Uridine Metabolism in the Goldfish Retina During Optic Nerve Regeneration: Cell-Free Preparations

The activities of uridine kinase (EC 2.7.1.48), uridine monophosphate (UMP) kinase (EC 2.7.1.3.14), and uridine diphosphate (UDP) kinase (EC 2.7.4.6) were measured in retinal high-speed supernatant fractions following unilateral optic nerve crush in the goldfish. The enzyme activities followed a similar time course, with initial increases 2-3 days following nerve crush, peak activity at 4 days, and a gradual return to basal levels by day 21. The magnitude of the stimulation on day 4 was about 35% in each case. Activities of two enzymes of intermediary metabolism, pyruvate kinase (EC 2.7.1.40) and lactic dehydrogenase (EC 1.1.1.27), were not altered, indicating that the coordinate increases in nucleoside and nucleotide kinase activities were specific responses to the nerve injury. The increased labeling could not be explained by altered phosphohydrolytic activities. The nature of the enhancement was further studied in UDP kinase, the most active of the kinases examined. Neither low-molecular-weight components nor substrate availability could account for the observed increase in UDP kinase in the 4 day post-crush retinas. The Km for UDP was unaltered, and a mixing experiment did not support the possibility that stimulatory or inhibitory factors played a role. The enhancement of UDP kinase activity was blocked by injection of actinomycin D following nerve crush. The results suggest that the observed increases in enzymes of uridine metabolism result from their increased formation following nerve crush.     

Metabolism of Uridine 5′-Diphosphate-Glucose in Golgi Vesicles from Pea Stems

Uridine 5′-diphosphate-glucose (UDP-Glc) is transported into the lumen of the Golgi cisternae, where is used for polysaccharide biosynthesis. When Golgi vesicles were incubated with UDP-[³H]Glc, [³H]Glc was rapidly transferred to endogenous acceptors and UDP-Glc was undetectable in Golgi vesicles. This result indicated that a uridine-containing nucleotide was rapidly formed in the Golgi vesicles. Since little is known about the fate of the nucleotide derived from UDP-Glc, we analyzed the metabolism of the nucleotide moiety of UDP-Glc by incubating Golgi vesicles with [α-³²P]UDP-Glc, [β-³²P]UDP-Glc, and [³H]UDP-Glc and identifying the resulting products. After incubation of Golgi vesicles with these radiolabeled substrates we could detect only uridine 5′-monophosphate (UMP) and inorganic phosphate (Pi). UDP could not be detected, suggesting a rapid hydrolysis of UDP by the Golgi UDPase. The by-products of UDP hydrolysis, UMP and Pi, did not accumulate in the lumen, indicating that they were able to exit the Golgi lumen. The exit of UMP was stimulated by UDP-Glc, suggesting the presence of a putative UDP-Glc/UMP antiporter in the Golgi membrane. However, the exit of Pi was not stimulated by UDP-Glc, suggesting that the exit of Pi occurs via an independent membrane transporter.     

Topology of UDP-galactose cleavage in relation to N-acetyl-lactosamine formation in Golgi vesicles. Translocation of activated galactose.

UDP-galactose appears to be produced on one side of a membrane barrier, opposite the galactosyltransferases that use it as a sugar donor. The translocation of activated galactose across membranes was studied in rat submaxillary-gland microsomal vesicles and in rat liver Golgi vesicles. When these intact vesicles containing the acceptor, N-acetylglucosamine, were incubated in the presence of UDP-galactose and two inhibitors of galactosyltransferase activity, the product, N-acetyl-lactosamine, formed within the vesicles. Thus at least the galactose moiety of UDP-galactose crossed the membranes. When intact Golgi vesicles were incubated with UDP-galactose labelled in both the uridine and the galactose moieties, labelled N-acetyllactosamine was again produced in the vesicles, but less than stoichiometric amounts of the uridine label was found there. Calculation of internal and external concentrations of UMP, a major product released from the cleaved uridine moiety, showed that the vesicles were actually enriched in UMP. When free UMP was incubated with the vesicles, this enrichment did not occur. This result was direct evidence for facilitated transport of UDP-galactose into the Golgi for use by galactosyltransferase.     

