Preparation of a Plasma Membrane Fraction from Rat Liver by Zonal Centrifugation

Abstract

The behaviour of heavy subcellular particles from rat liver during centrifugation in an A-XII zonal rotor has been studied with the help of enzymic markers and light and electron microscopy. A simple and reproducible method, not entailing hypotonic conditions, was developed for the isolation of plasma membrane fragments from liver homogenates, after study of factors affecting the yield and purity of such fragments. The livers were perfused with warm 0.25 M sucrose–5 mM NaHCO3, pH 7.5. Homogenisation and resuspension of the nuclear fraction were carried out in the same medium. Zonal centrifugation gave a band of plasma membrane fragments which showed a 14-fold increase in 5′-nucleotidase specific activity over the homogenate. This purification was increased to 30-fold by vigorous rehomogenisation in sucrose of density 1.19 followed by flotation of the plasma membrane fragments. These were now almost free of succinate-dehydrogenase activity although still possessing some glucose-6-phosphatase and acid ß-glycerophosphatase activity.