Regulation of the synthesis of nucleoside diphosphate sugars in reticulo-endothelial tissues

The kinetic and regulatory properties of enzymes involved in the biosynthesis of UDP-D-galactose, UDP-N-acetylglucosamine. GDP-alpha-D-mannose and GDP-beta-L-fucose from D-glucose 6-phosphate in various reticulo-endothelial tissues was studied. The tissues examined include bovine liver, thyroid, spleen, salivary gland, lung, intestine and mesenteric; pulmonary, portal and sub-maxillary lymphnodes. The maximum rates of specific enzymes in these pathways which were slow enough to be rate-limiting in the formation of glycoproteins in these tissues was determined. UDP-D-galactose 4-epimerase was consistently the rate-limiting reaction in the conversion of -d-glucose 6-phosphate to UDP-D-galactose in all of the tissues examined. The series of reactions leading to the formation of GDP-alpha-D-mannose and GDP-beta-L-fucose were limited by the activity of GDP-alpha-D-mannose pyrophosphorylase and GDP-alpha-D-mannose oxidoreductase, respectively. The formation of UDP-N-acetylglucosamine was limited by the rate of the amination reaction which converts -d-fructose 6-hosphate to D-glucosamine 6-phosphate in the presence of glutamine. Several of these rate-limiting enzymes were partially purified from mesenteric lymph node extracts, and their regulatory properties were examined. GDP-alpha-D-mannose was found to be a competitive inhibitor of GDP-alpha-D-mannose pyrophosphorylase. The apparent Km for GTP was 0.06 mM and the Ki for GDP-alpha-D-mannose was 0.03 mM. The concentrations of GTP and GDP-alpha-D-mannose in lymph node extracts were determined to be 0.095 and 0.012 mumol per g, respectively. UDP-N-acetylglucosamine and UDP-D-glucose inhibited D-fructose 6-phosphate amidotransferase in a manner competitive with D-fructose 6-phosphate. The Km for fructose 6-phosphate was 0.3 mM, while the Ki for UDP-D-glucose and UDP-N-acetyglucosamine were determined to be 0.4 mM and 0.045 mM, respectively. The concentrations of these metabolites in lymph node tissue were: UDP-D-glucose, 0.42; UDP-N-acetylglucosamine 0.095; and D-fructose 6-phosphate, 0.073 mumol per g wet weight of tissue. The results obtained in these studies show that specific rate-limiting enzymes in the pathways for the biosynthesis of nucleoside diphosphate sugars in reticulo-endothelial tissues may be subject to cumulative feedback inhibition by the nucleoside diphosphate sugars which are the final products of these systems and the initial precursors of the oligosaccharide units of glycoproteins in these tissues.