A study of the fatty acid-binding protein of rat liver
A study of the fatty acid-binding protein of rat liver
A fluorescent fatty acid derivative, dansyl undecanoic acid, has been used as a means for the rapid detection and quantification of rat liver fatty acid-binding protein (FABP) during purification procedures. At the emission maximum of the protein bound fatty acid probe there was a considerable fluorescence enhancement compared with the probe in buffer and as a result the method allowed the detection of μg amounts of FABP in column fractions. The use of this technique combined with high performance gel permeation chromatography has allowed the rapid estimation of FABP in cytosols prepared from rat liver. Using this technique, it was demonstrated that administration of the hypolipidaemic drugs clofibric acid, clobuzarit and tiadenol resulted in an increase in the cytosolic levels of FABP concomitant with an enhancement of peroxisomal β-oxidation activity. This finding supports the hypothesis that peroxisome proliferators, irrespective of their chemical structure, produce increases in the cytosolic level of liver FABP. In addition, the analysis of livers from rats which had been maintained on a 12-h light, 12-h dark cycle did not show any significant diurnal variation in cytosolic FABP levels. Detailed analysis of the binding of dansyl undecanoic acid to FABP indicated that there was a single binding site per protein molecule. However, the observed dissociation constant for probe binding was found to be dependent on protein concentration and non-linear Scatchard plots were observed. At low protein concentrations the Scatchard plot was resolved to give primarily a high affinity binding site (Kd 0.03 μM) whilst at high protein concentration (above 1 μM) a low affinity site predominated (Kd 0.5 μM). Competition experiments, in which the decrease in fluorescence emission due to the displacement of probe by competing ligands was measured, indicated that fatty acids were bound more strongly than their corresponding CoA-esters and that oleic acid and arachidonic acid were the most potent inhibitors tested. In addition, cholesterol was found to be completely ineffective in displacing the probe. Finally, the purified FABP has been used in order to raise polyclonal antibodies in rabbits. The antibodies obtained were shown to be monospecific by Ouchterlony immunodiffusion analysis and by Western blotting. The titre of antibodies, however, was found to be low indicating that native FABP is only weakly immunogenic. (D72718/87)
University of Southampton
Wilkinson, Trevor Charles Ian
1986
Wilkinson, Trevor Charles Ian
Wilkinson, Trevor Charles Ian
(1986)
A study of the fatty acid-binding protein of rat liver.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
A fluorescent fatty acid derivative, dansyl undecanoic acid, has been used as a means for the rapid detection and quantification of rat liver fatty acid-binding protein (FABP) during purification procedures. At the emission maximum of the protein bound fatty acid probe there was a considerable fluorescence enhancement compared with the probe in buffer and as a result the method allowed the detection of μg amounts of FABP in column fractions. The use of this technique combined with high performance gel permeation chromatography has allowed the rapid estimation of FABP in cytosols prepared from rat liver. Using this technique, it was demonstrated that administration of the hypolipidaemic drugs clofibric acid, clobuzarit and tiadenol resulted in an increase in the cytosolic levels of FABP concomitant with an enhancement of peroxisomal β-oxidation activity. This finding supports the hypothesis that peroxisome proliferators, irrespective of their chemical structure, produce increases in the cytosolic level of liver FABP. In addition, the analysis of livers from rats which had been maintained on a 12-h light, 12-h dark cycle did not show any significant diurnal variation in cytosolic FABP levels. Detailed analysis of the binding of dansyl undecanoic acid to FABP indicated that there was a single binding site per protein molecule. However, the observed dissociation constant for probe binding was found to be dependent on protein concentration and non-linear Scatchard plots were observed. At low protein concentrations the Scatchard plot was resolved to give primarily a high affinity binding site (Kd 0.03 μM) whilst at high protein concentration (above 1 μM) a low affinity site predominated (Kd 0.5 μM). Competition experiments, in which the decrease in fluorescence emission due to the displacement of probe by competing ligands was measured, indicated that fatty acids were bound more strongly than their corresponding CoA-esters and that oleic acid and arachidonic acid were the most potent inhibitors tested. In addition, cholesterol was found to be completely ineffective in displacing the probe. Finally, the purified FABP has been used in order to raise polyclonal antibodies in rabbits. The antibodies obtained were shown to be monospecific by Ouchterlony immunodiffusion analysis and by Western blotting. The titre of antibodies, however, was found to be low indicating that native FABP is only weakly immunogenic. (D72718/87)
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Published date: 1986
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Local EPrints ID: 460811
URI: http://eprints.soton.ac.uk/id/eprint/460811
PURE UUID: 1619051a-261f-4615-95fa-7fef2d457a8f
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Date deposited: 04 Jul 2022 18:30
Last modified: 04 Jul 2022 18:30
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Author:
Trevor Charles Ian Wilkinson
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