Uridine phosphorylase from Acholeplasma laidlawii: purification and kinetic properties.

Uridine phosphorylase was purified 1,370-fold from sonicated extracts of Acholeplasma laidlawii by ammonium sulfate precipitation, DEAE-Sephadex column chromatography, hydroxylapatite chromatography, and Sephadex G-200 fractionation. The molecular weight of the enzyme as determined by gel filtration was approximately 65,000. [U-14C]ribose-1-phosphate (Rib-1-P), prepared enzymatically from [U-14C]inosine, was utilized in initial velocity studies of uridine synthesis, which indicated a sequential reaction with a KmUra of 110 microM and a KmRib-1-P of 17 microM. The kinetics of uridine cleavage were assessed at a saturating cosubstrate concentration, resulting in a KmUrd of 170 microM and a KmPi of 120 microM. These results indicate that an intracellular flux from uracil to uridine is kinetically feasible. However, such flux would be metabolically unproductive, since the low affinity of uridine kinase (KmUrd = 3.2 mM) precludes the operation of uridine phosphorylase and uridine kinase in tandem to convert uracil to UMP. We conclude that uridine phosphorylase performs only a catabolic function in A. laidlawii.     

Facilitated transport of inosine and uridine in cultured mammalian cells is independent of nucleoside phosphorylases

The zero-trans uptake of uniformly and base-labeled inosine and uridine was measured a 25 degrees C in suspensions of Novikoff rat hepatoma cells, Chinese hamster ovary cells, mouse L cells, mouse S49 lymphoma cells and a purine-nucleoside phosphorylase-deficient subline thereof (NSU-1), and in monolayer culture of mouse 3T3 and L cells. The initial velocities of uptake of both nucleosides were about the same in all cell lines investigated, regardless of the position of the label or of the substrate concentration between 3 and 300 microM or whether or not the cells possessed uridine or purine-nucleoside phosphorylase activity. The kinetic parameters for the facilitated transport of uridine and inosine were also similar in phosphorylase positive and negative cell lines (K = 120--260 microM and V = 6--40 pmol/microliters cell water per s) and the transport activities of the cells exceeded their total phosphorylase activities by at least 10-fold for uridine and 1--2-fold for inosine. Chromatographic fractionation of the intracellular contents and of the culture fluid showed that the free nucleosides appeared intracellularly prior to and more rapidly than their phosphorolysis products. During the initial 20--60 s of uptake of U-14C-labeled nucleosides the rates of intracellular appearance of ribose-1-P and base were about the same. After several minutes of incubation, on the other hand, the main intracellular component was ribose-1-P whereas the base attained a low intracellular steady-state concentration and accumulated in the medium due to exit transport. Other nucleosides, dipyridamole and nitrobenzylthioinosine, specifically inhibited the transport of uridine and inosine, and depressed the intracellular accumulation of ribose-1-P and the formation of base commensurate with that inhibition. The data indicate that the metabolism of inosine and uridine by the various cell lines can be entirely accounted for by the facilitated transport of unmodified nucleoside into the cell followed by intracellular phosphorolysis.     

Uridine phosphorylase from Escherichia coli B.: kinetic studies on the mechanism of catalysis

Using a highly purified enzyme preparation of uridine phosphorylase from Escherichia coli B, we have performed detailed kinetic studies which include initial-velocity and product-inhibition experiments in the forward and reverse directions of the reaction. These studies indicate a rapid-equilibrium random mechanism for this enzyme with the formation of an enzyme . uracil phosphate abortive complex. Lack of formation of the enzyme . uridine . ribose-1-phosphate abortive complex suggests that the ribosyl moiety of the two ligands compete for the same binding site. The random mechanism is different from the ordered addition of substrates found for uridine phosphorylase from other sources. All the kinetic constants in the forward and reverse directions and the Keq of reaction for E. coli uridine phosphorylase are reported herein.     

Metabolisms of Nucleosides in Bacteria

The mechanism of trans-N-ribosylation in Corynebacterium sepedonicum was investigated. Using the DEAE-cellulose colum chromatography, this enzyme activity was divided into two fractions. One cleaved uridine to uracil and ribose phosphate, and the other decomposed inosine into hypoxanthine and ribose phosphate, in the presence of inorganic phosphate. The ribose phosphate was isolated and crystallized.

Several analytical data indicated that the ribose phosphate was ribose-1-phosphate. These two enzyme fractions catalyzed the formation of nucleosides from ribose-1-phosphate and bases.

