Purification and characterization of two glutamate dehydrogenase isoenzymes from Brassica napus |
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| Institution: | 1. Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM), Nicolás Cabrera 9, 28049 Madrid, Spain;2. Tecnologico de Monterrey, Escuela de Ingeniería y Ciencias. Av General Ramón Corona 2514, 45138 Zapopan, Jalisco, Mexico;1. Uppsala University, Solid State Electronics, Department of Engineering Sciences, Box 534, SE 75121 Uppsala, Sweden;2. Uppsala University, Tribomaterials Group, Department of Engineering Sciences, Box 534, SE 75121 Uppsala, Sweden |
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| Abstract: | Glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase, EC 1.4.1.2) was purified from Brassica napus leaves. Isoenzyme 1 (GDH1), with the lowest, and isoenzyme 7 (GDH7) with the highest electrophoretic mobility were characterized. The native GDH was estimated to have a molecular mass of about 239 kDa and consisted of six identical 41.4-kDa subunits for GDH1 and 42.4-kDa subunits for GDH7. The pH optima of both isoenzymes in amination and deamination reactions were 9.0 and 9.5, respectively. At optimum pH, the Km values for ammonium, 2-oxoglutarate, NADH, NAD and glutamate did not differ between the two isoenzymes. Addition of 10 mM EGTA inhibited the amination activity of GDH1, but that of GDH7 remained at about 30 %. Cellular fractionation experiments showed that both GDH1 and GDH7 localized in mitochondria with a loose association with the mitochondrial membrane. |
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