title: Glutamate decarboxylase from Lactobacillus brevis: activation by ammonium sulfate.
abstract: In this study, the glutamate decarboxylase (GAD) gene from Lactobacillus brevis IFO12005 (Biosci. Biotechnol. Biochem., 61, 1168-1171 (1997)), was cloned and expressed. The deduced amino acid sequence showed 99.6% and 53.1% identity with GAD of L. brevis ATCC367 and L. lactis respectively. The His-tagged recombinant GAD showed an optimum pH of 4.5-5.0, and 54 kDa on SDS-PAGE. The GAD activity and stability was significantly dependent on the ammonium sulfate concentration, as observed in authentic GAD. Gel filtration showed that the inactive form of the GAD was a dimer. In contrast, the ammonium sulfate-activated form was a tetramer. CD spectral analyses at pH 5.5 revealed that the structures of the tetramer and the dimer were similar. Treatment of the GAD with high concentrations of ammonium sulfate and subsequent dilution with sodium glutamate was essential for tetramer formation and its activation. Thus the biochemical properties of the GAD from L. brevis IFO12005 were significantly different from those from other sources.
authors.affiliation: Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Hiraga
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Ueno
authors.forename: Kohei
authors.initials: K
authors.lastname: Oda
mesh_terms: D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000494:pharmacology*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D052196:Levilactobacillus brevis / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase; D000645:Ammonium Sulfate