Activating -6 Polyunsaturated Fatty Acids and Inhibitory Purine Nucleotides Are High Affinity Ligands for Novel Mitochondrial Uncoupling Proteins UCP2 and UCP3*

Human UCP2 and UCP3, expressed in yeast, were studied to establish their high affinity regulatory ligands. UCPn were reconstituted into liposomes and assayed for fatty acid (FA)-induced H efflux. All natural long chain FAs activated UCP2and UCP3-mediated H translocation. Coenzyme Q10 had no further significant activating effect. Evaluated parameters of FA activation (FA cycling) kinetics revealed the highest apparent affinity to UCP2 (the lowest Km values: 20 and 29 M, respectively) for -6 polyunsaturated FAs (PUFAs), allcis-8,11,14-eicosatrienoic and all-cis-6,9,12-octadecatrienoic acids, which are also the most potent agonists of the nuclear PPAR receptor in the activation of UCP2 transcription. -3 PUFA, cis-5,8,11,14,17-eicosapentaenoic acid had lower affinity (Km, 50 M), although as an -6 PUFA, arachidonic acid exhibited the same low affinity as lauric acid (Km, 200 M). These findings suggest a possible dual role of some PUFAs in activating both UCPn expression and uncoupling activity. UCP2 (UCP3)dependent H translocation activated by all tested FAs was inhibited by purine nucleotides with apparent affinity to UCP2 (reciprocal Ki) decreasing in order: ADP > ATP GTP > GDP >> AMP. Also [H]GTP ([H]ATP) binding to isolated Escherichia coli (Kd, 5 M) or yeast-expressed UCP2 (Kd, 1.5 M) or UCP3 exhibited high affinity, similar to UCP1. The estimated number of [H]GTP high affinity (Kd, <0.4 M) binding sites was (in pmol/mg of protein) 182 in lung mitochondria, 74 in kidney, 28 in skeletal muscle, and 20 in liver mitochondria. We conclude that purine nucleotides must be the physiological inhibitors of UCPn-mediated uncoupling in vivo.