Photoreactive fatty acid analogues that bind to the rat liver fatty-acid binding protein: 11-(5'-azido-salicylamido)-undecanoic acid derivatives.
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)
Atlasovich
F
F
Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
Santomé
J A
JA
Fernández
H N
HN
D000818:Animals; D001386:Azides / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism*; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010777:Photochemistry; D051381:Rats
D001386:Azides; D002352:Carrier Proteins; C493320:Fabp1 protein, mouse; C493321:Fabp1 protein, rat; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D007202:Indicators and Reagents; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; C080433:11-(5'-azidosalicylamido)undecanoic acid