STUDIES ON ISOLATED CELL COMPONENTS XVII. The Distribution of Cytochrome Oxidase Activity in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugafion. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the m:.crosomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge. This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc. I N T R O D U C T I O N Centrifugal fractionation of homogenates prepared from cells and tissues is one of the most widely used tools in contemporary experimental biology. Most of these procedures are based on the differential centrifugation scheme originally proposed by Claude (9), according to which subcellular components are separated into so called soluble-phase, microsomal, mitochondrial, and nuclear fractions. These separations depend upon the simultaneous sedimentation, though at differing rates, of all species of particles present. With complex mixtures such as cellular homogenates, 317 on A uust 4, 2017 jcb.rress.org D ow nladed fom repeated centrifugations are required to obtain acceptably purified fractions (4, 12). The technique of zonal centrifugation in density gradients has greatly improved the obtainable resolution, since all the particles are layered near the top of the tube at the outset and each species of particles is allowed to move a distance from the starting level or zone, proportional to its sedimentat ion rate and density (3, 8, 11, 13, 29). This approach, utilizing swinging-bucket rotors such as the SW-39, has been hampered by the small volume of the tubes, difficulties in loading the tube with the gradient, difficulties in maintaining gradient stability during acceleration and deceleration, and difficulties in the recovery of the gradient with its separated zones of particles after centrifugation (13). These difficulties have been largely overcome by the development of the zonal ultracentrifuge (5, 6). This device is capable of swinging a 1.6 liter gradient at speeds up to 30,000 RPM with a force in excess of 51,000 g at the rotor edge, and has achieved high resolution separation of subcellular components from gram quantities of tissue during a single centrifugation