Glycoprotein reglucosylation and nucleotide sugar utilization in the secretory pathway: identification of a nucleoside diphosphatase in the endoplasmic reticulum.

UDP is generated in the lumen of the endoplasmic reticulum (ER) as a product of the UDP-glucose-dependent glycoprotein reglucosylation in the calnexin/calreticulin cycle. We describe here the identification, purification and characterization of an ER enzyme that hydrolyzes UDP to UMP. This nucleoside diphosphatase is a ubiquitously expressed, soluble 45 kDa glycoprotein devoid of transmembrane domains and KDEL-related ER localization sequences. It requires divalent cations for activity and hydrolyzes UDP, GDP and IDP but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate. By eliminating UDP, which is an inhibitory product of the UDP-Glc:glycoprotein glucosyltransferase, it is likely to promote reglucosylation reactions involved in glycoprotein folding and quality control in the ER.     

Characterization of nucleoside diphosphatase in the onion root tip. III. Solubilization and activation of membrane-bound enzyme(s)

The latency of nucleoside diphosphatase (NDPase) in onions root homogenates has been examined by comparing the activation of NDPase activity resulting from detergent treatment with that due to storage of homogenates for several days in the cold. Both detergent treatment and cold storage activated NDPase approximately two-fold. In both cases this activation was paralleled by the loss of enzyme activity from the membrane fractions and its appearance in the supernatants. Electrophoresis of these supernatants revealed an identifical isoenzyme pattern of 5 NDPase bands for both preparations. Enzyme kinetic studies demonstrated that NDPase from the detergent-treated homogenate and the homogenate stored in the cold as well as NDPase from the membrane and supernatant fractions from each of the homogenates all had the same Km value. These data suggest that latency of NDPase is the result of a breakdown of cellular membranes and subsequent release of NDPase. Abbreviations: DOC, deoxycholate; NDPase, nucleoside diphosphatase; IDP, inosine 5'-diphosphate; UDP, uridine 5'-diphosphate; GDP, guanosine 5'-diphosphate.     

Thiamine pyrophosphatase (nucleoside diphosphatase) in the Golgi apparatus is distinct from microsomal nucleoside diphosphatase

The properties of thiamine pyrophosphatase in the Golgi apparatus of rat liver were studied. Thiamine pyrophosphatase in an extract of the Golgi apparatus was separated into 6 bands of between pH 5.4 and 6.3 by isoelectric focusing on polyacrylamide gel. On the gels all these subforms catalyzed the hydrolyses of GDP, IDP, UDP, and CDP as well as that of thiamine pyrophosphate. The characteristics resembled those of Type B nucleoside diphosphatase of rat brain, though the enzyme did not have 3 subforms of Type B nucleoside diphosphatase in the higher pH region on isoelectric focusing. Thiamine pyrophosphatase of the Golgi apparatus was separated from microsomal nucleoside diphosphatase by DEAE-cellulose column chromatography. The properties of the enzyme were quite similar to those of Type B nucleoside diphosphatase with respect to its substrate specificity, optimum pH for activity, and inhibition by ATP. These findings suggest that thiamine pyrophosphatase in the Golgi apparatus is different from microsomal nucleoside diphosphatase and that it might be basically the same enzyme as Type B nucleoside diphosphatase except for different extents of modification.     

Phosphoglycolate phosphatase from human red blood cells

The nucleotide profile of rat liver Golgi vesicles isolated using sucrose gradients has been determined by high-pressure liquid chromatography. The nucleotide composition of this Golgi preparation, probably modified by osmotic shock, differs from that of liver supernatant fraction and from isolated rough microsomes. The major nucleotides present in the Golgi have been tentatively identified as uridine diphosphate and a peak containing uridine monophosphate plus cytidine monophosphate at 1.6 and 0.5 nmol/mg protein, respectively. In order to minimize osmotic shock, we have modified the isolation of Golgi using D₂O-sucrose gradients. Intact Golgi from these gradients were extracted directly and analyzed. Higher levels of nucleotides were found in the unshocked preparation, and the profile was also altered, although it was still distinct from that of liver supernatant. Four major peaks were found, tentatively identified as uridine monophosphate plus cytidine monophosphate, adenosine monophosphate, UDP, and uridine diphosphogalactose plus uridine diphosphoglucose, at 6.4, 6.4, 6.1, and 3.3 nmol/mg protein. These results indicate that the membrane of the Golgi apparatus is not freely permeable to nucleotides but that selective mechanisms exist for the uptake or exclusion of specific nucleotides from this organelle. The fact that UDP is selectively retained in shocked Golgi vesicles may indicate the presence of a binding protein which would prevent interference of Golgi function by UDP, a highly inhibitory product of galactosyltransferase.     