Most of bacteria, which had the activity to transfer N-ribosyl group between purine and pyrimidine, could synthesize the nucleoside from base and ribose-1-phosphate.     

Acholeplasma laidlawii B-PG9 adenine-specific purine nucleoside phosphorylase that accepts ribose-1-phosphate, deoxyribose-1-phosphate, and xylose-1-phosphate.

An adenylate-specific purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC12.4.2.1) (PNP) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (820-fold). This partially purified PNP could only ribosylate adenine and deribosylate adenosine and deoxyadenosine. The A. laidlawii partially purified PNP could not use hypoxanthine, guanine, uracil, guanosine, deoxyguanosine, or inosine as substrates, but could use ribose-1-phosphate, deoxyribose-1-phosphate, or xylose-1-phosphate as the pentose donor. Mg2+ and a pH of 7.6 were required for maximum activity for each of the pentoses. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 108,000 and a sedimentation coefficient of 6.9, and in gel filtration experiments it had an approximate molecular weight of 102,000 and a Stoke's radius of 4.1 nm. Nondenaturing polyacrylamide tube gels of the enzyme preparation produced one major and one minor band. The major band (Rf, 0.57) corresponded to all enzyme activity. The Kms for the partially purified PNP with ribose-1-phosphate, deoxyribose-1-phosphate, and xylose-1-phosphate were 0.80, 0.82, and 0.81 mM, respectively. The corresponding Vmaxs were 12.5, 14.3, and 12.0 microM min-1, respectively. The Hill or interaction coefficients (n) for all three pentose phosphates were close to unity. The characterization data suggest the possibility of one active site on the enzyme which is equally reactive toward each of the three pentoses. This is the first report of an apparently adenine-specific PNP activity.     

Transport of adenine, hypoxanthine and uracil into Escherichia coli.

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.     

Uridine phosphorylase from Schistosoma mansoni

Uridine phosphorylase is the only pyrimidine nucleoside cleaving activity that can be detected in extracts of Schistosoma mansoni. The enzyme is distinct from the two purine nucleoside phosphorylases contained in this parasite. Although Urd is the preferred substrate, uridine phosphorylase can also catalyze the reversible phosphorolysis of dUrd and dThd, but not Cyd, dCyd, or orotidine. The enzyme was purified 170-fold to a specific activity of 2.76 nmol/min/mg of protein with a 16% yield. It has a Mr of 56,000 as determined by molecular sieving on Sephadex G-100. The mechanism of uridine phosphorylase is sequential. When Urd was the substrate, the KUrd = 13 microM and the KPi = 533 +/- 78 microM. When dThd was used as a substrate, the KdThd = 54 microM and the KPi = 762 +/- 297 microM. The Vmax with dThd was 53 +/- 9.8% that of Urd. dThd was a competitive inhibitor when Urd was used as a substrate. The enzyme showed substrate inhibition by Urd, dThd (greater than 0.125 mM) and phosphate (greater than 10 mM). 5-(Benzyloxybenzyloxybenzyl)acyclouridine was identified as a potent and specific inhibitor of parasite (Ki = 0.98 microM) but not host uridine phosphorylase. Structure-activity relationship studies suggest that uridine phosphorylase from S. mansoni has a hydrophobic pocket adjacent to the 5-position of the pyrimidine ring and indicate differences between the binding sites of the mammalian and parasite enzymes. These differences may be useful in designing specific inhibitors for schistosomal uridine phosphorylase which will interfere selectively with nucleic acids synthesis in this parasite.     

Ribose 1-phosphate and inosine activate uracil salvage in rat brain

The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.     

Regulation of purine utilization in bacteria. VII. Involvement of membrane-associated nucleoside phosphorylase in the uptake and the base-mediated loss of the ribose moiety of nucleosides by Salmonella typhimurium membrane vesicles.

