Purification of frog muscle glycogen synthetase

Abstract

Purification of Frog Muscle Glycogen Synthetase. Rev. esp. Fisiol., 30, 159-166. 1974.

A detailed method for purification of frog muscle glycogen synthetase (GS) is presented in this paper. In this tissue GS is present apparently in a unique form whose activity is dependent on the presence of glucose-6-P (D-form).

The first step in the purification procedure consisted in the isolation of the glycogen­ bound GS by centrifugation at 25,000 X g. At this gravitacional force maximum specific activity was obtained in the sediment. The relatively low value of this figure indicated an easy sedimentability of the glycogen-enzyme complex from frog muscle.

The enzyme could not be separated from glycogen by gel filtration through Se­ pharose. Treatment of the particulate glycogen fractions with a-amylase liberated the enzyme protein. The interference caused by a-amylase on glycogen synthetase activity determination could be suppersed by increasing the concentration of glycogen in the assay mixture to 4 %. Chromatography on DEAE Sephadex of the x-amylase-treated preparations yielded a considerable purification of glycogen synthetase. The eluted enzyme was unstable at 4° C but Mg=+ stabilized the enzymatic activity. However sediments obtained by centrifugation at 100,000 Xg after addition of glycogen were stable at —20° C for months.

The specific activity of the final preparation was 4.6 /anoles of glucose incorporated per minute and mg of protein. A purification of 740 fold was achieved with a yield of 21 %.