Abstract
Directions are detailed for preparing the totally glucose-6-P dependent (D) form of glycogen synthase (UDPG:glycogen α-4-glucosyltransferase, EC 2.4.1.11) from rabbit skeletal muscle from which the glycogen has been removed. The conversion is carried out in the cold (3°) for several days in order to achieve full phosphorylation. Glycogen is removed by digestion with α-amylase. The enzyme is then isolated by gel filtration over Sepharose 4-B and Sephadex G-200 columns. The molecular weight by sedimentation diffusion is determined as 250 000. The enzyme is totally dependent on glucose-6-P and on added glycogen for activity. The residual carbohydrate content is about 10 μ/mg protein. The two substrate kinetics (glycogen, UDPG) was examined. In reciprocal plots parallel lines were obtained with either substrate, indicating a ping pong mechanism of catalysis.
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