Although uridine and adenosine are converted by membrane-associated nucleoside phosphorylases to ribose-1-phosphate (ribose-1-P) and the corresponding bases (uracil and adenine), only ribose -1-P is accumulated within Salmonella typhimurium LT2 membrane vesicles. In accordance with these observations, no uptake is observed when the vesicles are incubated with the bases or nucleosides labeled in their base moieties. The vesicles lack a transport system for ribos-1-P, since excess ribose-1-P does not inhibit the uptake of the ribose moiety of uridine. In addition, there is no exchange with preaccumulatedribose-1-P. Thus, uridine, rather than ribose-1-P, must serve as the initially transported substrate. The uptake of the ribose portion of uridine is coupled to electron transport, and the levels to which ribose-1-P are accumulated may be reduced by adding various bases to the reaction mixtures. The bases appear to inhibit the uridine phosphorylase reaction and/or cause an efflux of ribose-1-P from the vesicles. This loss of ribose-1-P reflects the accumulation of nucleosides in the external medium after being synthesized within the membranes. Synthesis of the nucleosides from intravesicular ribose-1-P and exogenous base proceeds even though the bases are not accumulated by the vesicles. Furthermore, ribose-1-P cannot significantly inhibit uridine phosphorylase activity unless the membranes are disrupted. These observations indicate that the membrane-associated nucleoside phosphorylases may have a transmembranal orientation with their base and ribose-1-P binding sites on opposite sides of the membranes. Such an asymmetric arrangement of these enzymes may facilitate the uptake of the ribosyl moiety of nucleosides by a group translocation mechanism. Thus, nucleosides may be cleaved during the membrane transport process, with the resultant bases delivered to the external environment while ribose-1-P is shunted to the intravesicular space.     

The crystal structure of Streptococcus pyogenes uridine phosphorylase reveals a distinct subfamily of nucleoside phosphorylases

Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 ? resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an α/β monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is ～7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.     

Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to β-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.     

Uridine transport in Novikoff rat hepatoma cells and other cell lines and its relationship to uridine phosphorylation and phosphorolysis.

Time courses of [³H]uridine uptake as a function of uridine concentration were determined at 25° in untreated and ATP-depleted wild-type and uridine kinase-deficient Novikoff cells and in mouse L and P388 cells, Chinese hamster ovary cells and human HeLa cells. Short term uptake was measured by a rapid sampling technique which allows sampling of cell suspensions in intervals as short as one and one-half seconds. The initial segments of the time courses were the same in untreated, wild-type cells in which uridine is rapidly phosphorylated and in cells in which uridine phosphorylation was prevented due to lack of ATP or uridine kinase. The initial rates of uptake, therefore, reflected the rate of uridine transport. Uridine uptake, however, was approximately linear for only five to ten seconds at uridine concentrations from 20–160 μM and somewhat longer at higher concentrations. In phosphorylating cells the rate of uridine uptake (at 80 μM) then decreased to about 20–30% of the initial rate and this rate was largely determined by the rate of phosphorylation rather than transport. At uridine concentrations below 1 μM, however, the rate of intracellular phosphorylation in Novikoff cells approached the transport rate. The apparent substrate saturation of phosphorylation suggests the presence of a low K_m uridine phosphorylation system in these cells. The “zero-trans” (zt) K_m for the facilitated transport of uridine as estimated from initial uptake rates fell between 50 and 240 μM for all cell lines examined. The zero-trans V_max values were also similar for all the lines (4–15 pmoles/μ1 cell H₂O.sec). The time courses of uridine uptake by CHO cells and the kinetic constants for transport were about the same whether the cells were propagated (and analyzed for uridine uptake) in suspension or monolayer culture. When Novikoff cells were preloaded with 10 μM uridine the apparent K_m and V_max values (infinite-trans) were two to three times higher than the corresponding zero-trans values. Uridine transport was inhibited in a simple competitive manner by several other ribo- and deoxyribonucleosides. All nucleosides seem to be transported by the same system, but with different efficiencies. Uridine transport was also inhibited by hypoxanthine, adenine, thymine, Persantin, papaverin, and o-nitrobenzylthioinosine, and by pretreatment of the cells with p-chloromercuri-benzoate, but not by high concentrations of cytosine, D-ribose or acronycin. The inhibition of uridine transport by Persantin involved changes in both V and K. Because of the rapidity of transport, some loss of intracellular uridine occurred when cells were rinsed in buffer solution to remove extracellular substrate, even at 0°. This loss was prevented by the presence of a transport inhibitor, Persantin, in the rinse fluid or by separating suspended cells from the medium by centrifugation through oil. Metabolic conversion of intracellular uridine were also found to continue during the rinse period. The extent of artifacts due to efflux and metabolism during rinsing increased with duration of the rinse.     

