The functional logic of cytosolic 5'-nucleotidases.

Adenosine- and uridine-cytidine kinases, purine-nucleoside phosphorylase, hypoxanthine-guanine phosphoribosyl transferase, and several related enzymes, are components of the salvage pathways which reduce the loss of intracellular purine and pyrimidine rings. Although this could explain the role of these enzymes, it poses a problem of the role of the cytosolic 5'-nucleotidase. Why are nucleosides produced from nucleoside-monophosphates, only to be converted back to the same compounds? To date, it is well established that a cross talk exists between the extracellular and intracellular nucleoside metabolism. In districts, such as brain, which are dependent on salvage nucleotide synthesis, nucleosides are produced through the action of the ecto-5'-nucleotidase, the last component of a series of plasma-membrane bound enzyme proteins, catalyzing the successive dephosphorylation of released nucleoside-triphosphates. Both nucleosidetriphosphates (mainly ATP and UTP) and nucleosides (mainly adenosine), act as extracellular signals. Once transported into cell cytosol, all nucleosides are salvaged back to nucleoside-triphosphates, with the exception of inosine, whose salvage is limited to IMP. Intracellular balance of nucleosides is maintained by the action of several enzymes, such as adenosine deaminase, uridine phosphorylase and cytidine deaminase, and by at least three 5'-nucleotidases, the ADP activated AMP preferring cN-IA, the ATP-ADP activated IMP-GMP preferring cN-II, and the UMP-CMP preferring cN-III. Here we are reviewing the mechanisms whereby cytosolic 5'-nucleotidases control changes in nucleoside and nucleotide concentration, with the aim to provide a common basis for the study of the relationship between biochemistry and other related disciplines, such as physiology and pharmacology.

The purine nucleoside cycle in cell-free extracts of rat brain: evidence for the occurrence of an inosine and a guanosine cycle with distinct metabolic roles.

The purine nucleoside cycle is a cyclic pathway composed of three cytosolic enzymes, hypoxanthine-guanine phosphoribosyltransferase, IMP-GMP specific 5'-nucleotidase, and purine-nucleoside phosphorylase. It may be considered a 'futile cycle', whose net reaction is the hydrolysis of 5-phosphoribosyl-1-pyrophosphate to inorganic pyrophosphate and ribose 1-phosphate. The availability of a highly purified preparation of cytosolic 5'-nucleotidase prompted us to reconstitute the purine nucleoside cycle. Its kinetics were strikingly similar to those observed when dialyzed extracts of rat brain were used. Thus, when the cycle is started by addition of inorganic phospate (Pi) and hypoxanthine or inosine (the 'inosine cycle'), steady-state levels of the intermediates are observed and the cycle 'turns over' as far as 5-phosphoribosyl-1-pyrophosphate is being consumed. In the presence of ATP, which acts both as an activator of IMP-GMP-specific 5'-nucleotidase and as substrate of nucleoside mono- and di-phosphokinases, no IDP and ITP are formed. The inosine cycle is further favored by the extremely low xanthine oxidase activity. Evidence is presented that ribose 1-phosphate needed to salvage pyrimidine bases in rat brain may arise, at least in part, from the 5-phosphoribosyl-1-pyrophosphate hydrolysis as catalyzed by the inosine cycle, showing that it may function as a link between purine and pyrimidine salvage. When the cycle is started by addition of Pi and guanine (the 'guanosine cycle'), xanthine and xanthosine are formed, in addition to GMP and guanosine, showing that the guanosine cycle 'turns over' in conjunction with the recycling of ribose 1-phosphate for nucleoside interconversion. In the presence of ATP, GDP and GTP are also formed, and the velocity of the cycle is drastically reduced, suggesting that it might metabolically modulate the salvage synthesis of guanyl nucleotides.

Activation pathways of 5-fluorouracil in rat organs and in PC12 cells.

