An improved procedure for the assay of glycogen synthase and phosphorylase in rat liver homogenates

Abstract

An improved rapid procedure for the separation of labeled glycogen formed in the assay of glycogen synthase (EC 2.4.1.11) in liver homogenates is described. The glycogen formed from UDPG-glucose is separated from phosphate compounds and other electrolytes by passage through a small column of an anion-exchange resin in the acetate or chloride form. The effluent contains glycogen and other polysaccharides and sugars. When the synthase activity is low about half of the labeled glycogen formed in the assay is broken down by amylases or glucosidases, causing an underestimation of the activity by conventional methods of glycogen precipitation. The use of anion-exchange resin avoids this error. From 30 to 70% of the a form of glycogen synthase in rat liver homogenates is in a particulate form found in the pellet centrifuged at 10,000 to 20,000 g. the apparent K m ̂ for UDPG, for both the a and b forms in rat liver homogenates is 0.9 m m. A rapid procedure to assay the two forms of glycogen phosphorylase by separating radioactive glycogen from glucose 1-phosphate with ion-exchange resins is described.