An electronmicroscopic study of the distribution of myelin basic protein in multilayer dispersions of diacylphosphatidylserine

Abstract

Although dispersions containing lipid and protein are widely used as model systems to explore the properties of biomembranes, the extent of mixing of the two components has generally not been determined. Here, the distribution of bovine myelin basic protein in dispersions with bovine brain L-α-diacylphosphatidylserine (PS) has been examined electronmicroscopically. Dispersions of PS were prepared by hydrating a known amount of dried lipid with buffer or with buffer containing an equal weight of myelin basic protein or lysozyme. The lipid-protein complexes were separated from unbound protein by centrifugation in 0–60% sucrose density gradients. In both systems only a few percent of the protein was unbound and the resultant recombinants, which gave single bands on the gradients, contained about 50% protein by weight. After removal of the sucrose by dialysis the dispersions were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide, dehydrated and embedded in epoxy resin. Thin sections cut from these blocks were incubated, after removal of osmium tetroxide, with antiserum raised in rabbits against human myelin basic protein. Excess antiserum was removed and the antigen-antibody complexes on the thin sections were labelled with 13 nm diameter colloidal gold particles stabilized with protein A. The distribution of these gold particles were examined under an electronmicroscope. Comparison of the labelling patterns for PS, PS-lysozyme and PS-basic protein demonstrated specific labelling in the last, and showed the gold partciles to be uniformly dispersed. It was concluded that in these dispersions the protein and lipid werre intimately mixed at the molecular level.