The Oxidation of Malate by Isolated Plant Mitochondria
Authors: Coleman Julian O. D. , Palmer John M. 
Published: March 2005

The functional relationship between NAD+‐linked oxidations of L‐malate and the operation of the exogenous and endogenous NADH dehydrogenases of isolated plant mitochondria have been investigated.

Mitochondria isolated from tubers of the Jerusalem artichoke (Helianthus tuberosus) oxidised L‐malate via an electron transport pathway which was inhibited by rotenone, piericidin A and added oxaloacetate. Phosphorylation linked to this pathway yielded an APD/O ratio of approx. 3.

In the presence of rotenone, piericidin A or added oxaloacetate, malate was oxidised by a pathway which was dependent on the presence of added NAD+ and low levels of Ca2+. Phosphorylation linked to this oxidation had an ADP/O ratio of approx. 2.

Oxidation of malate via the rotenone‐sensitive pathway was inhibited by 2‐n‐butylmalonate whereas oxidation via the rotenone‐insensitive pathway was unaffected by this compound.

By following the reduction of ferricyanide by malate in intact mitochondria, it was possible to identify two separate malate: NAD oxidoreductases: one located outside the inner membrane permeability barrier, the other inside this barrier.

Oxaloacetate was the primary product when malate was oxidised by the enzyme in the inner compartment which was therefore identified as L‐malate: NAD oxidoreductase whereas pyruvate was the primary product of malate oxidation by the enzyme in the outer compartment, which was identified as L‐malate : NAD oxidoreductase (decarboxylating) or malic enzyme.

The malic enzyme was extracted and partially purified from isolated mitochondria. The isolated enzyme showed a requirement for Mn2+ or Mg2+, required NAD+ as coenzyme, although activity to a lesser extent was obtained with NADP+. and was inhibited by NADH.

It was concluded that in intact plant mitochondria there were two pathways by which malate can be oxidised: in the inner compartment via malate dehydrogenase in association with the endogenous NADH dehydrogenase system and in the intermembrane compartment via NAD+‐linked malic enzyme associated with the exogenous NADH dehydrogenase system. The implications these findings are discussed.

L‐Malate is poorly oxidised by malate dehydrogenase in animal mitochondria unless the oxaloacetate formed as a product is removed, by either transamination with glutamate or by condensation with acetyl‐CoA [1–3]. In contrast isolated plant mitochondria will rapidly oxidise malate in the absence of added oxaloacetate‐removing agents [4–7].

The formation of some pyruvate during the oxidation of malate has been observed in avocado fruit mitochondria [9] and apple peel mitochondria [10] and increased rates of malate oxidation have been reported in the presence of CoA and thiamine pyrophosphate [8–9], suggesting that pyruvate gave rise to acetyl‐CoA which could remove the oxaloacetate formed. Recently it was shown that in cauliflower bud mitochondria, pyruvate was a direct product of malate oxidation by a NAD+‐requiring malic enzyme, which showed a dependence on added NAD+ and was linked to the electron transport chain [11].