Studies on isolated cell components. VIII. High resolution gradient differential centrifugation.

THE purpose of the studies described in this paper has been to lay the groundwork for the development of a technique for analytical medium-speed centrifugation in which quantitative separation of nuclei, whole cells, mitochondria, mirrosomes, soluble proteins, and lipid inclusions may be achieved in one or t\\-o steps. Since it is not possible, at present, to make quantitative measurements of all particulate fractions during centrifugation in a manner analogous to that used in the ultracentrifuge, it is necessary to rely on methods \\-hich allow fractions to be recovered, free of cross-contamination, for analysis at the end of the centrifugation. Fortunately, the procedures which are necessary to achieve convection-free sedimentation in suspensions of cell components are readily adapted to do this. The method to be described has been developed over a period of several years and is based in part on previous work done on starch gradients (N. G. Anderson, 1949, unpublished) and on techniques described by Behrens [l] and by Brakke [3, 41. It appears not to have been generally realized that the separation of tissue brei constituents in a centrifugal field cannot be predicted from a simple uncritical application of Stokes’ law. If one assumes, for example, equivalent radii of 4 k for nuclei and 1 p for mitochondria, their sedimentation velocities should cliffer by a factor of approximately lG, depending on the densities of the particles. Actually, no such clear-cut difference in sedimentation rate is observed, and a few nuclei are often found still in suspension after many of the mitochondria have sedimented. Jn methods wherein a brei layer is used over a denser clear layer of solution such as the one described by Hogeboom et crl. [8], one observes mitochondrial contamination of sedimented nuclei even when many nuclei are still to be found in the upper layer. This and other anomalous effects may be sho\vn to be caused by several artefacts which are common to nearly all isolation procedures.