LM fibroblast plasma membrane subfractionation by affinity chromatography on con A-sepharose.
Affinity chromatography was used to determine the heterogeneity and orientation of plasma membrane vesicles isolated from LM fibroblasts subjected to Dounce homogenization. Two plasma membrane subfractions were obtained by Con A-Sepharose affinity chromatography of LM fibroblast plasma membranes prepared by Dounce homogenization. The desmosterol-phospholipid molar ratio, the phospholipid composition, and the phospholipid fatty acid composition were almost identical between the two fractions. However, the lipid to protein ratio was almost 2-fold greater in the nonadherent fraction A. The binding of fluorescein-concanavalin A was the same in both fractions indicating a right-sided-out orientation of the vesicles. Similarly and asymmetric distribution of phosphatidylethanolamine in both membrane fractions was the same. In contrast, sialic acid content, 5'-nucleotidase activity, and (Na+ + K+)-ATPase activity were 47%, 3.7-fold, and 2.5-fold greater, respectively, in the nonadherent, lipid-rich fraction A. Structural properties of the two membrane fractions determined by fluorescence polarization and arrhenius plots of trans-parinaric acid fluorescence were similar. These results indicate that concanavalin-A affinity chromatography separates two membrane fractions differing in sialic acid content, lipid content, and enzyme profile but having the same right-side-out orientation.
D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000648:ultrastructure*; D002846:Chromatography, Affinity / Q000379:methods; D005347:Fibroblasts / Q000648:ultrastructure; D007739:L Cells / Q000648:ultrastructure; D008563:Membrane Lipids / Q000032:analysis; D051379:Mice; D008854:Microscopy, Electron; D010042:Ouabain / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis; D012685:Sepharose / Q000031:analogs & derivatives
D008563:Membrane Lipids; D010743:Phospholipids; C028326:concanavalin A-sepharose; D010042:Ouabain; D012685:Sepharose