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

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 (480 g for 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 similar Km values for the two GDH fractions for the substrates μ‐ketoglutarate, ammonia, and glutamate; however, there were small but significant differences in Km values 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 mM ADP, with apparent Ki values of 18.5 and 9.0 μM GTP 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 mM phosphate buffer.

Different Antigenic Reactivities of Bovine Brain Glutamate Dehydrogenase Isoproteins

“…Our work also led to the finding that GDH is present in bovine brain in "heatlabile" (GDH I) and "heat-stable'' (GDH 11) forms . It has been reported that the activities of the GDH isotypes differ in their relative resistance to thermal inactivation, detergent extractability, and allosteric regulation characteristics (Colon et al, 1986;Shashidharan et al, 1997). Similar results were reported by other investigators showing that reduction in GDH activity in patients with neurodegenerative disorders was largely limited to the heat-labile form (Plaitakis et al, 1984).…”

“…Similar studies also showed the presence of two GDH activities in rat brain differing in their relative resistance to thermal inactivation, solubility, and allosteric regulation characteristics (Colon et al, 1986). Recent studies in our laboratory have shown that two different GDH isoproteins (GDH I and GDH 11) are also present in bovine brain .…”

Further characterization of mammalian ceramide kinase: substrate delivery and (stereo)specificity, tissue distribution, and subcellular localization studies

“…Other markers (not shown) included catalase, acid phosphatase, glucose-6-phosphatase, 5V-nucleotidase, and lactate dehydrogenase, with the highest relative specific activity in the L, L, P, P, and S fractions, respectively. Although glutamate dehydrogenase is generally considered a marker for mitochondria, a soluble activity has been reported in nonhepatic tissues (65), likely explaining the rather high amount in the S fraction of testis.…”

Mechanism of age-dependent susceptibility and novel treatment strategy in glutaric acidemia type I

“…Since α-ketoglutarate is a precursor of glutamate synthesis (19) and the product of glutamate dehydration (20), depletion of α-ketoglutarate suggests that glutamate may be depleted as well. Additionally, GABA is produced from glutamate decarboxylation and was previously shown to be depleted in human GA-I (21).…”