Brush borders isolated from the epithelial cells of hamster jejunum have been dissociated by treatment with 1 M Tris(hydroxymethyl)aminomethane into several subfractions which can be separated by means of centrifugation on glycerol density gradients. Investigation of the chemical specificity of disrupting agents suggests that the amino group of Tris, in its positively charged state, is involved. Five individual bands or fractions have been routinely recovered from density gradients. The distribution of alkaline phosphatase and maltase activities among these fractions has been studied and the results indicate that both enzymes are predominantly associated with one fraction which has been identified in a companion paper as being composed of the membranes of the brush border microvilli. A fibrillar material of unidentified origin has also been obtained from Tris-disrupted brush borders.

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Vibration of hamster small intestinal segments in hypotonic media containing PVP is a rapid method for obtaining quantitative yields of viable intestinal epithelial cells. This preparation of epithelial cells offers a unique system for the study of epithelial cell function in vitro. The method for cell separation combines hypoosmotic swelling of cells, which separates them at the desmosomes, with mechanical agitation which releases the cells from the lamina propria. No chemical agents known to affect cell proteins and cell surfaces are employed in this procedure. Only a short time is elapsed between in vivo and in vitro conditions, i.e., a preparation time of approximately 75 minutes. Although the technique yields a pure population of epithelial cells, the cells are of different morphologies, are removed from different areas of the crypts and villi, and therefore presumably have different functions. Examination of the intestinal tissue remaining after several vibration intervals by light and scanning electron microscopy indicates that the sequence of release of cells is removal of: (1) cells from the villus bases, (2) cells from the lower one-half to two-thirds of the villi, (3) cells from the villus tips (and some crypts), and (4) cells from the crypts. When pools of a+b cells are compared to pools of c+d cells, it is found that villus cells can be characterized by: (1) processes, such as monosaccharide absorption, associated with the brush border, and (2) synthesis of components (e.g., glycoproteins) of the brush border. Surprisingly, disaccharide hydrolytic activity is found in cells which transport monosaccharides poorly. The subpopulations of cells synthesize proteins equally.