Preparation and characterization of the inner coat material associated with fat globule membranes from bovine and human milk

Milk fat globules of many species are characterized by a dense 10–50 nm thick layer sandwiched between the milk fat globule membrane (MFGM) and the outer shell of the fat droplet. This coat material is tightly associated with the membrane and survives isolation and extensive washing of the isolated MFGM. We have prepared these MFGM-associated coat structures from bovine and human milk by removal of membrane and loosely associated material using extractions in low and high salt buffers, non-ionic detergents such as Triton X-100, and/or solutions of lithium diiodosalicylate. Residual fractions obtained after such treatments are devoid of identifiable membrane structures but are enriched in MFGM coat material which appears in the form of densely stained plaques of a finely filamentous texture. MFGM fractions are enriched in some polypeptide bands seen after electrophoresis two of which are especially prominent in both species (band 3, apparent mol. wt 155 000; band 12, apparent mol. wt 67 000). Human and bovine MFGM coat fractions and isolated bovine band 12 polypeptide material separated after dissociation in sodium dodecylsulfate (SDS) by gel filtration, chromatography on hydroxylapatite or preparative electrophoresis in SDS-polyacrylamide gels are intimately associated with small amounts of phospholipids and gangliosides of a pattern different from that of total MFGM, contain carbohydrates (relatively high contents of mannose, glucosamine, galactose, and galactosamine; low levels of fucose and sialic acids) and show similar amino acid compositions. The relationship of band 12 polypeptide to components of MFGM coat preparations from various other species and to components present in other membrane fractions has been examined by immunodiffusion techniques and immunofluorescence microscopy using rabbit, mouse and guinea pig antibodies against purified band 12 polypeptide. Evidence is presented for the occurrence of related polypeptides in MFGM coat preparations from different species. The unusual structure and resistance of the MFGM coat material, especially the occurrence of glycopeptides in association with the cytoplasmic side of a membrane structure, are discussed in relation to the stabilization of the emulsified state of milk fat and the process of milk fat globule budding as well as a general model for local differentiation of membrane character.