Continuous measurement of oxaloacetate in purified mitochondria from the leaves of Kalanchoë blossfeldiana

Abstract

A new method for the continuous assay of oxaloacetate released or taken up by plant mitochondria during malate oxidation is described. It is based on the continuous spectrophotometric recording of the reduction level of externally added NAD+ (0.4 mM) to a mitochondrial preparation. In the presence of 20 mM malate and of externally added malate dehydrogenase (EC 1.1.1.37), an equilibrium is reached instantaneously, bringing about a partial reduction of NAD+ and the production of a proportional amount of oxaloacetate (OAA). Owing to the presence of a very active OAA carrier on the inner mitochondrial membrane, the concentration at the equilibrium position of the reactants of the external MDH is permanently displaced by the OAA released or taken up by the mitochondria. Therefore, changes in OAA concentration can be followed from the measurement of the reduction level of the external NAD+. This method appears as sensitive as the classical enzymatic method, but is much more rapid and requires much less mitochondrial protein. The proposed method was applied to Percoll‐purified mitochondria from the leaves of a CAM plant, Kalanchoë blossfeldiana Poelln. cv. Tom Thumb. The simultaneous recording of the change in OAA concentration and of oxygen uptake during malate oxidation emphasizes the major control exerted by OAA on the rate of malate oxidation.