Modified assays to detect activation of glycogen synthase following exercise

Abstract

Glycogen levels and glycogen synthase activity were measured in red vastus lateralis muscle of male Sprague-Dawley rats killed at rest, immediately after swimming to exhaustion, or 4 h postexhaustion. Glycogen levels were very low immediately after the exercise but returned to preexercise levels after 4 h of recovery. Activation of glycogen synthase by glucose 6-phosphate (G6P) was determined in the standard assay of Thomas et al. (Anal. Biochem. 25: 486-499, 1968) using 4.4 mM uridine diphosphate glucose (UDPG) (pH 7.8) or in a modified assay using 0.03 mM UDPG (pH 6.9) in the absence or presence of inhibitor (inorganic phosphate or uridine 5'-diphosphate). Activation of glycogen synthase was determined by measuring activity ratio (activity in the absence of G6P divided by activity in the presence of G6P), A0.5 for G6P (concentration of G6P producing half-maximal activation), and fractional velocity (activity with low G6P divided by activity with high G6P). All three measurements indicated glycogen synthase was significantly activated immediately after exhaustion. Activation after 4 h of recovery was not detected using activity ratio but was readily apparent when fractional velocity of A0.5 for G6P were measured.