Molecular and Cellular Biochemistry

, Volume 120, Issue 1, pp 15–23 | Cite as

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

  • Fabián Atlasovich
  • José A. Santomé
  • Horacio N. Fernández


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, 1.34×10−7 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 gmM; oleic acid, in turn, displaced the radiolabelled probe. Irradiation at 366 nm of125I-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. Both results, taken together, suggested that the derivatives are linked to the protein through the sites for fatty acids. When cross-linking of125I-5′ ASU was performed after incubation with delipidated cytosol and products were analyzed by SDS-PAGE, a band was visualized in a position similar to that of purified L-FABP.

Key words

fatty acid liver fatty acid-binding protein photoactivatable fatty acid cross-linking 



Fatty Acid-Binding Protein


Hepatic FABP


Intestinal FABP


Cardiac FABP

5′ ASU-11

(5′-azido-salicylamido)-undecanoic acid

Ac5′ ASU-11

(O-acetyl-5′-azido-salicylamido)-undecanoic acid


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Kaikaus RM, Bass NM, Okner RK: Functions of fatty acid binding proteins. Experientia 46:617–630, 1990Google Scholar
  2. 2.
    Clarke SD, Armstrong MK: Cellular lipid binding proteins: expression, function, and nutritional regulation. FASEB J 3:2480–2487, 1989Google Scholar
  3. 3.
    Bernier I, Jollès P: A survey on cytosolic non-enzymic proteins involved in the metabolism of lipophilic compounds: from organic anion binders to new protein families. Biochimie 69:1127–1152, 1987Google Scholar
  4. 4.
    Waggoner DW, Bernlhor DA: In situ labeling of the adipocyte lipid binding protein with 3-[125I]iodo-4-azido-N-hexadecylsalicylamide. J Biol Chem 265:11417–11420, 1990Google Scholar
  5. 5.
    McCormack M, Brecher P: Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes. Biochem J 244:717–723, 1987Google Scholar
  6. 6.
    Storch J: A comparison of heart and liver fatty acid-binding proteins: Interactions with fatty acids and possible functional differences studied with fluorescent fatty acid analogues. Mol Cell Biochem 98:141–147, 1990Google Scholar
  7. 7.
    Wilkinson TCI, Wilton DC: Studies on fatty acid-binding proteins. The binding properties of rat liver fatty acid-binding protein. Biochem J 247:485–488, 1987Google Scholar
  8. 8.
    Sheridan M, Wilkinson TCI, Wilton DC: Studies on fatty acid binding proteins. Changes in the concentration of hepatic fatty acid-binding protein during development in the rat. Biochem J 242:919–922, 1987Google Scholar
  9. 9.
    Storch J, Bass NM, Kleinfeld AM: Studies of the fatty acid-binding site of rat liver fatty acid-binding protein using fluorescent fatty acids. J Biol Chem 264:8708–8713, 1989Google Scholar
  10. 10.
    Waggoner DW, Manning JA, Bass NM, Bernlohr DA: In situ binding of fatty acids to the liver fatty acid binding protein: Analysis using 3-[125I]iodo-4-azido-N-hexadecylsalicylamide. Biochem Biophys Res Commun 180:407–415, 1991Google Scholar
  11. 11.
    Kinnunen PM, Klopf FH, Bastiani CA, Gelfman CM, Lange LG: 12-[(5-iodo-4-azido-2-hydroxybenzoil)amino]dodecanoic acid. Biological recognition by cholesterol esterase and axyl-CoA: cholesterol O-acyltransferase. Biochemistry 29:1648–1654, 1990Google Scholar
  12. 12.
    Dempsey ME, McCoy KE, Baker HN, Dimitriadou-Vafiadou A, Lorsbach T, Howard JB: Large-scale purification and structural characterization of squalene and sterol carrier protein. J Biol Chem 256:1867–1873, 1981Google Scholar
  13. 13.
    Glatz JFC, Veerkamp JH: A radiochemical procedure for the assay of fatty acid binding by proteins. Anal Biochem 132:89–95, 1983Google Scholar
  14. 14.
    Fraser RDB, Suzuki E: The use of least squares in data anlysis. In: Physical principles and techniques of protein chemistry, Part C, Leach, S.J. (ed.) Academic Press, N. York, 1973Google Scholar
  15. 15.
    Atkins PW: The rates of chemical reactions. In: Physical Chemistry, 4th ed. WH Freeman and Co, New York, 1990, pp 785–786 and 798–799Google Scholar
  16. 16.
    Chen RF: Removal of fatty acids from serum albumin by charcoal treatment. J Biol Chem 242:173–181, 1967Google Scholar
  17. 17.
    Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685, 1970Google Scholar
  18. 18.
    Bass NM: Function and regulation of hepatic and intestinal fatty acid binding proteins. Chem Phys Lipids 38:95–114, 1985Google Scholar
  19. 19.
    Wilkinson TCI, Wilton DC: Studies on fatty acid-binding proteins. The detection and quantification of the protein from rat liver by using a fluorescent fatty acid analogue. Biochem J 238: 419–424, 1986Google Scholar
  20. 20.
    Levi AJ, Gatmaitan Z, Arias IM: Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophtalein and other anions. J Clin Invest 48:2156–2167, 1969Google Scholar
  21. 21.
    Sacchettini JC, Gordon JI, Banaszak LJ: The structure of crystalline Escherichia coli-derived rat intestinal fatty acid-binding protein at 2.5-Å resolution. J Biol Chem 263:5815–5819, 1988Google Scholar
  22. 22.
    Sacchettini JC, Gordon JI, Banaszak LJ: Refined apoprotein structure of rat intestinal fatty acid binding protein produced in Escherichia coli. Proc Natl Acad Sci USA 86:7736–7740, 1989Google Scholar
  23. 23.
    Müller-Fahrnow A, Egner U, Jones TA, Rüdel H, Spener F, Saenger W. Three-dimensional structure of fatty-acid-binding protein from bovine heart. Eur J Biochem 199:271–276, 1991Google Scholar
  24. 24.
    Cistola DP, Sacchettini JC, Banaszak LJ, Walsh MT, Gordon JI: Fatty acid interactions with rat intestinal and liver fatty acid-binding proteins expressed in E. coli. J Biol Chem 264:2700–2710, 1989Google Scholar
  25. 25.
    Takahashi K, Odani S, Ono T: A close structural relationship of rat liver Z-protein to cellular retinoid binding proteins and peripheral nerve myelin P2 protein. Biochem Biophys Res Commun 106:1099–1105, 1982Google Scholar
  26. 26.
    Scapin G, Spadon P, Mammi M, Zanotti G, Monaco HL: Crystal structure of chicken liver basic fatty acid-binding protein at 2.7 Å resolution. Mol Cell Biochem 98:95–99, 1990Google Scholar
  27. 27.
    Westerman J, Wirtz KWA, Berkhout T, van Deenen LLM, Radhakrishnan R, Khorana HG: Identification of the lipid-binding site of phosphatidylcholine-transfer protein with phosphatidylcholine anlogs containing photoactivable carbene precursors. Eur J Biochem 132:441–449, 1983Google Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Fabián Atlasovich
    • 1
  • José A. Santomé
    • 1
  • Horacio N. Fernández
    • 1
  1. 1.Facultad de Farmacia y BioquímicaInstituto de Química y Fisicoquímica Biológicas (UBA-CONICET)Buenos AiresArgentina

Personalised recommendations