Activation of the pyrimidine analogue 5-fluorouracil (5-FU) to the ribonucleotide level may occur through one of the following three pathways: 1) the 5-phosphoribosyl 1-pyrophosphate (PRPP)-mediated direct transfer of ribose 5-phosphate to 5-FU as catalysed by orotate phosphoribosyltransferase; 2) the ribose 1-phosphate (Rib1-P)-mediated addition of ribose by uridine phosphorylase, followed by the action of uridine kinase; and 3) the 2'-deoxyribose 1-phosphate (deoxyRib1-P)-mediated addition of deoxyribose, thought to be catalysed by thymidine phosphorylase, followed by the action of thymidine kinase. Many of the conclusions as to the precise pathways by which normal tissues and different cell lines activate uracil are indirectly derived from drug interactions affecting the availability of the substrates of the three pathways, or from measurement of activities of the enzymes metabolising 5-FU in normal tissues and tumours. In previous papers (Cappiello et al. Biochim Biophys Acta 1998;1425:273--81; Mascia et al. Biochim Biophys Acta 1999;1472:93--8), we assessed the molecular mechanisms by which the natural base uracil is salvaged in vitro to uracil ribonucleotides and deoxyribonucleotides in rat liver and brain. In this paper, we investigated the pathways of 5-FU activation to cytotoxic ribonucleotide and deoxyribonucleotide levels in normal rat tissues and PC12 cell extracts. The results clearly showed that normal rat tissues activated 5-FU mainly via the Rib1-P pathway, and to a lesser extent via the PRPP pathway. The deoxyRib1-P pathway was absent. PC12 cells activated 5-FU mainly via the PRPP pathway and to a lesser extent by the other two pathways.

Uracil salvage pathway in PC12 cells.

The salvage anabolism of uracil to pyrimidine ribonucleosides and ribonucleotides was investigated in PC12 cells. Pyrimidine base phosphoribosyl transferase is absent in PC12 cells. As a consequence any uracil or cytosine salvage must be a 5-phosphoribosyl 1-pyrophosphate-independent process. When PC12 cell extracts were incubated with ribose 1-phosphate, ATP and uracil they can readily catalyze the synthesis of uracil nucleotides, through a salvage pathway in which the ribose moiety of ribose 1-phosphate is transferred to uracil via uridine phosphorylase (acting anabolically), with subsequent uridine phosphorylation. This pathway is similar to that previously described by us in rat liver and brain extracts (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273; Mascia et al., Biochim. Biophys. Acta 1472 (1999) 93). We show using intact PC12 cells that they can readily take up uracil from the external medium. The analysis of intracellular metabolites reveals that uracil taken up is salvaged into uracil nucleotides, with uridine as an intermediate. We propose that the ribose 1-phosphate-dependent uracil salvage shown by our in vitro studies, using tissues or cellular extracts, might also be operative in intact cells. Our results must be taken into consideration for the comprehension of novel chemotherapeutics' influence on pyrimidine neuronal metabolism.

In vitro recycling of alpha-D-ribose 1-phosphate for the salvage of purine bases.

In this paper, we extend our previous observation on the mobilization of the ribose moiety from a purine nucleoside to a pyrimidine base, with subsequent pyrimidine nucleotides formation (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273-281). The data show that, at least in vitro, also the reverse process is possible. In rat brain extracts, the activated ribose, stemming from uridine as ribose 1-phosphate, can be used to salvage adenine and hypoxanthine to their respective nucleotides. Since the salvage of purine bases is a 5-phosphoribosyl 1-pyrophosphate-dependent process, catalyzed by adenine phosphoribosyltransferase and hypoxanthine guanine phosphoribosyltransferase, our results imply that Rib-1P must be transformed into 5-phosphoribosyl 1-pyrophosphate, via the successive action of phosphopentomutase and 5-phosphoribosyl 1-pyrophosphate synthetase; and,in fact, no adenosine could be found as an intermediate when rat brain extracts were incubated with adenine, Rib-1P and ATP, showing that adenine salvage does not imply adenine ribosylation, followed by adenosine phosphorylation. Taken together with our previous results on the Rib-1P-dependent salvage of pyrimidine nucleotides, our results give a clear picture of the in vitro Rib-1P recycling, for both purine and pyrimidine salvage.

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.

In vitro assessment of salvage pathways for pyrimidine bases in rat liver and brain.

