneity of lactate dehydrogenase isozymes in tissues of Lampetra japonica. Korean J. Zool. 36, 319-328
  8. Cho, S. K. and J. J. Yum. 1996. The adaptational phenotype of lactate dehydrogenase isozymes in Pseudogobio esocinus by the environmental variation. J. Ind. Sci., Cheongju Univ. Korea 14, 333-343
  9. 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
  10. 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
  11. 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
  12. 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.
  13. 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
  14. Fields, P. A. 2001. Protein function at thermal extremes: balancing stability and flexibility. Comp. Biochem. Physiol. A 129, 417-431 https://doi.org/10.1016/S1095-6433(00)00359-7
  15. Fields, P. A. and D. E. Houseman. 2004. Decreases in activation energy and substrate affinity in cold-adapted A4-lactate dehydrogenase: evidence from the Antarctic notothenioid fish Chaenocephalus aceratus. Mol. Biol. Evol. 21, 2246-2255 https://doi.org/10.1093/molbev/msh237
  16. Fields, P. A. and G. N. Somero. 1998. Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes. Proc. Natl. Acad. Sci. USA 95, 11476-11481 https://doi.org/10.1073/pnas.95.19.11476
  17. Hochachka, P. W. and G. N. Somero. 2002. Biochemical adaptation. pp. 466, Oxford Univ. Press Inc., New York
  18. 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
  19. Holland, L. Z., M. McFall-Ngai, and G. N. Somero. 1997. Evolution of lactate dehydrogenase-A homologs of barracuda fishes (genus Sphyraena) from different thermal environments: differences in kinetic properties and thermal stability are due to amino acid substitutions outside the active site. Biochemistry 36, 3207-3215 https://doi.org/10.1021/bi962664k
  20. Johns, G. C. and G. N. Somero. 2004. Evolutionary convergence in adaptation of proteins to temperature: A4-lactate dehydrogenases of Pacific damselfishes (Chromis spp.). Mol. Biol. Evol. 21, 314-320 https://doi.org/10.1093/molbev/msh021
  21. Kim, J.-B., S. K. Cho, and J. J. Yum. 2004. Changes of activities and isozymes of lactate dehydrogenase in Coreoperca herzi to acute increase of temperature for short-term period. J. Ind. Sci. Cheongju Univ. Korea 22, 43-50
  22. 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. Korea 21, 37-44
  23. 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
  24. 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-1alpha) pathway, angiogenic factors production and poor prognosis. Ann. Surg. Oncol. 15, 2336-2344 https://doi.org/10.1245/s10434-008-9955-5
  25. Koukourakis, M. I., A. Giatromanolaki, C. Simopoulos, A. Polychronidis, and E. Sivridis. 2005. Lactate dehydrogenase 5 (LDH5) relates to up-regulated hypoxia inducible factor pathway and metastasis in colorectal cancer. Clin. Exp. Metastasis 22, 25-30 https://doi.org/10.1007/s10585-005-2343-7
  26. Koukourakis, M. I., M. Pitiakoudis, A. Giatromanolaki, A. Tsarouha, A. Polychronidis, E. Sivridis, and C. Simopoulos. 2006. Oxygen and glucose consumption in gastrointestinal adenocarcinomas: correlation with markers of hypoxia, acidity and anaerobic glycolysis. Cancer Sci. 97, 1056-1060 https://doi.org/10.1111/j.1349-7006.2006.00298.x
  27. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. 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, H. Y. et al. 1995. Fish Biology. pp. 478, Jungmunkag Co., Korea
  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. 1995. Acclimation of lactate dehydrogenase isozymes in Coreoperca herzi by environmental variation. Korean J. Environ. Biol. 13, 121-130
  36. Pesce, A., T. P. Fondy, F. Stolzenbach, F. Castillo and N. O. Kaplan. 1967. The comparative enzymology of lactic dehydrogenases. III. Properties of the H4 and M4 enzymes from a number of vertebrates. J. Biol. Chem. 242, 2151-2167 https://doi.org/10.1016/0305-0491(83)90193-1
  37. Pesce, A., R. H. Mckay, F. Stolzenbach, R. D. Cahn, and N. O. Kaplan. 1964. The comparative enzymology of lactic dehydrogenases. I. Properties of the crystalline beef and chicken enzymes. J. Biol. Chem. 239, 1753-1761
  38. Sevinc, A., R. Sari, and E. Fadillioglu. 2005. The utility of lactate dehydrogenase isoenzyme pattern in the diagnostic evaluation of malignant and nonmalignant ascites. J. Natl. Med. Assoc. 97, 79-84
  39. Sidell, B. D. and K. F. Beland. 1980. Lactate dehydrogenases of Atlantic hagfish: physiological and evolutionary implications of a primitive heart isozyme. Science 207, 769-770 https://doi.org/10.1126/science.7352286
  40. 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
  41. Yancey, P. H. and J. F. Siebenaller. 1987. Coenzyme binding ability of homologs of M4-lactate dehydrogenase in temperature adaptation. Biochim. Biophys. Acta 924, 483-491 https://doi.org/10.1016/0304-4165(87)90164-4
  42. 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
  43. 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

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