Maura Turriani

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 > 2deoxyadenosine. 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.

Cytosolic 5′-Nucleotidase/Phosphotransferase of Human Colon Carcinoma

Cytosolic 5′-nucleotidase acting preferentially on IMP and GMP has been purified from many sources1,2,3. The enzyme resulted to be allosterically activated by ATP, ADP and 2,3-diphosphoglycerate and inhibited by phosphate. Cytosolic 5′-nucleotidase appears to be widely distributed in mammalian tissues suggesting that it may have an essential function in cell metabolism. Furthermore the enzyme activity appears to be higher in tissues in active DNA synthesis or with a high turnover rate of nucleic acids and their precursors4.

8-Azaguanosine-5′-monophosphate synthesis via nucleoside kinase in cultured chinese hamster lung fibroblasts

Cultured chinese hamster lung fibroblasts, and a variant clone selected for resistance to 8-azaguanine, that lacks hypoxanthine-guanine phosphoribosyl transferase (EC 2.4.2.8), have been tested for the ability to convert 8-azaguanine into 8-azaguanosine-5-monophosphate via purine nucleoside phosphorylase and nucleoside kinase. Purine nucleoside phosphorylase of both cell types is able to synthesize 8-azaguanosine from 8-azaguanine with the same efficiency. Wild type cells possess a nucleoside kinase activity acting on 8-azaguanosine, but this activity is considerably lower in the cells displaying resistance to the base analog. Our lines of evidence demonstrate that purine nucleoside phosphorylase and nucleoside kinase constitute a possible way of synthesis of the cytotoxic mononucleotide of 8-azaguanine, and, in fact, cells selected for resistance to the base analog show an impairement in the nucleoside kinase activity.

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.

Regulation of Calf Thymus Cytosolic 5’-Nucleotidase/Nucleoside Phosphotransferase

Many phosphatases with different specificity and cellular location have been described to be able to catalyze a phosphotransferase reaction1, 2 as well as several enzymes named phosphotransferases have been demonstrated to hydrolyze phosphoesters3, 4. Even though a number of papers and very accurate reviews have been published on phosphohydrolases, particularly on nucleotidases classified following their substrate specificity or cellular location, still additional information is needed on the molecular characteristics and on the regulation of the phosphohydrolase/phosphotransferases in order to understand the role, if any, of their potential bifunctionality.

Cytotoxicity of Deoxycoformycin on Human Colon Carcinoma Cell Lines

Deoxycoformycin (DCF), a powerful inhibitor of adenosine deaminase1, by virtue of its immunosuppresive action2, has found clinical application for the treatment of several types of lymphatic leukemia3, 4. In fact, the prevention of the catabolism of deoxyAdo to deoxylno, results both in the accumulation of intracellular deoxyATP5, an inhibitor of ribonucleotide reductase, and in the depletion of ATP6. Indeed, it has been reported that human T lymphocyte cells are very sensitive to the cytotoxic action of deoxyAdo, and that their sensitivity to DCF is higher than B lymphocyte cells probably as a result of a reduced cytosolic 5’deoxynucletidase activity, with a concomitant preservation of the deoxyATP pool7. While many studies have been performed on the cytotoxicity of DCF on human lymphocytes and several reports describe its possible mechanism of action in this type of cells8, 9, the effect of this drug on different mammalian cells has not been thoroughly examined so far, also on account of the reported inefrkacy of DCF on the growth of cultured cells 10. Human colon carcinoma cultured cells are a model for the study of the responsiveness of this solid tumor to possible Chemotherapic agents. In view of the involvement of the purine salvage enzymes in the activation of purine analogs, the knowledge of their program in tumor cells might be of great help not only for the understanding of the analog mechanism of action, but also for the design of specific pro-drugs. In this line is the present study on the cytotoxicity of DCF in combination with deoxyAdo on both two human colon carcinoma (LOVO and HT29) and a Chinese hamster ovary (CHO K-l) cell Unes. The different sensitivity of the three cell lines to DCF is well related to their distinct purine salvage enzyme pattern, thus supporting, in a general point of view, the enzymatic approach to the choice of a suitable Chemotherapic agent.