In this paper we extend our previous observation on the mobilization of the ribose moiety from guanosine to xanthine catalyzed by rat liver extracts (Giorgelli et al., Biochim. Biophys. Acta 1335 (1997) 16-22). The data show that in rat liver and brain extracts the activated ribose, stemming from inosine and guanosine phosphorolysis as ribose 1-phosphate, can be used to salvage uracil to uracil nucleotides. Uridine is an intermediate. The salvage process occurs even in the presence of excess inorganic phosphate suggesting that uridine phosphorylase may function in vivo as an anabolic enzyme. Ribose 5-phosphate cannot substitute for inosine, guanosine or ribose 1-phosphate as ribose donor. When inorganic phosphate was substituted with arsenate, hindering the formation of ribose 1-phosphate, no ribose transfer could be observed. A similar pathway occurs at the deoxy level. The deoxyribose moiety of deoxyinosine can be used to salvage thymine to thymine nucleotides, again in the presence of excess inorganic phosphate. Our results introduce a novel aspect of the salvage pathway, in which ribose 1-phosphate seems to play a pivotal role.

Recycling of alpha-D-ribose 1-phosphate for nucleoside interconversion.

Mobilization of the ribose moiety and of the amino group of guanosine may be realized in rat liver extract by the concerted action of purine nucleoside phosphorylase and guanase. Ribose 1-phosphate formed from guanosine through the action of purine nucleoside phosphorylase acts as ribose donor in the synthesis of xanthosine catalyzed by the same enzyme. The presence of guanase, which irreversibly converts guanine to xanthine, affects the overall process of guanosine transformation. As a result of this purine pathway, guanosine is converted into xanthosine, thus overcoming the lack of guanosine deaminase in mammals. Furthermore, in rat liver extract the activated ribose moiety stemming from the catabolism of purine nucleosides can be transferred to uracil and, in the presence of ATP, used for the synthesis of pyrimidine nucleotides; therefore, purine nucleosides can act as ribose donors for the salvage of pyrimidine bases.

The phosphotransferase activity of cytosolic 5'-nucleotidase; a purine analog phosphorylating enzyme.

Cytosolic 5'-nucleotidase is involved in the phosphorylation of several purine nucleoside analogs,used as antiviral and chemotherapeutic agents. In order to assess its role in the mechanisms of activation and inactivation of purine prodrugs, it is essential to study the regulation of both hydrolase and phosphotransferase activities of the enzyme. Using a zone capillary electrophoresis apparatus, we were able to separate substrates and products of the reactions catalyzed by cytosolic 5'-nucleotidase. The method overcomes the frequent unavailability of radiolabeled substrates, and allows the influence of possible effectors and/or experimental conditions on both enzyme activities to be evaluated simultaneously. Results showed that the enzyme was able to phosphorylate several nucleosides and nucleoside analogs with the following efficiency: inosine and 2'-deoxyinosine > 2',3'-dideoxyinosine > 6-chloropurineriboside > 6-hydroxylaminepurine riboside> 2,6-diaminopurine riboside > adenosine > cytidine > deoxycoformycin > 2'deoxyadenosine. This is the first report of deoxycoformycin phosphorylation catalyzed by a 5'-nucleotidase purified from eukaryotic cells. The optimum pH for nucleoside monophosphate hydrolysis was 6.5, slightly more acidic than the optimum pH for the transfer of the phosphate, which was 7.2. Finally, the presence of a suitable substrate for the phosphotransferase activity of cytosolic 5'-nucleotidase caused a stimulation of the rate of formation of the nucleoside. The results suggest the requirements for phosphorylation of nucleoside analogs are a purine ring and the presence of an electronegative group in the 6 position. The stimulation of the rate of nucleoside monophosphate disappearance exerted by the phosphate acceptor suggests that the hydrolysis of the phosphoenzyme intermediate is the rate-limiting step of the process.

Occurrence of inosine kinase as a distinct enzyme in Spirulina platensis.

