Large-Scale Purification and Some Properties of the Mitochondrial Aspartate Aminotransferase from Pig Heart

Barra, Donatella; Bossa, Francesco; Doonan, Shawn; Fahmy, Hisham M. A.; Martini, Filippo; Hughes, Graham J.

doi:10.1111/j.1432-1033.1976.tb10331.x

Cited by 48 publications

(31 citation statements)

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“…Mitochondria1 aspartate aminotransferase was purified from beef liver to apparent electrophoretic homogeneity by the procedure described in [14]. All other reagents were analytical grade commercial products from Merck and Boehringer.…”

Section: Methodsmentioning

confidence: 99%

“…These concentrations were calculated spectrophotometrically assuming absorption coefficients of 0.973 cm2 mg-' at 279 nm for dehydrogenase [16] and 1.4 cm2 mg-' at 280 nm for transaminase [14].…”

Section: Methodsmentioning

confidence: 99%

“…The steady-state velocity of NADP production by the overall reactions reported in Scheme 3 is given by Eqn (14).…”

Section: (Sd) S-' In Good Agreementmentioning

confidence: 99%

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Kinetics of Coupled Reactions Catalyzed by Aspartate Aminotransferase and Glutamate Dehydrogenase

Salerno¹,

Ovádi²,

Keleti³

et al. 1982

Eur J Biochem

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In the presence of a slight excess of dehydrogenase, the time course of NADPH oxidation resulting from the overall reaction goes through a lag phase and reaches a linear phase. The slope of the linear part of this curve is a linear function of transaminase concentration. At high concentration (E 10 pM) of both enzymes the lag phase, as observed after rapid mixing of the two enzymes in a Durrum stopped-flow spectrophotometer, is shorter than that predicted from the kinetic parameters determined for the separate reactions catalyzed by each enzymeLiver mitochondrial aspartate aminotransferase and glutamate dehydrogenase are mainly present in the soluble submitochondrial fraction [l]. They are involved in important metabolic processes such as amino acid dehydrogenation, ammonium production, and urea synthesis.Much physicochemical evidence [2-91 suggests that transaminase binds to dehydrogenase forming a heterologous enzyme-enzyme complex. In the presence of NADPH and ammonium ions, dehydrogenase can react with the pyridoxal 5'-phosphate form of transaminase to produce the pyridoxamine 5'-phosphate form of this enzyme and NADP [2,10,11]. In this reaction, the transaminase is not functioning as a catalyst but delivers the actual substrate, pyridoxal 5'-phosphate, to the active site of the dehydrogenase. If oxaloacetate is added to the system, it binds to and reacts with the pyridoxamine 5'-phosphate form of the transaminase, the latter does not accumulate and the corresponding amino acid is produced.It has been proposed [lo] that these reactions are important in mitochondria because the reductive amination of oxaloacetate by the transaminase-dehydrogenase complex could lower the levels of NADPH produced by the oxidation of glutamate. Oxaloacetate is a poor substrate of glutamate dehydrogenase [2] and it seems unlikely [lo] that the reductive amination of the keto acid by the dehydrogenase in the absence of transaminase could be significant. However, it is well known that glutamate dehydrogenase also catalyzes the reductive amination of 2-oxoglutarate. This compound is Dedicated to Professor Alessandro Rossi Fanelli for his 75th birthday. Enzymes. Aspartdtc aminotransferase or L-aspartate : 2-oxoglutaratc aminotransferase (EC 2.6.1 . l ) ; glutamate dchydrogenase or L-glutamate : NAD(P)+ oxidoreductase (deaminating) (EC 1.4.1.3). present in mammalian cell mitochondria [12,13] and it is produced by L-glutamate : oxaloacetate transamination. The question arises whether the dehydrogenase of a transaminasedehydrogenase complex catalyzes the reductive amination of the pyridoxal 5'-phosphate form of the transaminase and/or that of 2-oxoglutarate. In the latter case it is important to define the role of the heterologous enzyme-enzyme complex in the consecutive reaction catalyzed by transaminase and dehydrogenase, 2-oxoglutarate being a substrate and a product of both. A kinetic approach to the solution of these questions is described in this paper. MATERIALS AND METHODSBeef liver glutamate dehydrogenase was purchased from Bo...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Kinetics of Coupled Reactions Catalyzed by Aspartate Aminotransferase and Glutamate Dehydrogenase

Salerno¹,

Ovádi²,

Keleti³

et al. 1982

Eur J Biochem

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“…Pig heart mitochondrial aspartate aminotransferase was prepared as previously described [22]. Analytical columns of alkyl agaroses were from MilesYeda (Rehovot, Israel).…”

Section: Methodsmentioning

confidence: 99%

The Cytosolic and Mitochondrial Aspartate Aminotransferases from Pig Heart. A Comparison of their Primary Structures, Predicted Secondary Structures and Some Physical Properties

Barra¹,

Bossa²,

Martini³

et al. 1980

Eur J Biochem

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1.A primary structure is presented for mitochondrial aspartate aminotransferase from pig heart based on previously published results [Barra et al. (1977) FEBS Lett. 83, 241 -244; ( 1 979 3. Hydrophobic chromatography of the isozymes using alkyl agaroses suggests that the cytosolic but not the mitochondrial isozyme has a hydrophobic patch or pocket capable of binding to the alkyl side chains of the affinity medium.4. Evidence is presented to show that the cytosolic isozyme contains some basic amino acids whose ionizations are not expressed under native conditions. The mitochondrial isozyme does not show this effect.5. Predictions of the secondary structures of the isozymes are presented based on four predictive models. Minimum predictions are derived on the basis of agreement of three out of four of the individual predictions. These agregate predictions underestimate the amount of ordered secondary structure in both isozymes and suggest a relatively low degree of similarity between the isozymes. Individual predictive models, on the other hand, reveal greater similarities in secondary structures between the two isozymes.Aspartate aminotransferase, in common with a small number of other enzymes, exists in two distinct molecular forms one associated with the cell cytosol and the other with the mitochondria [l-31. These two isozymes differ in chemical and physical properties (for a review, see [4]) and indeed it is now known that, in humans, they are coded by genes on different chromosomes (I. Craig, quoted in [5]). Some time ago we [6] and another group [7] reported the complete primary structure of the cytosolic isozyme from pig heart and it seemed logical to extend these investigations to the corresponding mitochondrial form in order to provide a basis for understanding the comparative enzymology, evolutionary history and compartmen-

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“…Cytosolic and mitochondrial holo-aspartate aminotransferase (EC 2.6.1.1) from pig heart were prepared essentially as in [2,3] and [4], respectively. The pyridoxamine holoenzymes were obtained as in [5].…”

Section: Methodsmentioning

confidence: 99%

Cooperative effects in the binding of pyridoxal 5′‐phosphate to mitochondrial apo‐aspartate aminotransferase

1984

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Large-Scale Purification and Some Properties of the Mitochondrial Aspartate Aminotransferase from Pig Heart

Cited by 48 publications

References 36 publications

Kinetics of Coupled Reactions Catalyzed by Aspartate Aminotransferase and Glutamate Dehydrogenase

Kinetics of Coupled Reactions Catalyzed by Aspartate Aminotransferase and Glutamate Dehydrogenase

The Cytosolic and Mitochondrial Aspartate Aminotransferases from Pig Heart. A Comparison of their Primary Structures, Predicted Secondary Structures and Some Physical Properties

Cooperative effects in the binding of pyridoxal 5′‐phosphate to mitochondrial apo‐aspartate aminotransferase

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