The isolation of a brush border membrane fraction from rat kidney.

A method for isolating brush borders from rat kidney homogenates has been developed. Minced tissue, 27 g, is gently homogenized in 20 mu sodium or potassium bicarbonate, pH 8.1. The diluted homogenate, containing large fragments of brush borders, is filtered and then fractionated with the use of combined differential, isopycnic, and ratezonal centrifugations. The purity of the final brush border fraction is documented with both morphological and enzymatic studies. Electron micrographs of the final brush border fraction show microvilli profiles with few cytoplasmic contaminants. Alkaline phosphatase assays show a 15-fold increase in specific activity of the brush border fraction relative to the starting homogenate. Acid phosphatase, glucose 6-phosphatase, and succinate cytochrome c reductase studies are consistent with contamination rates of 0.04% for lysosomes, 0.2 % for microsomes, and 7% for mitochondria. Calculations based on recovery of total alkaline phosphatase activity indicate that the procedure recovers 21% of the alkaline phosphatase present in the starting homogenate. The 6na.l brush border pellet obtained from 27 g of whole kidney has a wet weight of 0.27 g and a protein content of 20 mg.