Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein.
The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated by a simple two step protocol combining ion exchange chromatography and gel filtration. Dissociation constants for binding of oleic acid, arachidonic acid, oleoyl-CoA, lysophosphatidic acid and the peroxisomal proliferator bezafibrate to L-FABP have been determined by titration calorimetry. All ligands were bound in a 2:1 stoichiometry, the dissociation constants for the first ligand bound were all in the micro molar range. Oleic acid was bound with the highest affinity and a Kd of 0.26 microM. Furthermore, binding of cholesterol to L-FABP was investigated with the Lipidex assay, a liposome binding assay and a fluorescence displacement assay. In none of the assays binding of cholesterol to L-FABP was observed.
Rolf
B
B
Department of Biochemistry, University of Münster, Germany.
Oudenampsen-Krüger
E
E
Börchers
T
T
Faergeman
N J
NJ
Knudsen
J
J
Lezius
A
A
Spener
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D003001:Cloning, Molecular; D003911:Dextrans; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D008024:Ligands; D008081:Liposomes / Q000737:chemistry; D008099:Liver / Q000378:metabolism; D008246:Lysophospholipids / Q000378:metabolism; D008969:Molecular Sequence Data; D018996:Myelin P2 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis; D013816:Thermodynamics
D002352:Carrier Proteins; D003911:Dextrans; C493320:Fabp1 protein, mouse; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D008024:Ligands; D008081:Liposomes; D008246:Lysophospholipids; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C010599:lipidex; D002784:Cholesterol