Abstract
The activity of bovine liver glutamate dehydrogenase is affected in several ways depending on substrate concentrations and pH. At ph 6.5 and below, both oxidative deamination and reductive amination reactions are inhibited by ADP. At pH 7.0 and above both activatory and inhibitory effects can be observed depending on substrate concentrations. The effects are explicable in terms of a model with ADP binding at both a regulatory site and competing with coenzyme at the active site. The activatory effects of ADP result from destabilization of various abortive complexes by ADP binding to its regulatory site. The concerted effects of pH and ADP lead to a potentiation of either activation effects or inhibition effects depending on conditions. A consideration of in vivo concentrations of the various substrates involved and intramitochondrial pH and adenine nucleotide levels suggests that in vivo the reductive amination reaction is favored. It is suggested that glutamate dehydrogenase may be intimately involved with regulation of the urea cycle by responding to changes in the mitochondrial ammonia levels.
Highlights
The activity of bovine liver glutamate dehydrogenaseextensively dialyzed versus 0.1 M sodium phosphate buffer, pH 7.0, is affected in several ways depending on substrate con- containing 10 p~ EDTA prior to use.Enzyme concentrations were centrations andpH
At pH6.5 and below, botohxidative estimated using an extinction coefficient of 0.93 for a 1 mg/ml soludeamination and reductive amination reactions are inhibited byADP.AtpH 7.0 and above both activatory and inhibitory effects can be observed depending on tion at 280 nm [16]
From deionized substrate concentrations.The effects are explicable in Enzyme assays were performed usingeither the appearanceor the terms of a model witAhDP binding at both a regulatorydecrease in absorbance at 340 nmin a Cary 219 recording spectrosite and competing with coenzyme at the active site. photometer, corresponding to the production of NAD(P)H
Summary
Regulation of the urea cycleby responding to changes in the mitochondrial ammonialevels. 7.0 and above are examined (Fig. L 4 ) a marked activation is Bovine glutamatedehydrogenase catalyzes the oxidative observed at ADP concentrationsbelow 0.1 mM. The activation which is observed a t high coenzyme of oxidative deamination are increased as the pH increases. A number of studied with a variety of substrate and coenzyme concentrapotential regulators of glutamate dehydrogenase have been tions for both theNAD’-linked and theNADP’-linked reacstudied, including GTP [9], leucine (lo), and ADP [11].With tions at pH 6.5 and pH 8.0. I IB shown to fluctuate with the metabolic stoaftethe mitochondrion [14, 15], we have examined the effects of pH on the activation effects of ADP in both the oxidative deamination reaction and the reductive amination reaction catalyzed by glutamate dehydrogenase
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