SIMULTANEOUS PREPARATION OF RABBIT INTESTINAL BRUSH-BORDER AND BASOLATERAL MEMBRANE-VESICLES

A highly reproducible method is described for simultaneous preparation of the rabbit intestinal BBMV and BLMV. When BLMV were purified on co ntinuous sorbitol gradient the final enrichment was over ten-fold for both Na+, K+-ATPase and K+-activated phosphatase. BBMV were separated using repeated precipitation with magnesium and EGTA. Marker enzymes o f the brush border membrane also showed over ten-fold enrichment. Ther e was no contamination with other cellular organelles. The characteris tics of the preparations were checked by SDS gel electrophoresis, whic h showed distinct protein patterns for both membranes; monoclonal anti bodies for villin reacted with the BBMV but not with BLMV, showing the absence of contamination of the BLMV fraction. The quality of the mem branes, assessed by D-glucose transport, showed good functional proper ties for both BBMV and BLMV. Orientation of the vesicles was checked b y permeabilizing with SDS buffered with BSA and showed that BLMV are o riented inside out and BBMV right side out. The model simulates normal transepithelial intestinal transport at the level of membrane vesicle s and could be applied in transport studies in physiology and pharmaco logy.