Different glycosidic probes upon binding to coronary endothelial luminal lectins allow the isolation of these proteins

Abstract

The coronary endothelial luminal membrane (CELM) has structures involved in blood flow transduction-physiological response; these flow-sensitive structures may be lectinic. We isolated the coronary endothelial luminal plasma membrane from various species. Lectinic proteins were identified using monosaccharide polymers (probes) formed either by Mannose, N-acetyl-Glucosamine or Galactose covalently linked to a 70kDa Dextran. We isolated CELM by coating with cationic silica, polymerizing with polyacrylic acid, homogenizing the tissue, and centrifuged in a density gradient. Proteins were solubilized by probe sonication in SDS, analyzed via polyacrylamide gel electrophoresis. Adherence of the silica colloid was assessed via electron microscopy and non-luminal coronary protein contamination during homogenization was discarded. A comparison of protein profiles obtained from guinea pig, rat and porcine hearts. The protein fraction purified in affinity columns made up by one of the probes and retained lectins were eluted with the corresponding free monosaccharide. From the guinea pig luminal coronary membrane protein extract we isolated one lectin using the mannose probe and two lectins using the galactose probe. Together with physiological studies these data suggest that these purified proteins may be involved in blood flow sensing. Funded by CONACyT SEP-42567, CONACyT-SALUD 2004-C01-156