Mechanism of Purine Arabinoside Synthesis by Bacterial Transarabinosylation Reaction

The mechanism of purine arabinoside synthesis from uracil arabinoside and purine bases via the bacterial transarabinosylation reaction was investigated. Arabinose-1-phosphate was isolated from the reaction mixture in the form of the barium salt and proved to be the intermediate of the reaction. Two enzyme fractions were obtained from Enterobacter aerogenes by means of heat treatment, ammonium sulfate fractionation and DEAE-cellulose column chromatography. One enzyme split uracil arabinoside into uracil and arabinose-1-phosphate in the presence of inorganic phosphate and the other synthesized hypoxanthine arabinoside from arabinose-1-phosphate and hypoxanthine. The substrate specificity of these enzymes indicated that the former was uridine phosphorylase and the latter was purine nucleoside phosphorylase, respectively. Hypoxanthine arabinoside was synthesized from uracil arabinoside and hypoxanthine only in the presence of both enzymes and inorganic phosphate.     

Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer''s and Parkinson''s, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications.     

Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to β-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.     

Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells

The anabolism of pyrimidine ribo- and deoxyribonucleosides from uracil and thymine was investigated in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in a Burkitt's lymphoma-derived cell line (Raji). We studied the ability of these cells to synthesize pyrimidine nucleosides by ribo- and deoxyribosyl transfer between pyrimidine bases or nucleosides and the purine nucleosides inosine and deoxyinosine as donors of ribose 1-phosphate and deoxyribose 1-phosphate, respectively: these reactions involve the activities of purine-nucleoside phosphorylase, and of the two pyrimidine-nucleoside phosphorylases (uridine phosphorylase and thymidine phosphorylase). The ability of the cells to synthesize uridine was estimated from their ability to grow on uridine precursors in the presence of an inhibitor of pyrimidine de novo synthesis (pyrazofurin). Their ability to synthesize thymidine and deoxyuridine was estimated from the inhibition of the incorporation of radiolabelled thymidine in cells cultured in the presence of unlabelled precursors. In addition to these studies on intact cells, we determined the activities of purine- and pyrimidine-nucleoside phosphorylases in cell extracts. Our results show that Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases, and possess a low uridine phosphorylase activity and markedly decreased (about 1% of peripheral blood lymphocytes) thymidine phosphorylase activity. Lymphocytes have higher pyrimidine-nucleoside phosphorylases activities, they can synthesize deoxyuridine and thymidine from bases, but at high an non-physiological concentrations of precursors. Neither type of cell is able to salvage uracil into uridine. These results suggest that pyrimidine-nucleoside phosphorylases have a catabolic, rather than an anabolic, role in human lymphoid cells. The facts that, compared to peripheral blood lymphocytes, lymphoblasts possess decreased pyrimidine-nucleoside phosphorylases activities, and, on the other hand, more efficiently salvage pyrimidine nucleosides, are consistent with a greater need of these rapidly proliferating cells for pyrimidine nucleotides.     

Enzymes of Pyrimidine Metabolism in Mycoplasma mycoides subsp. mycoides

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides have been proposed from studies on its use of radioactive purines and pyrimidines. To interpret more fully the observed pattern of pyrimidine usage, cell extracts of this organism have been assayed for several enzymes associated with the salvage synthesis of pyrimidine nucleotides. M. mycoides possessed uracil phosphoribosyltransferase, uridine phosphorylase, uridine (cytidine) kinase, uridine 5'-monophosphate kinase, and cytidine 5'-triphosphate synthetase. No activity for phosphorolysis of cytidine was detected, and no in vitro conditions were found to give measurable deamination of cytidine. Of the two potential pathways for incorporation of uridine, our data suggest that this precursor would largely undergo initial phosphorolysis to uracil and ribose-1-phosphate. Conversely, cytidine is phosphorylated directly to cytidine 5'-monophosphate in its major utilization, although conversion of cytidine to uracil, uridine, and uridine nucleotide has been observed in vivo, at least when uracil is provided in the growth medium. Measurements of intracellular nucleotide contents and their changes on additions of pyrimidine precursors have allowed suggestions as to the operation of regulatory mechanisms on pyrimidine nucleotide biosynthesis in M. mycoides in vivo. With uracil alone or uracil plus uridine as precursors of pyrimidine ribonucleotides, the regulation of uracil phosphoribosyltransferase and cytidine 5'-triphosphate synthetase is probably most important in determining the rate of pyrimidine nucleotide synthesis. When cytidine supplements uracil in the growth medium, control of cytidine kinase activity would also be important in this regard.     

