While investigating glycogen synthetase (UDPglucose: glycogen a-4glycosyl-transferase, EC 2.4.1.11) activity in tissue homogenates we remarked a consistent disparity between UDP formation from UDPG and the incorporation of ["Cl glucose from UDP-[I%] glucose into glycogen. In an effort to distinguish between synthetase activity and the possible cleavage of UDPglucose to UDP and glucose, exogenous glycogen primer was omitted from the incubation medium. Surprisingly, full activity was found without added primer in the absence of glucose-6-P, and more than half the maximal activity was found without added glycogen in the presence of glucose-6-P. Furthermore, there was no evidence of glucose accumulation paralleling the UDP formation.
The following report encompasses three areas of investigation:
(1) the relationship between UDP formation and glycogen synthesis in tissue homogenates, (2) the requirement for glycogen primer of glycogen synthetase, and (3) the development of a direct, one-step assay for glycogen synthetase.
METHODS UDP Formation Compared with ['"C] Glucose Incorporation and Glycogen Primer Studies
For crude tissue preparations, male Osborne-Mendel weanling rats were quick-frozen in liquid nitrogen, the tissues dissected and weighed at -2O", and the samples stored at -50" until use. Homogenates were made at 0" in appropriate volumes at 50 mM Tris-WC1 buffer, pH 7.5, containing 5 mM EDTA, 25 mM KF, and 5 mM dithiothreitol.
In the two-step assay, aliquots of tissue homogenates were added to 10 528