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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.
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The gene specifying a membrane-bound nucleoside diphosphate sugar hydrolase of Salmonella typhimurium was mapped near the metA locus by using intergeneric crosses between this bacterium and Escherichia coli.
A protein fraction [precipitate obtained between 40 and 65% (NH4)2SO4 satn.] prepared from cambial cells, differentiating xylem cells and differentiated xylem cells of pine and fir trees contained all the enzymes required for the nucleoside diphosphate sugar interconversions. By using UDP-D-[U-14C]glucose or UDP-D-[U-14C]galactose, UDP-D-[U-14C-]glucuronic acid and UDP-D-[U-14C]xylose as substrates, the activities of UDP-D-galactose 4-epimerase (DC 5.1.3.2), UDP-D-xylose 4-epimerase(EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D-glucuronate 4-epimerase (EC5.1.3.6), UDP-d-glucuronate decarboxylase (EC 4.1.1.35) were measured at different stages of cell-wall development. The specific activities and the activities per cell of these enzymes varied during differentiation of cambium to xylem according to the type polysaccharide synthesized. Variations were also found between the two species investigated. These data, compared with those obtained in out previous work on angiosperms [see the preceding paper, Dalessandro & Northcote (1977) Biochem. J. 162, 267-279], suggest that some control of polysaccharide synthesis operates at the level of the formation of the precursors of pectin and hemicellulose syntheses.
During the transition from primary wall formation to secondary thickening there is a marked shift in the synthesis of pectin, hemicellulose and cellulose. The activities of the enzymes [UDP-D-galactose 4-epimerase (EC 5.1.3.2)8 UDP-l-arabinose 4-epimerase (EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D--glucuronate decarboxylase (EC 4.1.1.35)] were measured in cambial cells, differentiating xylem cells and differentiated xylem cells isolated from sycamore and poplar trees, and phloem cells from poplar. At the final stage of the differentiation of cambium to xylem there was a decrease in activity of the enzymes directly involved in producing the soluble precursors of pectin (DUP-D-galactose 4-epimerase and UDP-L-arabinose 4-epimerase and an increase in those producing the precursors of hemicellulose (UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase). These results strongly suggest ahat the changes were correlated with the differences observed in the chemical composition of the wall during development. The changes found in the catalytic activity of the enzymes of nucleoside diphosphate sugar interconversion exert a coarse control over the synthesis of pectin and hemicelluloses. The tissues at all stages of development contained the necessary enzyme activities to produce all the precursors of pectin and hemicellulose, even at the final stage of differentiation when no pectin was formed.
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The metal ion catalysed decomposition of the nucleotide diphosphate sugars, uridine diphosphate glucose, uriding diphosphate galactose, uridine diphosphate N-acetylglucosamine, guanosine diphosphate mannose, and guanosine diphosphate fucose (UDPGlc, UDPGal, UDPGlc-NAc, GDPMan, and GDPFuc, respectively), has been studies as a function of pH. UDPDlc and UDPGal decompose readily to the a,2-cycle phosphate derivative of the sugar and uridine 5'-phosphoric acid (UMP) in the presence of Mn2+. Under all conditions tested, UDPGal decomposes two to three times more rapidly than does UDPGlc. GDPFuc is slowly degraded to free fucose under similar conditions; the other nucleotide diphosphate sugars are stable. The rate of reaction increases with increasing hydroxide ion concentration from pH 6.5 to 7.9 and with metal ion concentration from 10 to 200 mm. Several metal ions are effective catalysts; at pH 7.5 WITH 20 mM UDPGal and 20 mM metal ion, the following apparent first-order rate constants (min-1 x 10(4)) were obtained: Eu3+ 700; Mn2+, 70; Co2+ 27; Zn2+, 22; Ca2+, 3.0; Cu2+, 2.4; and Mg2+, 0. It appears that Mn2+ concentrations that have been used in studies with nucleotide diphosphate sugars at neutral pH can catalyze significant decomposition leading to erroneous interpretation of kinetic and incorporation experiments.
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The synthesis of alpha-D-glucopyranosyl-, alpha-D-galactopyranosyl-, and alpha-D-mannopyranosylphosphonate is described. Condensation of tris(trimethylsilyl) phosphite with 2,3,4,6-tetrakis-O-(phenylmethyl)-1-O-acetyl-alpha-D-glucopyranose generated 2,3,4,6-tetrakis-O-(phenylmethyl)-alpha-D-glucopyranosylphosphonic acetic anhydride (13). The benzyl blocking groups were removed by catalytic hydrogenation, and the anhydride bond was cleaved by alkaline hydrolysis to obtain alpha-D-glucopyranosylphosphonate (15). alpha-D-Galactopyranosylphosphonate (17) and alpha-D-mannopyranosylphosphonate (19) were also similarly synthesized. The anomeric configuration of 15 was assigned by single-crystal X-ray analysis, and the structural assignments of 17 and 19 were made on the basis of comparative NMR spectral studies. Compound 15 was then coupled with adenosine 5'-phosphoric di-n-butylphosphinothioic anhydride in dry pyridine to give adenosine 5'-phosphoric alpha-D-glucopyranosylphosphonic anhydride (23). Similarly, uridine 5'-phosphoric alpha-D-galactopyranosylphosphonic anhydride (24) and guanosine 5'-phosphoric alpha-D-mannopyranosylphosphonic anhydride (25) were synthesized from 17 and 19, respectively. With ovalbumin as an acceptor for [3H]galactose, provided by UDP-[3H]galactose, only uridine 5'-phosphoric alpha-D-galactopyranosylphosphonic anhydride (24) was shown to inhibit glycoprotein beta-D-galactosyltransferase (EC 2.4.1.38), with an apparent Ki equal to 165 microM. Even though these ionic compounds hardly penetrate the cell membrane, preliminary in vitro antitumor screening shows that compounds 23 and 25 are slightly active against human B-lymphoblastic leukemia and human T-lymphoblastic leukemia. None of these compounds show any antiviral activity.