Pathways for the Oxidation of Malate and Reduced Pyridine Nucleotide by Wheat Mitochondria.

Piericidin A and oxaloacetate have been used as inhibitors to investigate the operation and localisation of NAD+-linked dehydrogenases and the possible compartmentation of these enzymes within the matrix of the mitochondrion. 1. Mitochondria isolated from etiolated shoots of wheat (Triticum vulgare) were shown to oxidise a variety of NAD+-linked substrates. Oxaloacetate, when added to rapidly respiring preparations (i.e. state 3) resulted in a large but transient inhibition of the oxidation of both pyruvate and citrate, but had little effect on the rate of oxidation of malate. 2. The products of oxidation of malate, in the absence of added NAD+, were found to be a large quantity of pyruvate together with smaller quantities of oxaloacetate and citrate. Using potassium ferricyanide as a non-penetrating electron acceptor it was possible to show that the malate oxidation occurred in the matrix. This is consistent with the operation of both the malic enzyme and a low level of malate dehydrogenase within the matrix of the mitochondrion. 3. The low level of inhibition of malate oxidation by oxaloacetate was a function of the concentration of malate and appeared to be of a competitive nature. 4. Measurement of the redox state of the endogenous NAD+ pool showed that under phosphorylatig conditions both malate and citrate reduce approximately 15% of the total coenzyme. The addition of 0.2 mM oxaloacetate to phosphorylating aerobic mitochondria failed to oxidise any of the NAD+ reduced by malate but resulted in a partial oxidation of NAD+ reduced by citrate. The transition from the aerobic to anaerobic state in the presence of malate resulted in a biphasic reduction of the endogenous NAD+, whereas in the presence of citrate it was monophasic. 5. The oxidation of NAD+-linked subtrates, including malate, was found to be only partially inhibited by piercidin A. The piericidin-A-resistant pathway was found to be located internally on the basis of experiments using ferricyanide as a non-penetrating electron accepter. The first site of phosphorylation was not coupled to oxidation mediated by the piericidin-A-resistant pathway. 6. The presence of piericidin A altered the shape of the malate saturation curve. In the absence of the inhibitor it was found to be biphasic; saturating at 60 mM and in its presence to be monophasic saturing at 15 mM. At high concentrations of malate (60 mM) piericidin A caused approximately a 30% inhibition, similar to that found with other NAD+-linked substrates, while at lower concentrations of malate (15 mM) piericidin A caused a much higher inhibition (80%), which subsequently recovered to the much higher piericidin-A-resistant rate observed with higher concentrations of malate. 7. In the presence of piericidin A oxaloacetate caused severe but transient inhibition of malate oxidation, similar in magnitude to that observed during pyruvate oxidation in the absence of piericidin A. 8. It was concluded that the data were consistent with the spatial separation of both the internal malic enzyme and malate dehydrogenase together with the internally located piericidin-A-sensitive and resistent electron-transport pathways. [ABSTRACT FROM AUTHOR]