title: Molecular gene cloning, expression, and characterization of bovine brain glutamate dehydrogenase.
abstract: 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 Km and Vmax values for NAD+ were 0.1 mM and 1.08 micromol/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 microM, 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.
authors.affiliation: Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon 200-702, Korea.
authors.forename: Dae Won
authors.initials: DW
authors.lastname: Kim
authors.forename: Won Sik
authors.initials: WS
authors.lastname: Eum
authors.forename: Sang Ho
authors.initials: SH
authors.lastname: Jang
authors.forename: Chang Sik
authors.initials: CS
authors.lastname: Yoon
authors.forename: Young Hoon
authors.initials: YH
authors.lastname: Kim
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Choi
authors.forename: Hee Soon
authors.initials: HS
authors.lastname: Choi
authors.forename: So Young
authors.initials: SY
authors.lastname: Kim
authors.forename: Hyeok Yil
authors.initials: HY
authors.lastname: Kwon
authors.forename: Jung Hoon
authors.initials: JH
authors.lastname: Kang
authors.forename: Oh-Shin
authors.initials: OS
authors.lastname: Kwon
authors.forename: Sung-Woo
authors.initials: SW
authors.lastname: Cho
authors.forename: Jinseu
authors.initials: J
authors.lastname: Park
authors.forename: Soo Young
authors.initials: SY
authors.lastname: Choi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D005969:Glutamate Dehydrogenase