Mitochondrial-microsomal interactions in vitro: use of microsomal 7-ethoxycoumarin O-deethylase to study succinate-stimulated malate efflux from mitochondria.
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.
D015660:7-Alkoxycoumarin O-Dealkylase; D000818:Animals; D001692:Biological Transport; D066298:In Vitro Techniques; D008291:Malate Dehydrogenase / Q000378:metabolism; D008293:Malates / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013386:Succinates / Q000502:physiology*; D019802:Succinic Acid
D008293:Malates; D013386:Succinates; D009249:NADP; C030298:malic acid; D019802:Succinic Acid; D008291:Malate Dehydrogenase; D010105:Oxygenases; D015660:7-Alkoxycoumarin O-Dealkylase