Radiochemical Assessment of Glycogen Synthase Enzyme Activity in Animal Tissue

Our research investigates glycogen metabolism, particularly glycogen synthase in health and disease, using genetically-engineered mouse models. Research on glycogen storage diseases primarily centers on developing treatments that minimize buildup of toxic glycogen. Begin by taking the prepared murine skeletal muscle homogenate and the 14-carbon UDP-glucose reaction mix plate.

Using a multi-channel pipette, add 25 microliters of the sample supernatant to the designated reaction wells. Add 25 microliters of homogenization buffer to the blank and total wells. Record the exact time at which the reaction is initiated for each row.

Ensure that equal volumes are added across all wells. Afterward, mix the reaction in each well by pipetting up and down several times. Using a multi-channel pipette, aspirate 55 microliters from each well and dispense the liquid onto the corresponding chromatography papers prepared earlier.

Spot the liquid carefully at the center of each paper. Using forceps, remove the chromatography papers labeled as totals from the clamp after spotting the liquid. Place these total papers on aluminum foil to air-dry safely.

Drop all other chromatography papers into a stirring 66%ethanol bath. Quadriceps glycogen synthase activity in murine skeletal muscle was lower under basal conditions without glucose 6-phosphate and significantly higher under maximal conditions with glucose 6-phosphate, consistent with partial phosphorylation of glycogen synthase. This protocol describing high throughput assessment of tissue glycogen synthase enzymatic activity using radioisotopes currently has no published protocol.

Protocol advantages are a direct measurement of enzyme activity and high sensitivity, integral for use on samples with low enzyme activity.