Effects of Long-Chain Acyl-Coenzyme A′s on the Activity of the Soluble Form of Nicotinamide Adenine Dinucleotide Phosphate-Specific Isocitrate Dehydrogenase from Lactating Bovine Mammary Gland

The cytosolic form of NADP +:isocitrate dehydrogenase, a primary source of the NADPH required for de novo fatty acid synthesis in lactating bovine mammary gland, was studied to determine possible mechanisms of regulation by fatty acyl-coenzyme A (CoA). The reduction of NADP + by the enzyme is inhibited by palmitoyl-CoA. In steady-state experiments, when added enzyme is used to start the reaction, analyses of velocity versus palmitoyl-CoA concentration as a binding isotherm, following the assumptions of Wyman′s theory of thermodynamic linkage, suggested that binding of palmitoyl-CoA produced two different inhibitory effects on the enzyme. This analysis suggested inhibition first through binding to sites with an average dissociation constant of 3.3 μM, then by binding to sites with an average dissociation constant of 294 μM. When the enzyme is preincubated with palmitoyl-CoA there is an induction of a significant lag-burst reaction rate (hysteretic kinetics). Preincubation of the enzyme with its substrate, metal-isocitrate complex, nearly abolished the lag time and decreased the degree of inhibition. Changes in lag time and percentage inhibition as a function of concentration of palmitoyl-CoA followed patterns, similar to those observed in steady-state reactions, where the enzyme is not preincubated. Examination of the effect of acyl chain length at 300 μM demonstrated that only long-chain CoA′s with carbon numbers >14 have pronounced effects on kinetics. CoA alone has little or no effect, while stearoyl-CoA completely inhibited the enzyme. Other C 18 acyl groups produced varying effects depending on the degree of unsaturation and cis-trans isomerism. NADP +:Isocitrate dehydrogenases, from other sources including that from Escherichia coli, do not show such sensitivity to acyl chain character under these conditions. Concentration ranges observed for these transitions are compatible with physiological conditions. This suggests that complexes of acyl-CoA′s and NADP +:isocitrate dehydrogenase, in tissue rich in the cytoplasmic form of the enzyme, could be related to cytoplasmic events in the synthesis and secretion of lipid and possibly protein, since palmitoyl-CoA is known to promote secretory processes through acylation reactions which lead to vesicle fusion.