Pier Luigi Ipata

α-5-Phosphoribosyl-1-pyrophosphate-independent salvage of purines in cultured chinese hamster lung fibroblasts

A variant clone of cultured chinese hamster lung fibroblasts (V79), selected for resistance to 8-azaguanine (V79 azagrst), although lacking hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), is able to convert hypoxanthine into IMP via purine-nucleoside phosphorylase (EC 2.4.2.1) and nucleoside kinase. In addition to the phosphoribosylation pathway, we also present evidence for the occurrence of a kinase-mediated pathway of recovery of hypoxanthine in the wild-type cells. The lower rate of formation of IMP in the V79 azagrst cells, apparently correlated with the phosphorylation of the nucleoside, suggests possible differences in the catalytic and/or regulatory properties of nucleoside kinase in the two cell lines. This fact might be of particular relevance in evaluating the mechanisms of resistance to purine analogs displayed by several cell types.

Phosphorylase-mediated mobilization of the amino group of adenine in Bacillus cereus

Mobilization of the ribose moiety of purine nucleosides as well as of the amino group of adenine may be realized in Bacillus cereus by the concerted action of three enzymes: adenosine phosphorylase, adenosine deaminase, and purine nucleoside phosphorylase. In this pathway, ribose-1-phosphate and inorganic phosphate act catalytically, being continuously regenerated by purine nucleoside phosphorylase and adenosine phosphorylase, respectively. As a result of such a metabolic pathway, adenine is quantitatively converted into hypoxanthine, thus overcoming the lack of adenase in B. cereus.

Characterization of the specificity of intracellular phosphodiesterases in Bacillus subtilis.

Abstract Previously described methods for the spectrophotometric determination of phosphodiesterases (orthophosphoric diester phosphohydrolase, EC 3.1.4.1) [P.L. Ipata and R.A. Felicioli, Eur. J. Biochem., 8 (1969) 174] have been employed to characterize intracellular phosphodiesterase in Bacillus subtilis during sporulation. Both 3′- and 5′-nucleoside monophosphate producing phosphodiesterases were found in vegetative and sporulating forms. In free spores the 3′-nucleoside monophosphate producing phosphodiesterase activity could not be detected with the methods employed.

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.

Breakdown of adenosine and inosine nucleotides in bone at physiological pH

Abstract The breakdown of adenosine and inosine nucleotides and nucleotides have been studied in articular and epiphyseal cartilage, epiphyseal and metaphyseal cancellous bone, diaphyseal compact bone and periosteum. Dephosphorylating, deaminating, aminating and adenylate-kinase activities have been demonstrated.