A rapid method for the preparation of microvilli from rabbit kidney

Abstract

A simple method for the isolation of microvilli from kidney brush border is described. The method depends on the preferential aggregation of other subcellular structures by bivalent metal ions. MgCl₂ is added to a homogenate of cortical tissue prepared from frozen rabbit kidneys. Aggregated material is removed by a low-speed centrifugation and the supernatant centrifuged at 15000g to yield a pellet enriched in microvilli. This is resuspended and given a second treatment with Mg²⁺. The purified preparation is obtained after four short differential centrifugations. The six brush-border enzymes that were monitored were enriched 11–17-fold compared with the original homogenate and were obtained in about 10% yield. Marker enzymes for other subcellular components showed the preparation to be essentially free of mitochondria and to be less contaminated with endoplasmic reticulum and baso-lateral plasma membranes than are conventional brush-border preparations. The main contamination was of lysosomal origin, about half of which was attributable to adsorbed acid hydrolases rather than to intact lysosomes. The aggregated components in the low-speed pellet bound less Mg²⁺than did the microvillus fraction. A possible mechanism for the role of Mg²⁺is discussed.