A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose

A modified method for enzymic synthesis of UDP-14C-glucose in high yield is described. Labeled UDPglucose is isolated by high-voltage paper electrophoresis after an alkaline phosphatase treatment of the reaction products. The synthesis gives high yields and is reproducible, and the isolation of radioactive UDPglucose from the electrophoresis is quantitative. A new assay for UDPglucose: glycogen α-4-glucosyltransferase (EC 2.4.1.11) is also described. Enzyme reaction mixtures are spotted directly on filter paper squares and washed with 66% ( v v ) ethanol, and 14C-glycogen is counted in a liquid scintillation spectrometer. The method is accurate for all transferase preparations tested if corrections are applied for sample quenching. Assay time is greatly reduced and large numbers of assays can be processed with very little increase in assay time. The assay is also readily applicable to the determination of transferase-I kinase activity. It was also found that stopping the transferase-catalyzed reaction by precipitation of trichloroacetic acid insoluble protein resulted in a loss of radioactive glycogen. When crude preparations of transferase were assayed, up to 20% of the radioactivity bound to glycogen was precipitated by 6% trichloroacetic acid.