Photoreactive fatty acid analogues that bind to the rat liver fatty-acid binding protein: 11-(5?-azido-salicylamido)-undecanoic acid derivatives

Abstract

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)

Interaction of Unsaturated Fatty Acids with the Red Blood Cell Ca2+-ATPase. Studies with a Novel Photoactivatable Probe

“…For comparison purposes, cross-linking with a different fatty acid analogue, 11 -(O-acetyl-3'-[125I]iodo-5'-azidosalicylamido)undecanoic acid (Atlasovich et al, 1993), was also examined (lane 3). Experiments testing the latter probe (not shown) evidenced no effect on the Ca2+-ATPase activity Biochemistry, Vol.…”

“…Synthesis of 8-(5'-Azidosalicylamido)octanoic Acid. 5-Azidosalicylic acid was obtained as described previously (Atlasovich et al, 1993). The subsequent coupling reaction was similar to that used by Kinnunen et al (1990).…”

Synthesis of an unsaturated fatty acid analogue (18-(4′-azido-2′-hydroxybenzoylamino)-oleic acid) and its interaction with lysophosphatidylcholine: acyl-CoA-O-acyltransferase

“…Several attempts have been made in the past to characterize enzymes and proteins involved in membrane lipid biosynthesis using photoaffinity fatty acyl-CoAs as substrate analogues. Most of these studies have been carried out with a photolabel based on medium-chain (C 11-12 ) saturated fatty acids (24)(25)(26)(27)(28)(29)(30)(41)(42)(43)(44); fewer studies were done with substances based on long-chain (C 16-18 ), saturated (45,46) or unsaturated (41)(42)(43)47) fatty acids, where some reactive groups are positioned within the hydrophobic chain, which may affect the binding of the analogue. Several proteins involved in lipid biosynthetic pathways have been examined with these analogues, e.g., acyl-CoA:cholesterol acyltransferase and cholesterol esterase (24), N -acylphosphatidylethanolamine synthase (29), acyl-CoA binding protein (25,28), fatty acid binding protein (26,45), acyl-CoA oxidase (27,41), LAT (42,43), or monoacylglycerol:acyl-CoA acyltransferase and diacylglycerol:acyl-CoA acyltransferase (30), but only few attempts have been made to characterize proteins specifically utilizing unsaturated fatty acids or acyl-CoAs.…”

“…An additional possibility to characterize enzymes is the use of photoaffinity probes. Several attempts to analyze acyl-CoA binding proteins have been made using a photoaffinity analogue of a medium-chain saturated fatty acid (24)(25)(26)(27)(28)(29)(30). Because of the high specificity of LAT in human placenta and pig spleen (31) that we examined, the synthesis of an unsaturated fatty acid (oleic acid) analogue with facilities for -terminal coupling with a (iodinable) photoaffinity label has become a highly desirable goal.…”