UDP-sugars, activated forms of monosaccharides, are synthesized through de novo and salvage pathways and serve as substrates for the synthesis of polysaccharides, glycolipids, and glycoproteins in higher plants. A UDPsugar pyrophosphorylase, designated PsUSP, was purified about 1,200-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The apparent molecular mass of the purified PsUSP was 67,000 Da. The enzyme catalyzed the formation of UDP-Glc, UDPGal, UDP-glucuronic acid, UDP-L-arabinose, and UDPxylose from respective monosaccharide 1-phosphates in the presence of UTP as a co-substrate, indicating that the enzyme has broad substrate specificity toward monosaccharide 1-phosphates. Maximum activity of the enzyme occurred at pH 6.5-7.5, and at 45°C in the presence of 2 mM Mg 2؉ . The apparent K m values for Glc 1-phosphate and L-arabinose 1-phosphate were 0.34 and 0.96 mM, respectively. PsUSP cDNA was cloned by reverse transcriptase-PCR. PsUSP appears to encode a protein with a molecular mass of 66,040 Da (600 amino acids) and possesses a uridine-binding site, which has also been found in a human UDP-N-acetylhexosamine pyrophosphorylase. Phylogenetic analysis revealed that PsUSP can be categorized in a group together with homologues from Arabidopsis and rice, which is distinct from the UDP-Glc and UDP-N-acetylhexosamine pyrophosphorylase groups. Recombinant PsUSP expressed in Escherichia coli catalyzed the formation of UDP-sugars from monosaccharide 1-phosphates and UTP with efficiency similar to that of the native enzyme. These results indicate that the enzyme is a novel type of UDPsugar pyrophosphorylase, which catalyzes the formation of various UDP-sugars at the end of salvage pathways in higher plants.It is highly probable that the relative amounts and the architecture of cell wall polysaccharides and of the sugar moieties of glycolipids and glycoproteins in higher plants are regulated by the level of nucleotide sugars available, as well as by the levels of glycosyltransferases that incorporate monosaccharide units from respective nucleotide sugars into the polymers. Activated nucleotide sugars that serve as glycosyl donors for these polymers in higher plants are generated through de novo pathways, in which various UDP-and GDP-sugars are produced through sequential interconversions from UDP-Glc and GDP-Man as the starting substrates (1). In the salvage pathways, alternative routes to synthesize nucleotide sugars, free monosaccharides released during the degradation of polysaccharides and glycoconjugates are first phosphorylated by the action of monosaccharide kinases, then converted into nucleotide sugars by the action of pyrophosphorylases in the presence of the respective nucleotide triphosphates as co-substrates. Pyrophosphorylases principally catalyze both the following forward (synthesis of NDP-sugars) and reverse (pyrophospholysis) reactions (1): monosaccharides 1-phosphates ϩ NTP % NDP-sugars ϩ PP i , where NTP and NDP are nucleoside triphosphates and diphosphates, respectively. Var...
Organic acids in caries lesions play important roles in initiation and progress of dental caries. We investigated relationships between clinical types of dentin caries and acid profile or pH in the lesions. Caries lesions in dentin from 76 permanent teeth were classified into active, arrested, situated beneath a restoration, and unclassified types. The pH of carious dentin was distinctly lower than that of sound dentin (p < 0.001). Carious dentin with a high percentage of lactate had a lower pH than that with a high percentage of acetate and propionate (p < 0.001). Dentin from active lesions showed a mean pH of 4.9, and the dominant acid was lactate (mean percentage, 88.2). In contrast, carious dentin from arrested lesions showed a higher pH, 5.7, with acetate and propionate as the dominant acids (mean percentages of acetate and propionate, 64.0 and 18.2, respectively). The acid profile (mean percentages of acetate and propionate, 54.0 and 27.7, respectively) and pH (mean 5.8) of carious dentin sampled from lesions beneath a restoration were similar to those of dentin from arrested lesions. This study showed a clear relationship between clinical classification of dentin caries and acid profile and pH, suggesting that both factors are important in dentin caries etiology.
