The binding of glycogen and phosphorylase.

It has been calculated from kinetic measurements that the rate of the phosphorylase reaction is half-maximal when the glycogen concentration is 20 mg./lOO ml. (I). This K, (Micharlis-Menten constant) value can be expressed as a molar concentration, if the percentage of terminal glucose units (with which phosphorylase reacts) is known. For a glycogen with 9 per cent end groups, the K, would be 1 x 1O-4 M, a concentration much higher than the enzyme concentration (2 x lo-* tir) used in such kinetic measurements. It seemed of interest to study the combination of glycogen and phosphorylase by the ultracentrifugal separation method in order to determine how the binding constant obtained by this method would compare with the K, obtained by the kinetic method. For this purpose a glycogen of much larger molecular weight than,that of the enzyme was needed. A phytoglycogen prepared from mature sweet corn seemed suitable; it had a mean sedimentation coefficient (.s*~, W) of 200 S and contained only 1.2 per cent of material of an sZO, W of less than 100.’ Phosphorylase a from rabbit muscle has an szo, U of 13. The principle of the method was to centrifuge most of this glycogen from the upper layer in the centrifuge tube, leaving most of the phosphorylase in the upper layer. The extra amount of protein sedimented in tubes which contained glycogen (compared with tubes without glycogen) would be considered to be bound to the glycogen.