Isolation of native glycogen by combined rate-zonal and isopycnic centrifugation.

Rat liver glycogen was separated into fractions of uniform particle size by the rate-zonal centrifugation of homogenates through cesium chloride and sucrose density gradients. Glycogen was then isolated from other cell constituents having similar sedimentation coefficients by isopycnic-zonal centrifugation in CsCI density gradients to effect a density separation. A density of 1.62 to 1.65 was isopycnic for glycogen. A continuous spectrunl of particle sizes up to 150 to 200 m!, was observed, and sedimentation coefficients up to 10,000 were measured. Sedimentation coefficients of particles of different sizes indicated a decrease in molecular packing with increasing cluster size. Microscopically, three levels of glycogen structure were resolved, including fundamental 'Y particles 2.5 to 3.5 m!, in diameter, subclusters of {3 particles 20 to 25 m!, in diameter, and a clusters of increasing size up to maximum diameter of 200 m!'. The 20 to 25 m!, subclusters would accommodate the glucose residues required to explain the molecular weight of glycogen particles normally extracted in strong alkali. Membrane fragments were not observed microscopically and specific 260 to 280 m!, absorption was absent in isolated glycogen fractions. Zonal centrifugation combined with isopycnic banding, therefore, provides a method for isolating large amounts of nativc glycogen separated into fractions of uniform particle size with a high degree of purity. Such preparations are obviously needed for studies of enzyme binding to glycogen particles and studies of the free and fixed glycogen fractions in tissues.-Nat Cancer Inst Monogr 21: 285-302, 1966.