歯垢由来のFusobacteriumのGlutamate Dehydrogenaseに関する酵素化学的研究
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概要
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Fusobacterium was isolated from dental plaque with modified FM agar and cultured in BHI broth at 37℃ for 48 hours anaerobically. After the cells were harvested with centrifugation, the cells were washed three times with saline solution and stored at -20℃. The frozen cells of 100mg stored were suspended in 0.01M Tris-HCl buffer, pH 7.6, 2.0ml, and disrupted by sonication at 9KHz for 40 sec. This suspension was centrifuged for 10 min at 15,000×g, and supernatant was used as crude extract. Since glutamate dehydrogenase (GDH) catalyzes reversibly the interconversion of α-ketoglutarate and L-glutamate, the assay was performed for both forward and reverse reactions. The assay mixture of forward reaction was contained α-ketoglutarate 5mM, NH_4Cl 225mM, NADH 0.2mM, triethanolamine-HCl buffer, pH 8.6, 50mM, and the crude extract in 0.4ml. The assay mixture of reverse reaction was contained L-glutamate 20mM, NAD 1.5mM, triethanolamine-HCl buffer, pH 9.0, 50mM, and crude extract in 0.4ml. These assay mixtures were incubated for 30 min at 37℃, and the enzyme activities were determined with the mesured amounts of NAD or NADH by fluorometric technique. In the supernatant fraction, α-hydroxyglutarate dehydrogenase which used α-ketoglutarate for substrate may be contained, so that the supernatant fraction was partially purified by DEAE-cellulose column chromatography. After DEAE-cellulose was equilibrated with 0.01M Tris-HCl buffer, pH7.6, the elution was performed by stepwise addition of several NaCl concentrations in the same buffer. High GDH activities were observed in the fractions of 0.07M NaCl eluate and α-hydroxyglutarate dehydrogenase activities were observed in those of 0.1M NaCl. Therefore, the fractions of 0.07M NaCl eluate were used as sample for studying on some enzymatic properties. And following conclusions were obtained. 1. This enzyme was NAD-dependent GDH. 2. The optimal pH of this enzyme activities for forward reaction was 8.6, while for reverse reaction was 9.0. 3. The Km values of this enzyme for each substrate were 0.9mM for α-ketoglutaratae, 45.5mM for NH_4C1, 0.067mM for NADH, 2.5mM for L-glutamate, and 0.3mM for NAD, respectively, and kinetic mechanism of this enzyme was "Ordered Ter Bi" type. 4. This enzyme activities were inhibited by p-chloromercuribenzonic acid and was completely recovered by glutathione. This inhibition was noncompetitive with α-ketoglutarate, NH_4C1, and NADH. 5. This enzyme activities were also inhibited by divalent metal ions, Cu^<2+>, Zn^<2+>, and Ni^<2+>. 6. This enzyme retained 50% of its activities after heating at 75℃ for 30 min. 7. The insignificant effect of AMP, ADP, and ATP on this enzyme activities were observed. 8. Molecular weight of this enzyme by Sephadex G-150 gel-filtration was about 400,000.
- 特定非営利活動法人日本歯周病学会の論文
- 1981-03-28