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...
Homer family proteins are encoded by three genes, homer1, 2 and 3. Most of these proteins are expressed constitutively in nervous systems and accumulated in postsynaptic regions. However, the functional significance of these proteins, especially the significance of the distinction among the proteins encoded by homer1, 2 and 3, is still obscure. In the present study, we isolated a cDNA clone encoding a novel protein by two-hybrid system screening using the C-terminal half of Homer2b as the bait. This protein, termed 2B28, has 297 amino acid residues and contains three major domains: a UBA domain, a coiled-coil region, and a UBX domain. When expressed in HEK293T cells, 2B28 showed colocalization with uniquitin and enhanced the expression levels of IkappaB or Homer1a proteins, which are known to be degraded by proteasomes, indicating that 2B28 is involved in ubiquitin-proteasome functions. 2B28 specifically interacted and colocalized with Homer2 proteins, but not with Homer1 proteins. So far, we have identified no counterpart of 2B28 for Homer1 experimentally or in the protein databases. These results suggest that the specific interaction of 2B28 with Homer2 may play a role in regulation of protein degradation by ubiquitin-proteasome systems and that this function may be specific to Homer2 proteins among Homer family proteins.
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