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Molecular Gene Cloning, Expression, and Characterization of Bovine Brain Glutamate Dehydrogenase

  • Kim, Dae-Won (Department of Genetic Engineering, Division of Life Sciences, Hallym University) ;
  • Eum, Won-Sik (Department of Genetic Engineering, Division of Life Sciences, Hallym University) ;
  • Jang, Sang-Ho (Department of Genetic Engineering, Division of Life Sciences, Hallym University) ;
  • Yoon, Chang-Sik (Department of Genetic Engineering, Division of Life Sciences, Hallym University) ;
  • Kim, Young-Hoon (Department of Genetic Engineering, Division of Life Sciences, Hallym University) ;
  • Choi, Soo-Hyun (Department of Genetic Engineering, Division of Life Sciences, Hallym University) ;
  • Choi, Hee-Soon (Department of Genetic Engineering, Division of Life Sciences, Hallym University) ;
  • Kim, So-Young (Department of Genetic Engineering, Division of Life Sciences, Hallym University) ;
  • Kwon, Hyeok-Yil (Department of Physiology, College of Medicine, Hallym University) ;
  • Kang, Jung-Hoon (Department of Genetic Engineering, Chongju University) ;
  • Kwon, Oh-Shin (Department of Biochemistry, College of Natural Science, Kyungpook National University) ;
  • Cho, Sung-Woo (Department of Biochemistry, University of Ulsan College of Medicine) ;
  • Park, Jin-Seu (Department of Genetic Engineering, Division of Life Sciences, Hallym University) ;
  • Choi, Soo-Young (Department of Genetic Engineering, Division of Life Sciences, Hallym University)
  • 투고 : 2003.05.03
  • 심사 : 2003.06.02
  • 발행 : 2003.11.30

초록

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The $K_m$ and $V_{max}$ values for $NAD^+$ were 0.1 mM and $1.08\;{\mu}mol/min/mg$, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - $100\;{\mu}M$, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.

