EFFECTS OF LONG-CHAIN ACYL-COENZYME AS ON THE ACTIVITY OF THE SOLUBLEFORM OF NICOTINAMIDE ADENINE-DINUCLEOTIDE PHOSPHATE-SPECIFIC ISOCITRATE DEHYDROGENASE FROM LACTATING BOVINE MAMMARY-GLAND

The cytosolic form of NADP(+):isocitrate dehydrogenase, a primary sour ce 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(+) b y the enzyme is inhibited by palmitoyl-CoA. In steady-state experiment s, when added enzyme is used to start the reaction, analyses of veloci ty 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 effec ts on the enzyme. This analysis suggested inhibition first through bin ding to sites with an average dissociation constant of 3.3 mu M, then by binding to sites with an average dissociation constant of 294 mu M. When the enzyme is preincubated with palmitoyl-CoA there is an induct ion of a significant lag-burst reaction rate (hysteretic kinetics), Pr eincubation 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 conce ntration of palmitoyl-CoA followed patterns, similar to those observed in steady-state reactions, where the enzyme is not preincubated, Exam ination of the effect of acyl chain length at 300 mu M demonstrated th at only long-chain CoA's with carbon numbers >14 have pronounced effec ts on kinetics. CoA alone has little or no effect, while stearoyl-CoA completely inhibited the enzyme. Other C-18 acyl groups produced varyi ng effects depending on the degree of unsaturation and cis-trans isome rism, 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 t hese transitions are compatible with physiological conditions. This su ggests that complexes of acyl-CoA's and NADP(+):isocitrate dehydrogena se, in tissue rich in the cytoplasmic form of the enzyme, could be rel ated to cytoplasmic events in the synthesis and secretion of lipid and possibly protein, since palmitoyl-CoA is known to promote secretory p rocesses through acylation reactions which lead to vesicle fusion. (C) 1995 Academic Press, Inc.