High-performance affinity chromatography for the purification of heparin-binding proteins from detergent-solubilized smooth muscle cell membranes.
Heparin and heparan sulfates are regulators of cellular events including adhesion, proliferation and migration. In particular, the antiproliferative effect of heparin on smooth muscle cell (SMC) growth is well described. However, its mechanism of action remains unclear. Numerous results suggest an endocytosis mediated by a still unknown heparin receptor on vascular SMCs. In order to identify a putative heparin receptor on SMCs that could be involved in heparin signalling, affinity chromatography supports were developed. In this paper, we describe high-performance liquid affinity chromatography (HPLAC) supports obtained from silica beads coated with dextran polymer substituted by a calculated amount of diethylaminoethyl functions. With a polysaccharide dextran layer, this type of support can be grafted with specific ligands, such as heparin, using conventional coupling methods. In a previous work, we demonstrated, using butanedioldiglycidyl ether, that silica stationary phases coupled to heparin could be used for the fast elution and good peak resolution of heparin-binding proteins. In the present work, an affinity chromatographic fraction of SMC membrane extracts was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and six heparin-binding proteins from dodecyloctaethyleneglycol monoether-solubilized SMCs were observed. Their Mr values were between 40 and 70 kDa, with three major protein bands at 66, 45 and 41 kDa. These results indicate the usefulness of the chromatographic method for purifying heparin binding proteins from SMC membrane.
Clairbois
A S
AS
LRM, CNRS URA 502, Institut Galilée, University of Paris XIII, Villetaneuse, France.
Letourneur
D
D
Muller
D
D
Jozefonvicz
J
J
D000818:Animals; D001011:Aorta; D002462:Cell Membrane / Q000737:chemistry*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003432:Cross-Linking Reagents; D003902:Detergents; D003911:Dextrans; D006493:Heparin / Q000494:pharmacology; D008863:Microspheres; D009131:Muscle, Smooth, Vascular / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D012822:Silicon Dioxide; D012995:Solubility
D003432:Cross-Linking Reagents; D003902:Detergents; D003911:Dextrans; D011956:Receptors, Cell Surface; C037680:heparin receptor; D012822:Silicon Dioxide; D006493:Heparin