Monomeric sugars generated during the metabolism of polysaccharides, glycoproteins, and glycolipids are imported to the cytoplasm and converted to respective nucleotide sugars via monosaccharide 1-phosphates, to be reutilized as activated sugars. Because L-fucose (L-Fuc) is activated mainly in the form of GDP derivatives in seed plants, the salvage reactions for L-Fuc are expected to be independent from those for Glc, Gal, L-arabinose, and glucuronic acid, which are activated as UDP-sugars. For this study we have identified, in the genomic data base of Arabidopsis, the gene ( The monosaccharide L-fucose (L-Fuc) is found as a constituent of cell wall polysaccharides and sugar moieties of glycoproteins and has important physiological functions in seed plants.L-Fucosyl residues, for example, occur as nonreducing terminal residues attached through ␣-(132)-linkages to penultimate sugar residues in xyloglucan and arabinogalactan protein (1, 2). The L-fucosylated trisaccharide side chains of xyloglucan modulate the interaction of xyloglucan with cellulose microfibrils, thereby affecting the mechanical properties of plant cell walls (3, 4), whereas some N-glycans of plant glycoproteins have L-fucosyl residues attached through ␣-(133)-linkages to the proximal GlcNAc residues (sugars mentioned in this paper belong to the D-series unless otherwise noted.), which are characteristic of plants but are not found in mammals and are responsible for the immunogenicity of plant glycoproteins in mammals (5). L-Fucosyl residues are also attached through ␣-(134)-linkages to L-rhamnosyl residues in pectic rhamnogalacturonan II (RG-II) 2 in seed plants (6). These L-fucosyl residues are transferred onto the glycoconjugates by actions of respective L-fucosyltransferases, which use GDP-L-Fuc as the L-fucosyl donor. Recent studies have identified a xyloglucan-specific L-fucosyltransferase, AtFUT1 (7), and the related L-fucosyltransferase genes (AtFUT2-10) have also been found in the genome of Arabidopsis (8).GDP-L-Fuc, the activated form of L-Fuc, is generated through both de novo and salvage pathways (9, 10). Recently, the importance of levels of GDP-L-Fuc for the architecture of L-Fuc-containing cell wall polysaccharides was demonstrated through the study of the L-Fuc-deficient mutant of Arabidopsis, mur1. The mur1 mutant has reduced L-Fuc content in RG-II because of a defect in GDP-Man 4,6-dehydratase (EC 4.2.1.47) that catalyzes the first step of conversion of GDP-Man to GDP-L-Fuc in the de novo pathway (11,12). The loss of L-fucosyl residues indispensable for the boron-mediated dimer formation of pectic RG-II reduces the growth of rosette leaves in the mutant. The dwarf phenotype of mur1, however, can be rescued by exogenously applied monomeric L-Fuc, possibly because a compensating supply of GDP-L-Fuc is generated from L-Fuc via L-Fuc 1-phosphate (L-Fuc-1-P) in the salvage pathway (11). It is
Organic acids in carious dentin from 69 permanent teeth were analyzed by gas chromatography. Lactate, acetate, propionate, and butyrate were detected in most samples, and limited amounts of isobutyrate, valerate, isovalerate, caproate, and isocaproate were occasionally detected. Lactate, acetate, and propionate were major acids and altogether accounted for about 90% of total acid in most samples of carious dentin. However, the proportion of these three acids varied among the samples. Some samples contained over 85% lactate, while others contained mainly acetate and propionate. A high percentage of acetate was usually accompanied by an appreciable amount of propionate. All seven samples in carious dentin under fillings or restorations had little lactate, but a high percentage of acetate plus propionate. The differences in acid profiles of carious dentin may reflect differences in the microbial ecology of carious dentin, and a stage of progress of dentin caries or a type of dentin caries.
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