Biochemical and ultrastructural characterization of the molecular topography of the rat intestinal microvillous membrane. Asymmetric distribution of hydrophilic groups and anionic binding sites.

The topography of the intestinal microvillous membrane and its surface components was examined using biochemical and ultrastructural techniques. Microvillous membrane surface glycoproteins were labeled using everted intact intestinal sacs, prepared from rat proximal small intestine. Galactosyl residues were identified by labeling with galactose oxidase/sodium boro[3H]hydride and free amino groups with pyridoxal phosphate/sodium boro[3H]hydride. Membranes were purified, solubilized in sodium dodecyl sulfate, and protein labeling analyzed by acrylamide electrophoresis. Using the intact intestine, only microvillous membrane surface amino groups were labeled. However, when microvillous membrane vesicles were purified first and then labeled, radioligand binding to galactoproteins and free amino groups substantially increased. Ultrastructural and cytochemical studies with ruthenium red revealed the intact intestinal surface to have a strong electronegative charge due to the presence of anionic sites in both the thick mucopolysaccharide surface coat and its underlying glycocalyx. During purification of microvillous membrane, the mucous coat was detached from the membrane surface, leaving the glycocalyx directly exposed to the external environment. Enzymatic probing of microvillous membrane vesicles with papain left the vesicles intact and revealed the membrane to be asymmetric with the majority of its integral proteins located at the outer membrane surface. This orientation of galactosyl and amino groups towards the intestinal lumen plus the overlying thick mucopolysaccharide coat should theoretically afford a greater degree of protection against destruction by luminal proteases and bile acids. Moreover, the shedding of membrane-bound hydrolases into the mucous layer may allow the "membrane surface" phase of digestion to commence before nutrients have diffused completely through the surface coat to reach the enterocyte surface.