Francesco Sgarrella

A specific adenosine phosphorylase, distinct from purine nucleoside phosphorylase.

Several enzymes, catalyzing the reversible phosphorolysis of purine nucleosides have been described in eucaryotic cells and in micro-organisms [l-6] . The best known is the ‘purine nucleoside phosphorylase’ (EC 2.4.2.1) acting on the nucleosides of hypoxanthine and guanine. Adenosine is not used as substrate by this enzyme [6-81. Phosphorolysis of adenosine has been reported in Salmonella typhimurium [9], where a single enzyme protein appears to act on inosine, guanosine and adenosine. Adenine was found to be substrate for purine nucleoside phosphorylase of four mammalian sources, but its unfavourable kinetic parameters with respect to those of hypoxanthine and guanine are against the role of adenine as a physiological substrate [lO,l l] . In Mycoplasma both adenosine and inosine phosphorolysis have been observed, but no attempt has been made to correlate the two activities to different proteins [12]. The separation of adenosine phosphorylase from the purine nucleoside phosphorylase has never been reported so far, even though Miech and Coll. have presented indirect evidence that in Schistosoma Mansoni worms adenosine phosphorylase activity is a separate entity from purine nucleoside phosphorylase: this conclusion is based on differences in the pH-activity curves and studies with product and nucleoside analogues inhibitors [ 131. The data presented in this paper give the first direct evidence that at least in B. subtilis the phosphorolysis of adenosine and that of inosine and guanosine are catalyzed by distinct enzyme proteins, which can be

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.

Induction and repression of enzymes involved in exogenous purine compound utilization in Bacillus cereus

5-Nucleotidase, adenosine phosphorylase, adenosine deaminase and purine nucleoside phosphorylase, four enzymes involved in the utilization of exogenous compounds in Bacillus cereus, were measured in extracts of this organism grown in different conditions. It was found that adenosine deaminase is inducible by addition of adenine derivatives to the growth medium, and purine, nucleoside phosphorylase by metabolizable purine and pyrimidine ribonucleosides. Adenosine deaminase is repressed by inosine, while both enzymes are repressed by glucose. Evidence is presented that during growth of B. cereus in the presence of AMP, the concerted action of 5-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to formation of adenine, and thereby to induction of adenosine deaminase. The ionsine formed would then cause induction of the purine nucleoside phosphorylase and repression of the deaminase. Taken together with our previous findings showing that purine nucleoside phosphorylase of B. cereus acts as a translocase of the ribose moiety of inosine inside the cell (Mura, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol Chem. 253, 7905-7909), our results provide a clear picture of the molecular events leading to the utilization of the sugar moiety of exogenous AMP, adenosine and inosine as an energy source.

Spectrophotometric determination of ribose 1-phosphate

Abstract In the course of studies on nucleoside monophosphate metabolism, the need was encountered for a method to determine ribose-1-phosphate. Published assays for ribose-1-phosphate depend either on chromatographic separation of the sugarphosphate, or else on its acid lability which allows it to be determined as a phosphate. The present work describes a less laborious spectrophotometric assay which is both rapid and specific. The basis of the method is the absorbance change at 265 nm associated with the following two-stage enzymatic conversion: ribose-1-phosphate + adenine phosphate + adenosine (adenosine phosphorylase); adenosine + H 2 O → inosine + NH 3 (adenosine deaminase). The change in absorbance was proportional to ribose-1-phosphate concentration at least up to 25 μg/ml. In tests of the assay, it was possible to detect ribose-1-phosphate formation from inosine and phosphate catalyzed by purine nucleoside phosphorylase. Further, the degradation of ribose-1-phosphate by various commercial phosphatases and several tissues or microbial extracts was observed.

The standard gibbs free energy change of hydrolysis of α-d-ribose 1-phosphate☆

Abstract The standard Gibbs free energy change of hydrolysis of α- d -ribose 1-phosphate has been measured at pH 7.0, ionic strength 0.1 m , and 25 °C by combining the corresponding values of the two following reactions: adenosine + H2O ag adenine + ribose (ΔG0′ = −2.3 ± 0.1 kcal/mol), catalyzed by adenosine nucleosidase, and ribose 1-phosphate + adenine ag adenosine + Pi (ΔG0′ = −3.1 ± 0.1 kcal/mol), catalyzed by adenosine phosphorylase. The standard Gibbs free energy changes were calculated for both reactions from the equilibrium constant. A value of -5.4 ± 0.15 kcal/mol, comparable to that of other hemiacetal phosphoric esters, was obtained for the hydrolysis of ribose 1-phosphate.

Induction of phosphoribomutase in Bacillus cereus growing on nucleosides

In this paper we show that phosphoribomutase is induced in Bacillus cereus by the same metabolizable purine and pyrimidine ribonucleosides previously shown to induce the purine nucleoside phosphorylase (Tozzi, M.G., Sgarrella, F. and Ipata, P.L. (1981) Biochim. Biophys. Acta 678, 460-466). The mutase allows ribose 1-phosphate formed from nucleosides to be utilized by the cell through the pentose cycle, upon transformation to ribose 5-phosphate. The equilibrium constant of the mutase reaction is towards ribose-5-phosphate formation. The coordinate induction of the two enzymes completes the picture of the molecular events leading to the utilization of the sugar moiety of purine nucleotides and nucleosides as an energy source (Mura. U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol. Chem. 253, 7905-7909).

Spectrophotometric and radioenzymatic determination of ribose 5-phosphate

The present work describes an assay which is highly specific for ribose-5-phosphate. The method is based on the following three-stage enzymatic conversion: (1) ribose 5-phosphate in equilibrium ribose 1-phosphate (phosphopentomutase); (2) ribose 1-phosphate + adenine in equilibrium adenosine + Pi (adenosine phosphorylase); (3) adenosine + H2O----inosine + NH3 (adenosine deaminase). Ribose 5-phosphate may be determined either directly following the change in absorbance at 265 nm associated with the conversion of adenine to inosine, or radioenzymatically by measuring the radioactivity of inosine formed from [8-14C]adenine, after chromatographic separation of the nucleoside on polyethyleneimine-cellulose. The spectrophotometric assay was used to follow ribose 5-phosphate formation and ribose 1-phosphate consumption catalyzed by phosphopentomutase. Further, the ability of alkaline phosphatase, 5-nucleotidase and crude extract of Bacillus cereus cells to act on ribose 5-phosphate was tested. The radioenzymatic assay was proved useful in determining the levels of ribose 5-phosphate in rat tissues.

Enzymatic synthesis of [U-14C] ribose-labeled inosine

Abstract A convenient enzymatic method to synthesize inosine specifically labeled in the ribose moiety is presented. The synthesis is effected in two stages subjecting [U-14C]inosine to the action of: (1) purine-nucleoside phosphorylase, leading to the formation of [U-14C]ribose 1-phosphate, and then (2) adenosine phosphorylase and adenosine deaminase plus unlabeled adenine, which permits the reformation of inosine containing the labeled ribose formed in the first stage.

A coupled optical assay for determination of adenine in mixtures.

Abstract A rapid and specific spectrophotometric assay for the determination of adenine is described. The method is based on the absorbance change at 265 nm which accompanies the ribose 1-phosphate-dependent conversion of adenine into inosine, catalyzed by the successive action of adenosine phosphorylase and adenosine deaminase. Common purine and pyrimidine bases, nucleosides, and nucleotides do not interfere. The assay was tested in various biochemical situations, in which there was both adenine formation and utilization.