Subcellular compartmentation of uridine nucleotides and nucleoside-5' -diphosphate kinase in leaves

The subcellular compartmentation of nucleoside diphosphate kinase (EC 2.7.4.6) and the uridine nucleotides has been studied in leaves. Membrane filtration of barley (Hordeum vulgare L.) leaf mesophyll protoplasts and differential centrifugation of spinach (Spinacia oleracea L.) leaf extracts showed that about half the nucleoside diphosphate kinase is present in the cytosol. The activity is adequate to account for the turnover of UTP and UDP during photosynthetic sucrose synthesis. Nonaqueous density gradient centrifugation of freeze-stopped, lyophilized spinach leaf material showed that the uridine nucleotides are predominantly located in the cytosol and that the cytosolic UDP-glucose pool is considerably larger than the UTP or UDP pools.     

Alterations in the cytochemical activity of several phosphatases in hepatocytes from rats exposed prenatally to ethanol

The activities of acid phosphatase, alkaline phosphatase, glucose-6-phosphatase, uridine diphosphatase, inosine diphosphatase, thiamine pyrophosphatase and 5'-nucleotidase have been investigated cytochemically in hepatocytes of the offspring of alcohol-fed rats, using cerium ions as a capturing agent and qualitative and quantitative electron microscopy. All these enzyme activities were decreased in the experimental animals compared with controls not exposed to ethanol. The pattern of deposition of the product of glucose-6-phosphatase activity in the cisternae of the endoplasmic reticulum was also different in the two groups. The phosphatases analyzed are functional markers of different cell components, and the results suggest that prenatal exposure of rats to ethanol causes functional alterations in the endoplasmic reticulum, Golgi apparatus, lysosomes and plasma membrane of hepatocytes.     

Profiles of pyrimidine biosynthesis,salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.     

Uridine diphosphate glucose-sterol glucosyltransferase and nucleoside diphosphatase activities in etiolated pea seedlings.

1. UDP-glucose-sterol glucosyltransferase and nucleoside diphosphatases were isolated in a particulate fraction from 7-day-old etiolated pea seedlings. The glucosyltransferase and UDPase (uridine diphosphatase) are stimulated by Ca2+ cation, less so by Mg2+ cation, and inhibited by Zn2+. 2. Each activity has a pH optimum near 8. 3. The glucosyltransferase is specific for UDP-glucose as the glucosyl donor and is inhibited by UDP. Partial recovery from UDP inhibition is effected by preincubation of the enzyme. 4. Freeze-thaw treatment and subsequent sucrose-density-gradient centrifugation of the particulate fraction shows the glucosyltransferase to be widely distributed among cell fractions but to be most active in particles with a density of 1.15 g/ml. UDPase is most active in particulate material with a density of over 1.18 g/ml but an activity peak also appears at 1.15 g/ml. Of several nucleoside diphosphatase activities, UDPase activity is most enhanced by the freeze-thaw and sucrose-density-gradient-fractionation procedures. 5. Detergent treatment with 0.1% sodium deoxycholate allows the partial solubilization of the glucosyltransferase and UDPase. The two activities are similarly distributed between pellet and supernatant after high-speed centrifugation for two different time intervals. 6. A role for UDPase in the functioning of glucosylation reactions is discussed.     

Properties of chitin synthase in homogenates from Chironomus cells

Homogenates of Chironomus cells synthesize chitin as effectively as intact cells. Chitin is produced in a dose-dependent manner, when GlcN, GlcNAc, or UDP-GlcNAc is used as precursor. Due to the lability of UDP-GlcNAc incorporation of this substrate is underestimated. No allosteric effect is observed when GlcN or GlcNAc is used as a substrate. Chitin synthesis is stimulated by Mg²⁺ and inhibited by uridine monophosphate (UMP), uridine diphosphate (UDP), and uridine triphosphate (UTP). The apparent temperature optimum is 30°C, the apparent pH optimum is 5.5–6. Addition of the chitinase inhibitor allosamidin does not enhance chitin synthesis significantly. The time course of chitin formation reveals a lag period of about 12 h, which can be overcome by trypsin treatment. Addition of protease inhibitors prevents chitin synthesis.     

