Earlier gel
LY2228820 studies had suggested that UGPPase behaved as a dimer in solution [4]. We sought to confirm this observation by cross-linking the subunits using glutaraldehyde (Fig. 3A). As analyzed by SDS–PAGE, a species of apparent Mr ～ 54,000 was generated in the presence of glutaraldehyde in a concentration dependent manner, consistent with the presence of dimers. At higher glutaraldehyde concentrations, both the monomer and dimer tended to run as less discrete bands, possibly due to the occurrence of intra-subunit cross-linking producing more compact molecules even in the presence of SDS. Interestingly,
desmosome phenomenon was accentuated in the presence of Mg2+ ions (not shown), suggesting that Mg2+ binding may affect the protein structure to enhance intra-subunit cross-linking. Because of the observed lag in the UDP-glucose hydrolysis reaction at low substrate concentrations, we wondered whether the dimerization might be affected by the presence of reactants. Cross-linking was therefore performed in the presence of UDP-glucose and/or glucose-1-P together with Mg2+. Cross-linking was totally unaffected by the presence of these compounds (not shown).