Among a series of purine nucleosides, inosine was found to be phosphorylated at the highest rate by crude extracts of the cyanobacterium Spirulina platensis. The inosine phosphorylating activity could be separated from hypoxanthine-guanine phosphoribosyl transferase. This result shows that IMP formation may occur via the direct phosphorylation of inosine at its 5'-position, rather than via inosine phosphorolysis, followed by hypoxanthine phosphoribosylation, and provides unequivocal evidence for the occurrence of inosine kinase in nature.

The bifunctional cytosolic 5'-nucleotidase: regulation of the phosphotransferase and nucleotidase activities.

The cytosolic 5'-nucleotidase specific for IMP, GMP, and their deoxyderivatives has been purified approximately 1000 times from calf thymus. The enzyme, in the presence of a suitable nucleoside, can act as a phosphotransferase, catalyzing the transfer of the phosphate moiety from a nucleoside monophosphate donor to a nucleoside acceptor, thus operating as an interconverting activity. This phosphorylating activity has drawn the attention of several research groups because the cytosolic 5'-nucleotidase represents the only cellular enzyme able to phosphorylate inosine and guanosine analogs, which are not substrates of known cellular nucleoside kinases. In this paper, we report the kinetic parameters of the bifunctional enzyme and its response to variations in adenylate energy charge. The results seem to indicate that in the presence of physiological concentrations of ATP and phosphate, the enzyme behaves mainly as a phosphotransferase, its activity being dependent only on the availability of a suitable nucleoside.

Relationship between the levels of purine salvage pathway enzymes and clinical/biological aggressiveness of human colon carcinoma.

A large series of samples obtained after surgical resection of intestinal mucosa of patients affected by intestinal carcinoma was examined in order to define possible relationships between levels of enzymes involved in the purine salvage pathway and clinical/biological parameters of aggressiveness and invasiveness. The results confirm our previous observation on a different pattern of purine salvage enzymes in tumor as compared to normal colon tissues (Camici et al., 1990). In fact, we observed in human colon tumor tissues a significant enhancement of the three enzymes involved in the synthesis of IMP, hypoxanthine guanine phosphoribosyltransferase (HGPRT), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). On the other hand, no variation was observed in the 5'-nucleotidase and alkaline phosphatase activities. While we could not find a significant correlation between HGPRT, ADA and PNP activities and histologic grading or biological parameters of tumor aggressiveness, the significant correlation with the extent of disease, as expressed by the Dukes' stage, would demonstrate at least for human colon tumors, a relationship between enzyme activity and tumor invasiveness.

Cytosolic 5'-nucleotidase/nucleoside phosphotransferase: a nucleoside analog activating enzyme?

Nucleoside phosphotransferase acting on inosine and deoxyinosine has been partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5'-nucleotidase acting on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisphosphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2',3'-dideoxyinosine and 8-azaguanosine are substrates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GMP, and deoxyGMP are the best phosphate donors. The cytosolic 5'-nucleotidase/phosphotransferase substrate, 8-azaguanosine, was found to be very toxic for cultured fibroblasts (LD50 = 0.32 microM). Mutants resistant to either 8-azaguanosine and the correspondent base 8-azaguanine were isolated and characterized. Our results indicated that the 8-azaguanosine-resistant cells were lacking both cytosolic 5'-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine resistant cells were lacking only the latter enzyme. Despite this observation, both mutants displayed 8-azaguanosine resistance, thus indicating that cytosolic 5'-nucleotidase is not essential for the activation of this nucleoside analog.

Cytosolic 5'-nucleotidase/nucleoside phosphotransferase: a single assay for a bifunctional enzyme.

Cytosolic 5'-nucleotidase/nucleoside phosphotransferase has been purified from calf thymus. Since the same protein is able to catalyze both the hydrolysis and the interconversion of several nucleoside monophosphates, it is necessary to study the effect of different metabolites and assay conditions on both activities in order to elucidate their physiological roles. We describe herein a method which allowed us to follow both activities contemporaneously in the same assay mixture. The method takes advantage of the observation that deoxyGMP is both a good substrate for hydrolysis and a good phosphate donor for the phosphotransferase reaction, but its dephosphorylated product, deoxyguanosine, is not a phosphate acceptor. As a consequence, it is possible to follow both the deoxyguanosine production and the transfer of phosphate from deoxyGMP to the best phosphate acceptor, inosine, during the reaction, applying a method for the chromatographic separation on HPLC of both substrates (inosine and deoxyGMP) and both products (IMP and deoxyguanosine). The method was applied to the determination of the KM for inosine.