Assay of galactosyltransferase by high-performance liquid chromatography

Galactosyltransferase catalyzes transfer of galactose from UDP-galactose to glucose or N-acetylglucosamine with resultant formation of galactosides and UDP. In this new assay galactosyltransferase activity is measured by determining UDP by isocratic high-performance liquid chromatography on an amino-bonded column monitored spectrophotometrically. Concurrently, unreacted UDP-galactose and breakdown products arising from UDP-galactose (UMP and uridine) are also determined. The new technique does not require radioactive substrates, permits usage of saturating concentrations of UDP-galactose, and provides monitoring of side reactions.     

Extracellular metabolism of nucleotides in neuroblastoma x glioma NG108-15 cells determined by capillary electrophoresis

1. The metabolism of extracellular nucleotides in NG108-15 cells, a neuroblastoma × glioma hybrid cell line, was studied by means of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC).2. In NG108-15 cells ATP, ADP, AMP, UTP, UDP, and UMP were hydrolyzed to the nucleosides adenosine and uridine indicating the presence of ecto-nucleotidases and ecto-phosphatases. The hydrolysis of the purine nucleotides ATP and ADP was significantly faster than the hydrolysis of the pyrimidine nucleotides UTP and UDP.3. ATP and UTP breakdown appeared to be mainly due to an ecto-nucleotide- diphosphohydrolase. ADP, but not UDP, was initially also phosphorylated to some extent to the corresponding triphosphate, indicating the presence of an adenylate kinase on NG108-15 cells. The alkaline phosphatase (ALP) inhibitor levamisole did not only inhibit the hydrolysis of AMP to adenosine and of UMP to uridine, but also the degradation of ADP and to a larger extent that of UDP. ATP and UTP degradation was only slightly inhibited by levamisole.4. These results underscore the important role of ecto-alkaline phosphatase in the metabolism of adenine as well as uracil nucleotides in NG108-15 cells. Dipyridamole, a potent inhibitor of nucleotide breakdown in superior cervical ganglion cells, had no effect on nucleotide degradation in NG108-15 cells.5. Dipyridamole, which is a therapeutically used nucleoside reuptake inhibitor in humans, reduced the extracellular adenosine accumulation possibly by allosteric enhancement of adenosine reuptake into the cells.     

Precursors of pyrimidine nucleotide biosynthesis for gravid Angiostrongylus cantonensis (Nematoda: Metastrongyloidea).

Gravid Angiostrongylus cantonensis can utilize radiolabelled bicarbonate, orotate, uracil, uridine and cytidine but not cytosine, thymine and thymidine for the synthesis of RNA and DNA. In cell-free extracts of the worm, a phosphoribosyltransferase was shown to convert orotate to OMP and uracil to UMP. A similar reaction was not observed with cytosine and thymine. Uridine was readily phosphorylated by a kinase but a similar reaction for thymidine and deoxyuridine was not found. Cytidine could be phosphorylated by a kinase or be deaminated by a deaminase to uridine. No deaminase for cytosine was detected. There was also no phosphotransferase activity for pyrimidine nucleosides in the cytosolic or membrane fractions. Pyrimidine nucleosides were, in general, converted to the bases by a phosphorylase reaction but only uracil and thymine could form nucleosides in the reverse reaction. The activity of thymidylate synthetase was also measured. These results indicate that the nematode synthesizes pyrimidine nucleotides by de novo synthesis and by utilization of uridine and uracil and that cytosine and thymine nucleotides are formed mainly through UMP. The thymidylate synthetase reaction appears to be vital for the growth of the parasite.     

