Isolation and characterization of the basal cell membranes of the human term placenta.

ABSTRACT

A relatively simple and rapid method is described for the isolation of basal cell membranes (BCM) from the human placenta at term which showed considerable improvement in the yield, purity and membrane characteristics as compared to the earlier described methods. The method is based on thorough washings of the syncytium in balanced salt solution, selective grinding, hypotonic lysis, sonication, incubation with EDTA and then more conventional differential centrifugation and ultracentrifugation. The isolated material showed smooth surfaced vesicular structure of various sizes as revealed by both positive and negative staining and transmission electron microscopic analysis. The membrane was highly enriched in Na+/K+, Ca2+ and Mg2+ dependent ATPase activities while the cross contamination with brush border surfaces was low as revealed by the marker enzyme assays specific for the brush border membrane (BBM) such as the disaccharide hydrolases, aminopeptidase and alkaline phosphatase. The membrane showed a relatively low lipid/protein ratio and the lipid composition represented by a variety of phospholipids (phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl inositol and phosphatidyl serine), neutral lipids (cholesterol, triacyl glycerol, free fatty acids) and glycosphingolipids (ganglioside, cerebroside and sulfatide). It also contained plasmalogens. On SDS-PAGE analysis and Coomassie blue staining reaction, the isolated membrane showed 14 major bands with as many minor ones with a molecular weight ranging between 30-110 kDa.