Azido palmitic acid

Photoreactive probes for the hydrophobic pocket of the liver fatty acid-binding protein, 11-(5'-azido-salicylamido)-undecanoic acid (5' ASU) and its acetyl ester (Ac5' ASU), were synthesized and their interaction with the protein was assessed. Fatty acid-binding proteins are closely related proteins which are abundantly expressed in tissues with active lipid metabolism. A simple model that assumes that the protein possesses a single kind of sites fitted the binding of radioiodinated 5' ASU to L-FABP satisfactorily. The apparent dissociation constant, 1.34 x 10(-7) M, evidenced a slightly higher affinity than that reported for C16-C20 fatty acids. Consistent with the binding curve, 5' ASU effectively competed with palmitic acid for the hydrophobic sites and the effect was nearly complete for concentrations of 1 microM; oleic acid, in turn, displaced the radiolabelled probe. Irradiation at 366 nm of 125I-5' ASU bound to L-FABP caused the covalent cross-linking of the reagent. The amount of radioactivity covalently bound reached a maximum after 2 min thus agreeing with the photo-activation kinetics of the unlabelled compound that evidenced a t1/2 of 31.1 sec. The yield with which probes bound to L-FABP became covalently linked to the protein, appraised after SDS-PAGE of irradiated samples, was estimated as 23 and 26 per cent for 5' ASU and Ac5' ASU respectively. In turn, irradiation of L-FABP incubated with 5' ASU or Ac5' ASU resulted in the irreversible loss of about one fourth its ability to bind palmitic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

The suitability as FRET probes of two bichromophoric 1-deoxydihydroceramides containing a labelled spisulosine derivative as a sphingoid base and two differently ω-labelled fluorescent palmitic acids has been evaluated. The ceramide synthase (CerS) catalyzed metabolic incorporation of ω-azido palmitic acid into the above labeled spisulosine to render the corresponding ω-azido 1-deoxyceramide has been studied in several cell lines. In addition, the strain-promoted click reaction between this ω-azido 1-deoxyceramide and suitable fluorophores has been optimized to render the target bichromophoric 1-deoxydihydroceramides. These results pave the way for the development of FRET-based assays as a new tool to study sphingolipid metabolism.

The protonophoretic function of uncoupling protein (UCP) is activated by fatty acids. According to the "docking site" hypothesis (Jezek, P., and Garlid, K. D., J. Biol. Chem. 265, 19303-19311, 1990), the fatty acid binding site is identical with the anion channel of UCP. Skulachev (Skulachev, V. P. (1991) FEBS Lett. 294, 158-162) extended this hypothesis by suggesting that fatty acid anions are transported by UCP and that H+ are delivered by back-diffusion of the protonated fatty acid through the lipid bilayer. In this model, UCP does not transport H+ at all but rather enables fatty acids to act as cycling protonophores. New evidence supports this mechanism (Garlid, K. D., Orosz, D. E., Modriansky, M., Vassanelli, S., and Jezek, P. (1996) J. Biol. Chem. 271, 2615-2620). To help elucidate these hypotheses, we synthesized a photoreactive analog of dodecanoic acid, 12-(4-azido-2-nitrophenylamino)dodecanoic acid (AzDA), and studied its effect on transport in mitochondria and proteoliposomes. AzDA behaved in every respect like a typical fatty acid. In micromolar doses, AzDA activated H+ translocation and inhibited Cl- and hexanesulfonate uniport through UCP. After UV light exposure, however, activation of H+ transport was inhibited, whereas inhibition of anion transport was preserved. These effects were irreversible. Photolabeling of mitochondria with [3H]AzDA resulted in a prominent 32 kDa band of UCP, and few other proteins were labeled. The results indicate that AzDA can be ligated to the protein at or near the docking site, causing irreversible inhibition of both H+ and anion transport. The finding that fatty acid-induced H+ transport disappears along with anion transport supports the fatty acid-protonophore mechanism of H+ transport by UCP.