Glycoprotein synthesis in the adult rat pancreas: II. Characterization of Golgi-rich fractions

Golgi-rich fractions were prepared from homogenates of adult rat pancreas by discontinuous gradient centrifugation. These fractions were characterized by stacks of cisternae associated with large, irregular vesicles and were relatively free of rough microsomes, mitochondria, and zymogen granules. The Golgi-rich fractions contained 50% of the UDP-galactose: glycoprotein galactosyltransferase activity; the specific activity was 12-fold greater than the homogenate. Such fractions represented < 19% of thiamine pyrophosphatase, uridine diphosphatase, adenosine diphosphatase, and Mg²⁺-adenosine triphosphatase. Zymogen granules and the Golgi-rich fractions were extracted with 0.2 m NaHCO3, pH 8.2, and the membranes were isolated by centrifugation. The glycoprotein galactosyltransferase could not be detected in granule membranes, while the specific activity in Golgi membranes was 25-fold greater than the homogenate.At least 35 polypeptide species were detected in Golgi membranes by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. These ranged in molecular weight from 12,000 to <160,000. There were only minor differences between Golgi membranes and smooth microsomal membrane. In contrast, zymogen granule membranes contained fewer polypeptides. A major polypeptide, which represented 30–40% of the granule membrane profile, accounted for less than 3% of the polypeptides of Golgi membranes or smooth microsomal membranes.     

Mechanism of Apparent Transcription Inhibition by Methyl Mercury in Cerebellar Neurons

Abstract: We have investigated the mechanism of inhibition of RNA synthesis by methyl mercury (MeHg) in isolated neonatal rat cerebellar cells. Each of the three component steps involved in the incorporation of exogenous [³H]uridine into cellular RNA was examined separately in whole-cell and/or subcellular preparations. Nuclear RNA polymerase activity was measured in preparations containing both free nuclei and whole cells. Incorporation of [³H]UTP into nuclear RNA was found to be unimpaired at concentrations of MeHg that inhibited whole-cell incorporation of [³H]uridine by > 75%. Cellular uptake of [³H]uridine was assayed in cerebellar cells treated with KCN to deplete ATP levels and block subsequent phosphorylation reactions of transported uridine. Uptake activity under these conditions was unaffected by MeHg. Measurement of intracellular phosphorylation of [³H]uridine indicated that inhibition of this activity closely paralleled that of RNA synthesis. Quantitation of individual uridine nucleotides by polyethyleneimine-cellulose TLC revealed reduced levels of UTP and UDP whereas levels of UMP were elevated, suggesting that impairment of phosphorylation was not the result of cellular ATP depletion but, more likely, a direct effect on phosphouridine kinase enzymes. This mechanism of MeHg-induced inhibition of RNA synthesis was confirmed by assays of uridine phosphorylation using cell-free extracts in which exogenous ATP was supplied.     

Sucrose biosynthesis in Dunaliella

Sucrose phosphate synthetase (EC 2.4.1.14) is the key enzyme for sucrose synthesis in Dunaliella tertiolecta. It has been partially purified and characterized. The enzyme contains one binding site for uridine diphosphoglucose and two binding sites for fructose-6-phosphate; it is allosterically controlled by fructose-6-phosphate. Inorganic phosphate stimulates the enzymic activity, particularly in the presence of higher concentrations of fructose-6-phosphate. Sucrose phosphate synthetase is not halophilic or halotolerant. The temperature dependence of the enzymic activity cannot fully explain the observed increase in sucrose synthesis in Dunaliella by elevated temperature.Abbreviations F-6-P fructose 6-phosphate - UDP uridine biphosphate - UDPG uridine biphosphoglucose     

Distribution of ADPase activity in the lactating rat mammary gland and its possible role in an ATP cycle in the Golgi apparatus.

A Ca2+/Mg(2+)-stimulated ADPase has been found to occur in the lactating rat mammary gland. The enzyme is membrane associated and occurs in mitochondrial, microsomal, and Golgi apparatus fractions. The pH activity curves for the Golgi apparatus and microsomal fractions display two distinct maxima, one at pH 6.3 and one at pH 7.4. Studies with inhibitors and activators indicate that the enzyme is similar to ADPases found in other tissues and is distinct from the uridine nucleoside diphosphatase previously reported in the mammary Golgi apparatus. The occurrence of ADPase in the Golgi apparatus indicates a possible role for this enzyme in the milk secretory process, while the microsomal enzyme could be involved in extracellular activities.