Studies of native glycogen isolated from synchronized Tetrahymena pyriformis (HSM)

Abstract

Native glycogen was isolated from Tetrahymena pyriformis (HSM) by isopycnic centrifugation in cesium chloride density gradients. A density of 1.62 to 1.65 was isopycnic for glycogen. Most of the banded glycogen existed as 35 to 40 mmicro particles which had a sedimentation coefficient of 214. These particles were composed of aggregates of 2 to 3 mmicro spherical particles. Extraction of glycogen with hot alkali reduced the sedimentation coefficient of native glycogen from 214 to 64.7 and the particle diameter from approximately 40 to 20 mmicro and smaller. Cell division was synchronized by a repetitive 12-hour temperature cycle, and glycogen was measured at several times during the cell cycle. The temperature cycle consisted of 9.5 hours at 12 degrees C and 2.5 hours at 27 degrees C. Approximately 90 per cent of the cells divided during the last 1.5 hours of the warm period. The carbohydrate/protein ratio of cells at the end of the cold period was 0.27 and was reduced slightly during the warm period. Glucose was incorporated into glycogen during both periods, although the rate of incorporation was greater during the warm period. No preferential incorporation on the basis of particle size was noted. Incorporation was measured in both native glycogen and KOH-extracted glycogen. Tetrahymena glycogen is compared with rat liver glycogen previously isolated by similar procedures, and the significance of using combined rate-zonal and isopycnic centrifugation for isolating native glycogen is discussed.