키워드

참고문헌

  1. Ahn, J. Y., Cho, E. H., Lee, K. S., Choi, S. Y. and Cho, S. W.(1999a) Chemical modification of brain glutamatedehydrogenase isoproteins with phenylglyoxal. J. Biochem.Mol. Biol. 32, 515-520.
  2. Ahn, J. Y., Choi, S. Y. and Cho, S. W. (1999b) Identification oflysine residue involved in inactivation of brain glutamatedehydrogenase isoproteins by o-phthaladehyde. Biochimie 82,1-7.
  3. Bailey, J., Bell, E. T. and Bell, J. E. (1982) Regulation of bovineglutamate dehydrogenase. The effects of pH and ADP. J. Biol.Chem. 257, 5579-5583.
  4. Bradford, M. A. (1976) A rapid and sensitive method for thequantification of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72, 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  5. Brusilow, S. W. and Horwich, A. L. (1995) The metabolic andmolecular bases of inherited disease; in Urea cycle enzymes,Scriver, C. S., Beaudet, A. L., Sly, W. S., Valle, D. (eds.), pp.1187-1232, McGraw-Hill, New York, USA.
  6. Cho, S. W., Lee, J. and Choi, S. Y. (1995) Two soluble forms ofglutamate dehydrogenase isoproteins from bovine brain. Eur. J.Biochem. 233, 340-346. https://doi.org/10.1111/j.1432-1033.1995.340_1.x
  7. Choi, S. Y., Hong, J. W., Song, M. S., Jeon, S. G., Bahn, J. H.,Lee, B. R., Ahn, J. Y. and Cho, S. W. (1999) Differentantigenic reactivities of bovine brain glutamate dehydrogenaseisoproteins. J. Neurochem. 72, 2162-2169.
  8. Colman, R. F. (1991) Glutamate dehydrogenase; in A Study of Enzymes, Kuby, S. A. (ed.), pp. 173-192, CRC Press, New York, USA.
  9. Duvoisin, R. C., Chokroverty, S., Lepore, F. and Nicklas, W.(1983) Glutamate dehydrogenase deficiency in patients witholivopontocerebellar atrophy. Neurology 33, 1322-1326.
  10. Fang, J., Hsu, B. Y. L., MacMullen. C. M., Poncz, M., Smith, T.J. and Stanley, C. A. (2002) Expression, purification andcharacterization of human glutamate dehydrogenase (GDH)allosteric regulatory mutations. Biochem. J. 363, 81-87. https://doi.org/10.1042/0264-6021:3630081
  11. Fisher, H. F. (1985) L-glutamate dehydrogenase from bovine liver.Methods Enzymol. 113, 16-27. https://doi.org/10.1016/S0076-6879(85)13006-5
  12. Fonnum, F. (1984) Glutamate: a neurotransmitter in mammalianbrain. J. Neurochem. 42, 1-11. https://doi.org/10.1111/j.1471-4159.1984.tb09689.x
  13. Hanahan, D. (1983) Studies on transformation of Escherichia coliwith plasmids. J. Mol. Biol. 166, 557-580. https://doi.org/10.1016/S0022-2836(83)80284-8
  14. Hudson, R. C. and Daniel, R. M. (1993) L-Glutamatedehydrogenase: distribution, properties and mechanisms. Comp.Biochem. Physiol. 106B, 767-792.
  15. Hussain, M. H., Zannis, V. I. and Plaitakis, A. (1989)Characterization of glutamate dehydrogenase isoproteinspurified from the cerebellum of normal subjects and patientswith degenerative neurological disorders, and from humanneoplastic cell lines. J. Biol. Chem. 264, 20730-20735.
  16. Koh, E., Shim, K., Kim, H. K., Park, K., Kim, S. L., Yoo, S. D.,Choi, S. C. and Hong, S. (2001) Cloning, expression, andcharacterization of protein carboxyl a methyltrans ferase fromporcine brain. J. Biochem. Mol. Biol. 34, 559-565.
  17. Lee, E. Y., Yoon, H. Y., Ahn, J. Y., Choi, S. Y. and Cho, S. W.(2001a) Identification of the GTP binding site of humanglutamate dehydrogenase by cassette mutagenesis andphotoaffinity labeling. J. Biol. Chem. 276, 47930-47936.
  18. Lee, E. Y., Yoon, H. Y., Kim, T. U., Choi, S. Y., Won, M. H. andCho, S. W. (2001b) Inactivation of brain glutamatedehydrogenase isoproteins by MDL 29951. J. Biochem. Mol.Biol. 34, 268-273.
  19. Lee, J. W., Choi, S. Y. and Cho, S. W. (1995) A soluble form ofnovel glutamate dehydrogenase from bovine brain. Exp.Neurobiol. 4, 7-14.
  20. MacMullen, C., Fang, J., Hsu, B. Y. L., Kelly, A., deLonlay-Debeney, P., Saudubray, J. M., Ganguly, A., Smith, T. J. andStanley, C. A. (2001) Hyperinsulinism/hyperammonemiasyndrome in children with regulatory mutations in theinhibitory guanosine triphosphate-binding domain of glutamatedehydrogenase. J. Clin. Endocrinol. Metab. 86, 1782-1787. https://doi.org/10.1210/jc.86.4.1782
  21. Maurizi, M. R. and Rasulova, F. (2002) Degradation of L-glutamatedehydrogenase from Escherichia coli: allostericregulation of enzyme stability. Arch. Biochem. Biophys. 397,206-216. https://doi.org/10.1006/abbi.2001.2703
