S. Allegrini

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.

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.

On the Validity of Continuous Spectrophotometric Assays for Adenosine Deaminase Activity: A Critical Reappraisal

Kinetic investigations on adenosine deaminase from calf intestinal mucosa by spectrophotometric monitoring of the reaction at 264, 270, or 228 nm show that this method does not produce artifactual inhibition by substrate excess up to 0.7 mM concentration, when either adenosine or 2-deoxyadenosine are employed with calf adenosine deaminase. The evaluation of kinetic parameters for this system was carried out both by initial rate measurements and by numerical differentiation of time progress curves according to a recently published method (S. C. Koerber and A. L. Fink, 1987, Anal. Biochem. 165, 75-87). The following results were obtained by the latter method at pH 7.0 and 30 degrees C: for the conversion of adenosine to inosine, kcat = 251 +/- 15 s-1, KMs = 29.7 +/- 2.8 microM, KMp = 613 +/- 62 microM; for the conversion of 2-deoxyadenosine to 2-deoxyinosine, kcat = 283 +/- 17 s-1, KMs = 22.4 +/- 2.2 microM, KMp = 331 +/- 35 microM. At 285 nm, a slight negative deviation from Beers law was observed for adenosine at concentrations higher than 0.9 mM. No deviation was found for inosine up to 2.0 mM at the same wavelength.

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.

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.