Membrane-bound 5'-nucleotidase/nucleoside phosphotransferase from Bacillus cereus.

1. A search for nucleoside phosphotransferase activity in Bacillus cereus led to the following results: (i) The phosphotransferase activity was associated with a membrane bound 5'-nucleotidase. (ii) The enzyme phosphorylates both purine and pyrimidine nucleosides as well as 2',3'-dideoxyinosine. (iii) The enzyme was inhibited by adenylic nucleotide di- and triphosphates, and its nucleotidase activity was increased in the presence of inosine as phosphate acceptor. 2. Bacterial and vertebrate 5'-nucleotidases with phosphotransferase activity differ for several characteristics, such as cellular location, substrate specificity, magnesium requirement and regulation.

Purine nucleoside phosphorylase from bovine lens: purification and properties.

Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) was purified 38,750-fold to apparent electrophoretic homogeneity from bovine ocular lens. The enzyme appears to be a homotrimer with a molecular weight of 97,000, and displays non-linear kinetics with concave downward curvature in double-reciprocal plots with orthophosphate as variable substrate. The analysis of the kinetic parameters of bovine lens purine nucleoside phosphorylase, determined both for the phosphorolytic activity on nucleosides and for ribosylating activity on purine bases, indicates the occurrence of a rapid equilibrium random Bi-Bi mechanism with formation of abortive complexes. The effect of pH on the enzyme activity and on the sensitivity of the enzyme to photoinactivation, as well as the effect of thiol reagents on the enzyme activity and stability, strongly suggest the involvement of histidine and cysteine residues in the active site. From the measurements of the kinetic parameters at different temperatures, heats of formation of the enzyme-substrate complex for guanosine, guanine, orthophosphate and ribose 1-phosphate were determined. Activation energies of 15,250 and 14,650 cal/mol were obtained for phosphorolysis and synthesis of guanosine, respectively.

Purine salvage as a metabolite and energy saving mechanism in the ocular lens.

The ocular lens is an organ which depends mainly on anaerobic processes to obtain the metabolic energy required for the maintenance of its physiological functions. In these circumstances, the purine salvage pathway enzymes, by using preformed purine rings, and allowing the utilization of the activated ribose moiety of nucleosides, might be of relevance as an energy saving device. In this paper we show that the calf lens possesses many enzymes of the purine salvage pathway, with a particularly high specific activity of purine nucleoside phosphorylase (EC 2.4.2.1), and that the isolated lens epithelium can actively convert adenine and adenosine into adenine nucleotides. In addition, as in bacteria and red blood cells, inosine and adenosine in the lens, acting as ribose donors, exert a profound effect on the process of adenine conversion into ATP.

Nucleoside phosphotransferase activity of human colon carcinoma cytosolic 5'-nucleotidase.

A cytosolic 5'-nucleotidase, acting preferentially on IMP and GMP, has been isolated from human colon carcinoma extracts. This enzyme activity catalyzes also the transfer of the phosphate group of 5'-nucleoside monophosphates (mainly, 5'-IMP, 5'-GMP, and their deoxycounterparts) to nucleosides (preferentially inosine and deoxyinosine, but also nucleoside analogs, such as 8-azaguanosine and 2',3'-dideoxyinosine). It has been proposed that the enzyme mechanism involves the formation of a phosphorylated enzyme as an intermediate which can transfer the phosphate group either to water or to the nucleoside. The enzyme is activated by some effectors, such as ATP and 2,3-diphosphoglycerate. Results indicate that the effect of these activators is mainly to favor the transfer of the phosphate of the phosphorylated intermediate to the nucleoside (i.e., the nucleoside phosphotransferase activity). This finding is in accordance with previous suggestions that cytosolic 5'-nucleotidase cannot be considered a pure catabolic enzyme.