Mitochondrial-microsomal interactions in vitro: use of microsomal 7-ethoxycoumarin O-deethylase to study...

An assay system was developed utilizing microsomal 7-ethoxycoumarin O-deethylase to monitor the formation and efflux of malate from mitochondria in vitro. Malate was coupled by means of malic enzyme, NADP+, and the microsomal mixed-function oxidase system to the production of highly fluorescent 7-hydroxycoumarin. Under conditions of the assay, steady-state concentrations of NADPH could be maintained, and half-maximal rates of microsomal 7-ethoxycoumarin O-deethylase were observed with about 0.2 microM NADPH and 250 microM malate. Addition of succinate to mixtures of intact mitochondria and microsomes enhanced rates of 7-hydroxycoumarin formation by facilitating the generation and transport of malate from mitochondria (half-maximal stimulation was observed with 0.4 mM succinate). Stimulation of 7-hydroxycoumarin formation by succinate, under conditions where concentrations of cofactors and substrate were adjusted to resemble those in the intact cell, provides further evidence that reducing equivalents for mixed-function oxidation may arise via malate efflux from mitochondria.