Problems of uridine protection

Various routes to synthesize 5'-O-dimethoxytrityl-O4-p-nitrophenylethyl- 2'-O-p-nitrophenylethylsulfonyluridine (7) as a useful intermediate in oligoribonucleotide synthesis have been investigated. The direct sulfonylation of 5'-dimethoxytrityl-O4-p-nitrophenylethyluridine (4) gave the best results despite the fact that 7 is formed in almost equal amounts with its 3'-NPES isomer (8) and the 2',3'-di-O-NPES derivative (6).     

Phosphorylation of uridine with inorganic phosphates

Phosphorylation of uridine by heating with inorganic orthophosphates can take place through reaction of nucleoside with thermally produced polyphosphate or direct reaction of nucleoside with orthophosphate or a combination of both. The relative importance of the two pathways varies with the orthophosphate and temperature.     

Inhibition of UDP-N-acetylglucosamine pyrophosphorylase by uridine

UDP-N-acetylglucosamine pyrophosphorylases (UTP: 2-acetamido-2-deoxy-alpha-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.23) from baker's yeast and Neurospora crassa IFO 6178 were inhibited by uridine which is the nucleoside moiety of UDP-GlcNAc. The inhibition was shown in both directions of pyrophosphorolysis and of synthesis of UDP-GlcNAc. Kinetic analysis revealed that uridine demonstrated a noncompetitive type of inhibition with UDP-GlcNAc and competitive inhibition with PPi. The Ki values for the baker's yeast enzyme were 1.8 mM for UDP-GlcNAc and 0.16 mM for PPi, and the values for the Neurospora enzyme were 1.1 mM for UDP-GlcNAc and 0.15 mM for PPi, respectively. Uridine did not bind irreversibly to the enzyme, as the activity was restored with dialysis. No other nucleosides caused inhibition of the enzyme activity except uridine. Some uridine derivatives, such as 5-hydroxyuridine, 5,6-dihydrouridine and pseudouridine, also inhibited the enzyme activity. But doexyuridine showed only slight inhibition, and 5'-UMP and orotidine caused no inhibition of the enzyme activity.     

Enzymatic analysis of uridine diphosphate N-acetyl-D-glucosamine

The Methanococcus maripaludis MMP0352 protein belongs to an oxidoreductase family that has been proposed to catalyze the NAD⁺-dependent oxidation of the 3′′ position of uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc), forming a 3-hexulose sugar nucleotide. The heterologously expressed MMP0352 protein was purified and shown to efficiently catalyze UDP-GlcNAc oxidation, forming one NADH equivalent. This enzyme was used to develop a fixed endpoint fluorometric method to analyze UDP-GlcNAc. The enzyme is highly specific for this acetamido sugar nucleotide, and the procedure had a detection limit of 0.2 μM UDP-GlcNAc in a 1-ml sample. Using the method of standard addition, UDP-GlcNAc concentrations were measured in deproteinized extracts of Escherichia coli, Saccharomyces cerevisiae, and HeLa carcinoma cells. Equivalent concentrations were determined by both enzymatic and chromatographic analyses, validating this method. This procedure can be adapted for the high-throughput analysis of changes in cellular UDP-GlcNAc concentrations in time series experiments or inhibitor screens.     

Studies on uridine diphosphate-galactose pyrophosphatase and uridine diphosphate-galactose: glycoprotein galactosyltransferase activities in microsomal membranes.

Rat liver microsomes showed very active uridine diphosphate-galactose pyrophosphatase activity leading to the hydrolysis of uridine diphosphate-galactose into galactose1-phosphate and finally into galactose. The activity was observed in presence of buffers with wide ranges of pH. Different concentrations of divalent cations, such as Mn²⁺, Mg²⁺, and Ca²⁺ had no significant effect on the enzyme activity. A number of nucleotides and their derivatives inhibited the pyrophosphatase activity. Of these, different concentrations of uridine monophosphate, cytidine 5′-phosphate and cytidine 5′-diphosphate have slight or no effect; cytidine 5′-triphosphate, adenosine 5′-triphosphate, guanosine 5′-triphosphate, cytidine 5′-diphosphate-glucose and guanosine 5′-diphosphate-glucose showed strong inhibitory effect whereas cytidine 5′-diphosphate-choline showed a moderate effect on the pyrophosphatase. All these nucleotides also showed variable stimulatory effects on uridine diphosphate-galactose:glycoprotein galactosyltransferase activity in the microsomes which could be partly related to their inhibitory effects on uridine diphosphate-galactose pyrophosphatase. Among them uridine monophosphate, cytidine 5′-phosphate, and cytidine 5′-diphosphate stimulated galactosyltransferase activity without showing appreciable inhibition of pyrophosphatase, cytidine 5′-diphosphate-choline, although did not inhibit pyrophosphatase as effectively as cytidine 5′-triphosphate, guanosine 5′-triphosphate, adenosine 5′-triphosphate, cytidine 5′-diphosphate-glucose, and guanosine 5′-diphosphate-glucose but stimulated galactosyltransferase activity as well as those. The fact that cytidine 5′-diphosphate-choline stimulated galactosyltransferase more effectively than cytidine 5′-phosphate, cytidine 5′-diphosphate, and cytidine 5′-triphosphate suggested an additional role of the choline moiety in the system. It has been also shown that cytidine 5′-diphosphate-choline can affect the saturation of galactosyltransferase enzyme at a much lower concentration of uridine diphosphate-galactose. Most of the pyrophosphatase and galactosyltransferase activities were solubilized by deoxycholate and the membrane pellets remaining after solubilization still retained some galactosyltransferase activity which was stimulated by cytidine 5′-diphosphate-choline. In different membrane fractions a concerted effect of both uridine diphosphate-galactose pyrophosphatase and glycoprotein:galactosyltransferase enzymes on the substrate uridine diphosphate-galactose is indicated and their eventual controlling effects on the glycopolymer synthesis in vitro or in vivo need careful evaluation.     

Homeostatic control of uridine and the role of uridine phosphorylase: a biological and clinical update

Both a 25-hydroxylation and a 1alpha-hydroxylation are necessary for the conversion of vitamin D(3) into the calcium-regulating hormone 1alpha,25-dihydroxyvitamin D(3). According to current knowledge, the hepatic mitochondrial cytochrome P450 (CYP) 27A and microsomal CYP2D25 are able to catalyze the former bioactivation step. Substantial 25-hydroxylase activity has also been demonstrated in kidney. This paper describes the molecular cloning and characterization of a microsomal vitamin D(3) 25- and 1alpha-hydroxylase in kidney. The enzyme purified from pig kidney and the recombinant enzyme expressed in COS cells catalyzed 25-hydroxylation of vitamin D(3) and 1alpha-hydroxyvitamin D(3) and, in addition, 1alpha-hydroxylation of 25-hydroxyvitamin D(3). The cDNA encodes a protein of 500 amino acids. Both the DNA sequence and the deduced peptide sequence of the renal enzyme are homologous with those of the hepatic vitamin D(3) 25-hydroxylase CYP2D25. Genomic Southern blot analysis suggested the presence of a single gene for CYP2D25 in the pig. Immunohistochemistry experiments indicated that CYP2D25 is expressed almost exclusively in the cells of cortical proximal tubules. The expression of CYP2D25 in kidney, but not in liver, was much higher in the adult pig than in the newborn. These findings indicate a tissue-specific developmental regulation of CYP2D25. The results from the current and previous studies on renal vitamin D hydroxylations imply that CYP2D25 has a biological role in kidney.