Nucleoside transport in cultured mammalian cells. Multiple forms with different sensitivity to inhibition by nitrobenzylthioinosine or hypoxanthine

The zero-trans influx of 500 microM uridine by CHO, P388, L1210 and L929 cells was inhibited by nitrobenzylthioinosine ( NBTI ) in a biphasic manner; 60-70% of total uridine influx by CHO cells and about 90% of that in P388, L1210 and L929 cells was inhibited by nmolar concentrations of NBTI (ID50 = 3-10 nM) and is designated NBTI -sensitive transport. The residual transport activity, designated NBTI -resistant transport, was inhibited by NBTI only at concentrations above 1 microM (ID50 = 10-50 microM). S49 cells exhibited only NBTI -sensitive uridine transport, whereas Novikoff cells exhibited only NBTI -resistant uridine transport. In all instances NBTI -sensitive transport correlated with the presence of between 7 7 X 10(4) and 7 X 10(5) high-affinity NBTI binding sites/cell (Kd = 0.3-1 nM). Novikoff cells lacked such sites. The two types of nucleoside transport, NBTI -resistant and NBTI -sensitive, were indistinguishable in substrate affinity, temperature dependence, substrate specificity, inhibition by structurally unrelated substances, such as dipyridamole or papaverine, and inhibition by sulfhydryl reagents or hypoxanthine. We suggest, therefore, that a single nucleoside transporter can exist in an NBTI -sensitive and an NBTI -resistant form depending on its disposition in the plasma membrane. The sensitive form expresses a high-affinity NBTI binding site(s) which is probably made up of the substrate binding site plus a hydrophobic region which interacts with the lipophilic nitrobenzyl group of NBTI . The latter site seems to be unavailable in NBTI -resistant transporters. The proportion of NBTI -resistant and sensitive uridine transport was constant during proportion of NBTI -resistant and sensitive uridine transport was constant during progression of P388 cells through the cell cycle and independent of the growth stage of the cells in culture. There were additional differences in uridine transport between cell lines which, however, did not correlate with NBTI sensitivity and might be related to the species origin of the cells. Uridine transport in Novikoff cells was more sensitive to inhibition by dipyridamole and papaverine than that in all other cell lines tested, whereas uridine transport in CHO cells was the most sensitive to inactivation by sulfhydryl reagents.     

A novel structural mechanism for redox regulation of uridine phosphorylase 2 activity

Uridine phosphorylase (UPP) catalyzes the reversible conversion of uridine to uracil and ribose-1-phosphate and plays an important pharmacological role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil and capecitabine. Most vertebrate animals, including humans, possess two homologs of this enzyme (UPP1 & UPP2), of which UPP1 has been more thoroughly studied and is better characterized. Here, we report two crystallographic structures of human UPP2 (hUPP2) in distinctly active and inactive conformations. These structures reveal that a conditional intramolecular disulfide bridge can form within the protein that dislocates a critical phosphate-coordinating arginine residue (R100) away from the active site, disabling the enzyme. In vitro activity measurements on both recombinant hUPP2 and native mouse UPP2 confirm the redox sensitivity of this enzyme, in contrast to UPP1. Sequence analysis shows that this feature is conserved among UPP2 homologs and lacking in all UPP1 proteins due to the absence of a necessary cysteine residue. The state of the disulfide bridge has further structural consequences for one face of the enzyme that suggest UPP2 may have additional functions in sensing and initiating cellular responses to oxidative stress. The molecular details surrounding these dynamic aspects of hUPP2 structure and regulation provide new insights as to how novel inhibitors of this protein may be developed with improved specificity and affinity. As uridine is emerging as a promising protective compound in neuro-degenerative diseases, including Alzheimer’s and Parkinson’s, understanding the regulatory mechanisms underlying UPP control of uridine concentration is key to improving clinical outcomes in these illnesses.     

URIDINE PHOSPHORYLASE FROM RAT BRAIN: CHARACTERISTICS AND DEVELOPMENTAL COURSE

—Uridine phosphorylase (uridine: orthophosphate ribosyltransferase; EC 2.4.2.3) from rat brain was purified and its properties were studied. The enzyme resembled preparations made from other mammalian sources. Its pH optimum was between 7·6 and 8·0. An examination of its action on various substrates showed rates of reaction in the order: uridine > deoxyuridine > thymidine > cytidine. The enzyme showed a requirement for phosphate which could also be satisfied by arsenate. The activity of the enzyme was protected from heat inactivation by uridine and by phosphate. In brain and liver the activity of the enzyme increased five- to ten-fold between 10 and 20 days of life. Injections of cortisol or of uridine did not increase the enzymic activity.