Purification of basic fibroblast growth factor receptors from bovine brain.
Fibroblast growth factors are proteins which play a major role, in vitro and in vivo, in the control of cellular growth and differentiation of a large number of cells. Biological activities of these factors are mediated by the interaction with specific membrane receptors. Previous studies indicated that the apparent molecular weight of a family of these receptors for the basic form of Fibroblast Growth Factor (bFGF), ranges from 125 to 165 kDa according to cell species and types. We have purified this family of receptors from bovine brain. We first set up a radioreceptor assay to detect receptors throughout the purification by measuring its ability to inhibit the fixation of radiolabeled bFGF to insolubilized membranes from bovine brain. The purification was also monitored by using cross-linking reagents in order to allow the visualization of radiolabeled bFGF bound to its receptor. The first purification steps involved 2 anion-exchange chromatographic steps, DEAE Trysacryl and FPLC Mono Q, and yielded an enrichment over 500 fold. Affinity chromatography with bFGF immobilized on Sepharose 4B was then performed. Covalent fixation of bFGF to the Sepharose matrix was carried out in presence of N-acetylated heparin in order to protect the recognition site for bFGF on its receptor. These 3 chromatographic steps yielded only 2 bands of apparent molecular weight of 100 kDa and 135 kDa as detected by electrophoresis. These 2 bands are also detected after chromatography on immobilized wheat germ agglutinin hence confirming the presence of carbohydrates on bFGF receptors.
Mereau
A
A
Laboratoire de Biotechnologie des Cellules Eucaryotes, Université Paris XII, Créteil, France.
Pieri
I
I
Gamby
C
C
Courty
J
J
Barritault
D
D
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D005346:Fibroblast Growth Factors / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor; D012995:Solubility
D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D017468:Receptors, Fibroblast Growth Factor; D005346:Fibroblast Growth Factors