PURIFICATION OF INTESTINAL BRUSH-BORDER MEMBRANE-VESICLES BY THE USE OF CONTROLLED-PORE GLASS-BEADS COLUMN

A simple rapid method for obtaining highly purified and efficiently transporting intestinal brush border membrane vesicles was developed. The new method consists of two major steps: Ca-treatment of the homogenate of the scraped epithelium (rabbit ileum) and the subsequent chromatography of the supernatant of the Ca-treated homogenate through a controlled-pore glass beads column. The fraction showing the peak value of the optical density at 280 nm and two subsequent fractions were identified as those containing purified brush border membrane vesicles as judged from the activities of the marker enzymes (sucrase and alkaline phosphatase) and the rate of D-glucose uptake. Whole procedures could be completed in about 90 min. Specific activities of sucrase and alkaline phosphatase increased to 35.5 and 34 times, respectively, while Na, K-ATPase activity decreased to one tenth as compared with the initial homogenate. Overshoot uptake of D-glucose in the presence of a NaSCN gradient showed peak at 1-1.5 min after the start of incubation, when it reached 10-40 nmoles/mg protein. Electron microscopic examination revealed that the highly purified vesicles had fairly homogeneous size, the average diameter being about 80 nm. © 1979.