Simultaneous preparation of basolateral and brush-border membrane vesicles from sea bass intestinal epithelium

A method is described for simultaneous preparation of brush-border and basolateral sea bass enterocyte membranes using simple differential centrifugation and discontinuous sucrose gradient density centrifugation techniques. Basolateral membranes were purified with a Na+/K(+)-ATPase yield of about 11% of the original activity, with an enrichment factor of 12. The yield of maltase-glucoamylase, a specific marker of brush-border membranes, was also about 11% of the original activity, with 15-fold enrichment. The characteristics of these membrane preparations were determined. Electron microscopy analysis showed that these two membrane preparations were uniform in size and vesicular in nature. Orientation studies revealed that the luminal membrane vesicles were right-side out and 43% of the antiluminal membrane vesicles were sealed inside out. Investigation of D-glucose and L-leucine uptake showed that these two plasma membrane preparations retained their transport properties.