Abstract
Free fatty acids in plasma and cells are mainly bound to membranes and proteins such as albumin and fatty acid binding proteins (FABP), which can regulate their biological activities and metabolic transformations. We have investigated the effect of FABP and albumin on the peroxidation of linoleic acid (18:2) and arachidonic acid (20:4) by 15-lipoxygenase (15-LO). Rabbit reticulocyte 15-LO produced a rapid conversion of [1- 14C]18:2 to 13-hydroxyoctadecadienoic acid (13-HODE) and [ 3H]20:4 to 15-hydroxyeicosatetraenoic acid (15-HETE). 13-HODE formation was reduced when intestinal FABP (I-FABP), liver FABP (L-FABP) or albumin was added. The relative ability of these proteins to reduce 15-LO induced formation of 13-HODE and 15-HETE was BSA>L-FABP>I-FABP. Smaller reductions in activity were observed with 20:4 as compared to 18:2. The IC 50-values of I-FABP and L-FABP, using either 18:2 (3.4 μM) or 20:4 (3.4 μM), were 4.6±0.6 and 1.9±0.2 μM, respectively, for reduction of 13-HODE and 6.8±0.3 and 3.1±0.2 μM, respectively, for reduction of 15-HETE formation. The smaller 15-HETE reduction correlated with decreased binding of 20:4 to the FABP. Titration calorimetry also showed that the I-FABP IC 50 for 18:2, 0.25 μM, was lower then for 20:4, 0.6 μM. Thus the reduction in fatty acid lipid peroxidation relates to the binding capacity of each FABP. We also demonstrated that 18:2 rapidly diffuses (flip-flops) across the phospholipid bilayer of small unilamellar vesicles (SUV) and measured partitioning of 18:2 between proteins and SUV by the pyranin fluorescence method [Kamp, F. and Hamilton, J.A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11367–11370]. Addition of proteins to SUV in buffer resulted in a complete desorption of 18:2 from SUV with a relative effect of BSA>L-FABP>I-FABP. This suggests that the relative effects of these proteins on 18:2 peroxidation will not be altered by the presence of membranes. Our results indicate that FAPBs protect intracellular polyunsaturated fatty acids against peroxidation and, through differential binding of 18:2 and 20:4, they may modulate the availability of these polyunsaturated fatty acids to intracellular oxidative pathways. © 1997 Elsevier Science B.V. All rights reserved.
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