Large scale fractionation of calf brain by double run zonal centrifugation

A previously described method for the subfractionation of subcellular particles by zonal centrifugation (Spanner, 1972), gives an unsatisfactory resolution when applied to calf brain, particularly when large amounts of subcellular particles are required. We succeeded in subfractionating 30-45 g of gray matter by modifying the procedure for preparing the P2 fraction (crude mitochondrial fraction) and introducing a new stepwise sucrose gradient on double run zonal centrifugation. Satisfactory resolution can also be achieved by single run zonal centrifugation when the starting material does not exceed 15 g of gray matter. The following fractions were separated as characterized chemically morphologically and by their enzyme markers: a) light myelin (0.4-0.6 M sucrose); b) heavy myelin (0.7 M); c) plasmamembranes (0.9-1.0 M); d) nerve endings (1.0-1.2 M); e) mitochondria (1.3 M); f) lysosomes (1.4 M).