  22. McCarthy, A. D., Walker, J. M. and Tipton, K. F. (1980)Purification of glutamate dehydrogenase from ox brain andliver. Biochem. J. 191, 605-611.
  23. McPherson, M. J. and Wootton, J. C. (1983) Complete nucleotidesequence of the Escherichia coli gdhA gene. Nucleic AcidsRes. 11, 5257-5266. https://doi.org/10.1093/nar/11.15.5257
  24. Miki, Y., Tomohiko, T., Obura, T., Kato, H., Yanagisawa, M. andHayashi, Y. (2000) Novel misense mutations in the glutamatedehydrogenase gene in the congenital hyperinsulinism-hyperammonemiasyndrome. J. Pediatr. 136, 69-72. https://doi.org/10.1016/S0022-3476(00)90052-0
  25. Peterson, P. and Smith, T. J. (1999) The structure of bovineglutamate dehydrogenase provides insights into the mechanism of allostery. Structure 7, 769-782. https://doi.org/10.1016/S0969-2126(99)80101-4
  26. Peterson, P., Pierce, J. and Smith, T. J. (1998) Crystallization andcharacterization of bovine glutamate dehydrogenase. J. Struct.Biol. 120, 73-77.
  27. Plaitakis, A., Metaxari, M. and Shashidharan, P. (2000) Nervetissue-specific (GLUD2) and housekeeping (GLUD1) humanglutamate dehydrogenase are regulated by distinct allostericmechanisms: implications for biologic function. J. Neurochem.75, 1862-1869.
  28. Plaitakis, A., Berl, S. and Yahr, M. D. (1982) Abnormal glutamatemetabolism in an adult-onset degenerative neurologicaldisorder. Science 216, 193-196. https://doi.org/10.1126/science.6121377
  29. Rice, D. W., Hornby, D. P. and Engel, P. C. (1985) Crystallizationof an $NAD^+-dependent$ glutamate dehydrogenase fromClostridium symbiosum. J Mol. Biol. 181, 147-149. https://doi.org/10.1016/0022-2836(85)90334-1
  30. Sambrook, J., Fritsch, E. and Maniatis, T. (1989) Molecularcloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York, USA.
  31. Smith, E. L., Austin, B. M., Blumenthal, K. M. and Nyc, J. F.(1975) Glutamate dehydrogenase; in The enzymes. Vol. 11,Boyer, P. D. (ed.), pp. 293-367, Academic Press, New York,USA.
  32. Smith, E. L. (1979) The evolution of glutamate dehydrogenaseand a hypothesis for the insertion of deletion of multipleresidues in the interior of polypeptide chains. Proc. Am. Phil.Soc. 123, 73-84.
  33. Smith, T. J., Peterson, P., Schmidt, T., Fang, J. and Stanley, C.(2001) Structures of bovine glutamate dehydrogenasecomplexes elucidate the mechanism of purine regulation. J. Mol. Biol. 307, 707-720. https://doi.org/10.1006/jmbi.2001.4499
  34. Stanley, C. A., Lieu, Y. K., Hsu, B. Y., Burlina, A. B., Greenberg,C. R., Hopwood, N. J., Perlman, K., Rich, B. Y., Zammarchi,E. and Poncz, M. (1998) Hyperinsulinism andhyperammonemia in infants with regulatory mutations of theglutamate dehydrogenase gene. N. Engl. J. Med. 338, 1352-1357. https://doi.org/10.1056/NEJM199805073381904
  35. Stanley, C. A., Fang, J., Kutyna, K., Hsu, B. Y., Ming, J. E.,Glaser, B. and Poncz, M. (2000) Molecular basis andcharacterization of the hyperinsulinism/hyperammonemiasyndrome: predominance of mutation in exons 11 and 12 ofthe glutamate dehydrogenase gene. Diabetes 49, 667-673. https://doi.org/10.2337/diabetes.49.4.667
  36. Veronese, F. M., Nyc, J. F., Degani, Y., Brown, D. M. and Smith,E. L. (1974) Nicotinamide adenine dinucleotide-specificglutamate dehydrogenase of Neurospora. I. Purification andmolecular properties. J. Biol. Chem. 249, 7922-7928.
  37. Weinzimer, S. A., Stanley, C. A., Berry, G. T., Yudkoff, M.,Tuchman, M. and Thornton, P. S. (1997) A syndrome ofcongenital hyperinsulinism and hyperammonemia. J. Pediatr.130, 661-664. https://doi.org/10.1016/S0022-3476(97)70256-7
  38. Yoon, H. Y., Cho, E. H., Kwon, H. Y., Choi, S. Y. and Cho, S. W.(2002) Importance of glutamate 279 for the co-enzyme bindingof human glutamate dehydrogenase. J. Biol. Chem. 277, 41448-41454. https://doi.org/10.1074/jbc.M208208200
  39. Yorifuji, T., Muroi, J., Uematsu, A., Hiramatsu, H. and Momoe, T.(1999) Hyperinsulinism-hyperammonemia syndrome caused bymutant glutamate dehydrogenase accompanied by novelenzyme kinetics. Hum. Genet. 104, 476-479. https://doi.org/10.1007/s004390050990

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