Calsequestrin, Myosin, and the Components of the Protein‐Glycogen Complex in Rabbit Skeletal Muscle

The protein‐glycogen particle preparation isolated from rabbit skeletal muscle extracts by mild acidification and differential centrifugation shows eight major protein‐staining bands when examined by polyacrylamide gel electrophoresis, five of which have previously been identified as the glycogen debranching enzyme, the α and β subunits of phosphorylase kinase, glycogen phosphorylase and glycogen synthase [Taylor, C., Cox, A. J., Kernohan, J. C. & Cohen, P. (1975) Eur. J. Biochem. 51, 105–115]. The other three major proteins in this preparation have now been identified. Two of the proteins were identified as the heavy chain of myosin and actin respectively, based on their electrophoretic migration, their insolubility in buffers of low ionic strength, and their solubility in buffers containing 0.6 M KCl. The third protein has been found to be calsequestrin, a major calcium‐binding protein of the sarcoplasmic reticulum. This conclusion was based on the unusual chromatographic behaviour of the protein on DEAE‐cellulose at pH 7.5, its electrophoretic mobility, amino acid composition, and immunological cross reactivity. A simple method for the isolation of calsequestrin as a by‐product of the purification of glycogen synthase has been developed, by which 70–80 mg of the protein were routinely isolated from 5000 g of muscle (6 rabbits) within five days. The protein was homogeneous by the criterion of polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. The molecular weight determined by the latter technique was 41000 and the absorbance index A_{280 nm}^1%, was 11.7. The protein had a very low sedimentation coefficient of 1.97 S indicating that it is either very asymmetric or a largely unfolded protein, in the buffers used in these experiments, a conclusion supported by its abnormal gel filtration behaviour. Since the three hitherto unidentified components of the protein‐glycogen particle preparation are not enzymes of glycogen metabolism and merely constituents of particulate material (actomyosin and fragments of the sarcoplasmic reticulum) which have coprecipitated with the protein‐glycogen complex, over 95% of the protein attached to glycogen is accounted for by just four enzymes.