Characterization of soybean plasma membrane during development. Free sterol composition and concanavalin A binding studies

Plasma membrane preparations from soybean root and hypocotyl contained the following free sterols: cholesterol, campesterol, stigmasterol, and sitosterol. The cholesterol level was relatively low in root plasma membrane (less than 0.5%) but was 1.4 to 2.4% in hypocotyl membrane. The relative levels of the three other sterols fluctuated with cellular development and tissue source. Campesterol level decreased with the development of both root and hypocotyl membrane. With development, stigmasterol increased greatly in root membrane but remained constant in hypocotyl membrane, and sitosterol, the major free sterol component of all membrane preparations, decreased in root membrane but increased slightly in hypocotyl membrane. Electron microscope studies indicated that all root plasma membrane preparations were equivalent in terms of relative purity. Hypocotyl membrane preparations contained significantly greater levels of contaminating membrane components. Root plasma membrane fractions were between 70 and 80% pure as determined by staining with the phosphotungstic acid-chromic acid procedure (PACP). Staining was most definitive for vesicles present in complete cross-section. Electron micrographs showed that vesicles treated with concanavalin A (Con A)-ferritin were extensively labeled at the outer surface indicating the presence of mannosyl and/or glucosyl residues at the vesicle surface. Densities of ferritin were highest on vesicles present in oblique section. PACP and Con A-ferritin were thus complementary with respect to topological specificity. The percentage of Con A-ferritin-labeled and/or PACP-stained vesicles in plasma membrane root preparations was greater than 80%. Con A did not bind in purified tonoplast preparations, and binding was reduced in regions of low PACP reactivity in a root membrane fraction containing a lowered proportion of plasma membrane. Con A specificity for the plasma membrane in subcellular membrane preparations is discussed.