• BMB Reports
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• v.32 no.5
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• pp.515-520
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• 1999

Incubation of two types of glutamate dehydrogenase isoproteins from bovine brain with the arginine-specific dicarbonyl reagent phenylglyoxal resulted in a biphasic loss of enzyme activity. Reaction of the glutamate dehydrogenase isoproteins with phenylglyoxal caused a rapid loss of 53~62% of the enzyme activities and modification of two residues of arginine per enzyme subunit. Prolonged incubation of the glutamate dehydrogenase isoproteins with phenylglyoxal resulted in the modification of an additional four residues of arginine per enzyme subunit without further loss of the residual activities. Partial protection against inactivation was provided by the coenzyme NADH or substrate 2-oxoglutarate. The most marked decrease in the rate of inactivation was observed by the combined addition of NADH and 2-oxoglutarate, suggesting that the first two modified arginine residues are in the vicinity of the catalytic site. However, inactivation of the glutamate dehydrogenase isoproteins by phenylglyoxal appears to be partial with approximately 40% activity remained after an extended reaction time with excess reagent, suggesting that the modified arginine residues may not be directly involved in catalysis. The lack of complete protection by substrates also suggest the possibility that the modified arginine residues are not directly involved at the active site, and the partial loss of activity by the modification of arginine residues may be due to a conformational change. There were no significant differences between the two glutamate dehydrogenase isoproteins in sensitivities to inactivation by phenylglyoxal, indicating that the microenvironmental structures of the glutamate dehydrogenase isoproteins are very similar to each other.

• BMB Reports
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• v.30 no.5
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• pp.332-336
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• 1997

The stimulatory effects of leucine on the activities of two soluble forms of brain glutamate dehydrogenase isoproteins (GDH I and GDH II) have been studied at various conditions. There were significant differences between GDH I and GDH II in their sensitivities to the action of leucine. When the effects of varied leucine concentrations on GDH activities were studied in the direction of reductive amination of 2-oxoglutarate with NADPH as a coenzyme, a marked activation was observed for both isoproteins at leucine concentrations up to 10 mM, whereas both isoproteins showed activation to a lesser extent with NADH as a coenzyme. The stimulatory effects of leucine on GDH activities in the direction of the oxidative deamination of glutamate were also observed, but to a much lesser extent. Leucine relieved the inhibition of GDH I by GTP and this resulted in an increase in the apparent activation by leucine in the presence of GTP. 2-Oxoglutarate was found to give rise to high substrate inhibition and leucine significantly reduced the substrate inhibition in the presence of $200\;{\mu}M$ NADH. Thus, the effects of leucine might be composed of a direct effect on the enzyme together with a relief of high substrate inhibition.

• BMB Reports
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• v.34 no.3
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• pp.268-273
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• 2001

In addition to the recognition site for glutamate, the N-methyl-D-aspartate (NMDA)-preferring glutamate receptor subtype shows a binding site for glycine. In this paper, we present the effects of 3-(4,6-dichloro-2-carboxymethylamino-5,7-dichloroquinoline-2-carboxylic acid (MDL 29951), a potent inhibitor of glycine binding to the NMDA receptor, on glutamate dehydrogenase (GDH) from bovine brains. The incubation of GDH isoproteins from bovine brains with MDL 29951 resulted in a dose-dependent loss of enzyme activity Separately or together, 2-oxoglutarate and NADH did not give an efficient protection against the inhibition, indicating that GDH isoproteins saturated with NADH or 2-oxoglutarate are still open to attack by MDL 29951. MDL 29951 was an uncompetitive inhibitor with respect to both 2-oxoglutarate and NADH for GDH isoproteins. These results suggest that the binding site of MDL 29951 is not directly located at the catalytic site, and the inhibition of GDH isoproteins by MDL 29951 is probably due to a steric hindrance, or a conformational change altered upon the interaction of the enzyme with its inhibitor. The inhibitory effects of MDL 29951 on GDH isoproteins were significantly diminished in the presence of ADP. GDH I reacted more sensitively with ADP than GDH II on the inhibition by MDL 29951. Our results suggest a possibility that the two types of GDHs are differently regulated by MDL 29951, depending on the physiological concentrations of ADP.

• Animal cells and systems
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• v.2 no.2
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• pp.223-227
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• 1998

We isolated two soluble forms of glutamate dehydrogenase isoproteins, GDH I and GDH II, from bovine brain. The regulation of GDH I and GDH II by phosphorylation and dephosphorylation has been examined in various conditions. There were dose- and time- dependent activation of the GDH isoproteins when phosphorylated by cAMP-dependent protein kinase. The phosphorylated GDH had 1.1 mol of covalently bound phosphate/mol of subunit and a 2-fold increased specific activity. The phosphorylated amino acid was identified as serine. When treated with alkaline phosphatase, the activities of the phosphorylated GDH isoproteins were reduced in dose and time dependent manner and returned to those of unphosphorylated enzymes. There were no significant differences between GDH I and GDH II in their sensitivities to the action of phosphorylation and dephosphorylation demonstrating that the microenvironmental structures of the phosphorylation site in GDH isoproteins are similar to each other, These results results suggest that the inter-conversion between less active form of brain GDH isoproteins and more active form is regulated by phosphorylation through cAMP-dependent protein kineses.