title: Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO 12005.
abstract: Glutamate decarboxylase (GAD) [EC 4.1.1.15] was purified from a cell-free extract of Lactobacillus brevis IFO 12005 by chromatographies on Sephadex G-100, DEAE-Sepharose CL-6B, and Mono Q. About 9 mg of purified GAD was obtained from 90.2 g of wet cells. The purified preparation showed a single protein band on SDS-PAGE. The molecular weights of purified GAD by SDS-PAGE and gel filtration on Superdex 200 were 60,000 and 120,000, respectively, indicating that GAD from L. brevis exists as a dimer. The N-terminal amino acid sequence of the purified GAD was NH2-Met-Asn-Lys-Asn-Asp-Gln-Glu-Gln-Thr-. The optimum pH and temperature of GAD were at pH 4.2 and at 30 degrees C. The GAD activity was increased by the addition of sulfate ions in a dose-dependent manner. The order of effects was as follows: ammonium sulfate > sodium sulfate > magnesium sulfate, indicating that the increase of hydrophobic interaction between subunits causes the increase of GAD activity. The purified GAD reacted only with L-glutamic acid as a substrate and the K(m), kcat, and kcat/K(m) values were 9.3 mM, 6.5 S-1, and 7 x 10(2) M-1 S-1, respectively.
authors.affiliation: Kyoto Prefectural Comprehensive Center for Small and Medium Enterprises, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Ueno
authors.forename: K
authors.initials: K
authors.lastname: Hayakawa
authors.forename: S
authors.initials: S
authors.lastname: Takahashi
authors.forename: K
authors.initials: K
authors.lastname: Oda
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods; D005968:Glutamate Decarboxylase / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007778:Lactobacillus / Q000737:chemistry / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis / Q000379:methods; D013379:Substrate Specificity; D013431:Sulfates / Q000494:pharmacology; D013696:Temperature
chemical_list: D013431:Sulfates; D005968:Glutamate Decarboxylase