Purification and Characterization of a Soluble and a Particulate Glutamate Dehydrogenase from Rat Brain

Abstract

Abstract:Glutamate dehydrogenase (GDH) activity was determined in high‐speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates after removal of a low‐speed pellet (480gfor 10 min). Approximately 60% of the high‐speed GDH activity was particulate (associated with membrane) and the remaining was soluble (probably of mitochondrial matrix origin). Most of the particulate GDH activity resisted extraction by several commonly used detergents, high concentration of salt, and sonication; however, it was largely extractable with the cationic detergent cetyltrimethylammonium bromide (CTAB) in hypotonic buffer solution. The two GDH activities were purified using a combination of hydrophobic interaction, ion exchange, and hydroxyapatite chromatography. Throughout these purification steps the two activities showed similar behavior. Kinetic studies indicated similarKmvalues for the two GDH fractions for the substratesμ‐ketoglutarate, ammonia, and glutamate; however, there were small but significant differences inKmvalues for NADH and NADPH. Although the allosteric stimulation by ADP and L‐leucine and inhibition by diethylstilbestrol was comparable, the two GDH components differed significantly in their susceptibility to GTP inhibition in the presence of 1 mMADP, with apparentKivalues of 18.5 and 9.0 μMGTP for the soluble and particulate fractions, respectively. The HIll plot coefficient, binding constant, and cooperativity index for the GTP inhibition were also significantly different, indicating that the two GDH activities differ in their allosteric sites. In addition, enzyme activities of the two purified proteins exhibited a significant difference in thermal stability when inactivated at 45°C and pH 7.4 in 50 mMphosphate buffer.