Identification of two uridine binding domain peptides of the UDP-glucose-binding site of rabbit muscle glycogenin

Glycogenin, the autoglucosyltransferase that initiates the de novo biosynth esis of glycogen, photoaffinity labeled with [beta(32)P]5-azido-UDP-glucose . The photoinsertion of the azidouridine derivative showed activating ultra violet light dependency, saturation effects, and inhibition by UDP-glucose, thus demonstrating the specificity of the interaction, In the absence of M n2+, the requirement for the catalytic activity of glycogenin, the photolab eling decreased by 70%. Competitive binding experiments indicated that the pyrophosphate or a phosphate was the moiety of UDP-glucose implicated in th e strongest interaction at the binding site. Proteolytic digestion of photo labeled glycogenin resulted in the identification of two labeled fragments, 89-143 and 168-233, that carried the uridine binding sites, This is the fi rst report of the region of glycogenin that harbors the UDP-glucose-binding domain. (C) 1998 Academic Press.