Regulation of Phosphoenolpyruvate Synthesis from Succinate in Guinea Pig Liver Mitochondria 1

Abstract

The synthesis of phosphoenolpyruvate (PEP) from oxaloacetate (OAA) is catalyzed by phosphoenolpyruvate carboxykinase [GTP: oxaloacetate carboxy-lyase (transphos-phorylating EC 4.1.1.32)] and is one of the key reactions in the pathway of gluconeogenesis. This enzyme has been shown to be present exclusively in the mitochondria of chicken and rabbit liver while it is found almost exclusively in the cytosol of rat liver (1-3). In guinea pig liver the phosphoenolpyruvate carboxykinase is present both in the mitochondria and in the cytosol (2). The phosphoenolpyruvate carboxykinase (PEPcK) in the cytosol is readily responsive to metabolic, hormonal, and dietary modulation, while the activity of the mitochrondrial enzyme remains essentially unchanged (4, 5). Mitochondrial PEP synthesis requires GTP which can be generated by substrate level phosphorylation from a-ketoglutarate (6-8) or by nucleosidediphosphate kinase (EC 2.7.4.6) from ATP (9-11).

Major studies with guinea pig liver mitochondria concerning PEP synthesis from a variety of substrates have been previously reported (1, 2, 8, 12-16). The regulation and synthesis of PEP from succinate in the absence of substrate level phosphorylation which generates GTP has not yet been well studied. This investigation therefore deals with the above-mentioned regulation through control of the substrate equilibrium by malonate, 2,4-dinitrophenol (DNP), fatty acids, ATP, ADP, and rotenone.

Materials and Methods. Male albino guinea pigs (weighing 275-450 g) were used in all experiments. The isolation of intact liver mitochondria; incubation; processing of samples; and analysis for ¹⁴CO₂-trapped, malate, citrate, acetoacetate, β-hydroxybutyrate, phosphoenolpyruvate, and mitochondrial nitrogen were performed by methods previously described (17—19).

With the exception of the polarographic measurements shown in Table III all the incubation mixtures contained 6.7 mM succinate, 10.0 mM MgSO₄, 13.3 mM potassium phosphate, and 13.3 mM triethanolamine buffers at pH 7.3.