Rabbit skeletal muscle glycogen. A morphological and biochemical study of glycogen beta-particles isolated by the precipitation-centrifugation method

Abstract

Glycogen in its particulate beta-form is localized in the sarcoplasm close to the sarcoplasmic reticulum. Some particles are in close contact with the membranes, on the outer side of the vesicles. The mild technique of differential precipitation-centrifugation has been adapted to the preparation of glycogen from adult skeletal muscle. A preliminary low-speed centrifugation which eliminates the contractile protein structures and the cell debris is followed by a high-speed centrifugation which produces pellets containing glycogen mixed with smooth-walled vesicles, the glycogen-sarcovesicular fraction. The glycogen obtained after treatment of this fraction with deoxycholate and two washings contains 3% protein. A similar protein content contaminates glycogen banded in a linear sucrose gradient. The glycogen-sarcovesicular fraction and the purified glycogen have been examined, under the electron microscope, in sections of fixed and embedded material or with the negative staining technique. The glycogen beta-particles in negatively stained preparations have an average diameter of 39.4 mmicro. The largest particles present irregular outlines, suggesting the presence of conglomerated subunits, about 20 mmicro in diameter. These subunits seem to fall apart under the influence of concentrated potassium hydroxide. The mean sedimentation coefficients calculated for infinite dilution vary from 115 to 135S. The spectrophotometric analysis of the glycogen-iodine complex indicates the presence of long end-chains in the molecule.