The energy-linked transhydrogenase in rat liver in relation to the reductive carboxylation of 2-oxoglutarate.

Abstract

1 The energy-linked transhydrogenase reaction was studied in isolated mitochondria and in submitochondrial particles from rat liver. 2 The rate of the transhydrogenase reaction in intact mitochondria, estimated by measuring the rate of reduction of NADP+ with 3-hydroxybutyrate as hydrogen donor, was about 30 nmol · min−1· mg mitochondrial protein−1 at 25° C. This value was higher than that calculated from the rate of the energy-linked transhydrogenase measured in submitechondrial particles. From the ratio of the activities at 25° C and 37° C in submitochondrial particles and the rate at 25° C in intact mitochondria it was estimated that the rate of the transhydrogenase reaction in intact mitochondria at 37° C is about 80 nmol · min−1· mg protein−1. 3 In intact mitochondria the rate of reductive carboxylation of 2-oxoglutarate with 3-hydroxybutyrate as hydrogen donor was about 20 nmol · min−1· mg protein−1 at 37° C. The main product formed (> 95%) was citrate. 4 The concentration gradient of citrate across the mitochondrial membrane was the same during reductive carboxylation of 2-oxoglutarate as when citrate was added under non-flux conditions, indicating that transport of tricarboxylates does not limit citrate formation. 5 The activity of isocitrate dehydrogenase (NADP), measured in sonicated mitochondria in the direction of reductive carboxylation, was about 20 nmol · min−1· mg protein−1 at 37° C. 6 It is concluded that the rate of formation of citrate by reductive carboxylation of 2-oxoglutarate is limited by the activity of isocitrate dehydrogenase (NADP). However, the rate of citrate formation is sufficiently high to account for the transport of reducing equivalents needed for hydroxylation reactions in the extramitochondrial compartment in liver.