Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells.

Uptake and catabolism of purine nucleosides have been commonly considered as means to salvage the purine ring for nucleic acid synthesis, usually neglecting the destiny of the pentose moiety. With the aim to ascertain if deoxyribose derived from exogenous DNA can be utilised as a carbon and energy source, we studied the catabolism of exogenous deoxyinosine in a cell line derived from human amnion epithelium (WISH). Intact WISH cells catabolise deoxyinosine by conversion into hypoxanthine. The nucleoside enters the cell through a nitrobenzylthioinosine-insensitive equilibrative transport. Deoxyinosine undergoes a phosphorolytic cleavage inside the cell. The purine base diffuses back to the external medium, while the phosphorylated pentose moiety can be further catabolised to glycolysis and citric acid cycle intermediates. Our results indicate that the catabolism of the deoxynucleoside can be considered mainly as a means to meet the carbon and energy requirements of growing cells.

Bovine cytosolic 5'-nucleotidase acts through the formation of an aspartate 52-phosphoenzyme intermediate.

Cytosolic 5'-nucleotidase/phosphotransferase (cN-II), specific for purine monophosphates and their deoxyderivatives, acts through the formation of a phosphoenzyme intermediate. Phosphate may either be released leading to 5'-mononucleotide hydrolysis or be transferred to an appropriate nucleoside acceptor, giving rise to a mononucleotide interconversion. Chemical reagents specifically modifying aspartate and glutamate residues inhibit the enzyme, and this inhibition is partially prevented by cN-II substrates and physiological inhibitors. Peptide mapping experiments with the phosphoenzyme previously treated with tritiated borohydride allowed isolation of a radiolabeled peptide. Sequence analysis demonstrated that radioactivity was associated with a hydroxymethyl derivative that resulted from reduction of the Asp-52-phosphate intermediate. Site-directed mutagenesis experiments confirmed the essential role of Asp-52 in the catalytic machinery of the enzyme and suggested also that Asp-54 assists in the formation of the acyl phosphate species. From sequence alignments we conclude that cytosolic 5'-nucleotidase, along with other nucleotidases, belong to a large superfamily of hydrolases with different substrate specificities and functional roles.

Role of the phosphorolysis of deoxyadenosine in the cytotoxic effect of the combination of deoxyadenosine and deoxycoformycin on a human colon carcinoma cell line (LoVo).

In LoVo cells, phosphorolytic activity acting on deoxyadenosine plays a major role in the resistance to the cytotoxic effect of the combination of deoxynucleoside with deoxycoformycin. In fact, the observed dependence of toxicity on cell density appears to be related to the metabolic conversion of deoxyadenosine into adenine. The phosphorylation of the deoxynucleoside, which represents the first step towards the formation of the cytotoxic agent dATP, proceeds at a significantly lower rate as compared to the phosphorolysis of deoxyadenosine. The analysis of the levels of deoxyadenosine and its derivatives in the incubation media reveals that the rates of disappearance of deoxyadenosine and of formation of adenine increase in concert with the reduction of the effect on cell survival.

6-thioguanine resistance in a human colon carcinoma cell line with unaltered levels of hypoxanthine guanine phosphoribosyltransferase activity.

Cell populations resistant to high doses (30 microM) of 6-thioguanine (6-TG, 6-TG(r) cells) were selected from a human colon carcinoma cell line, LoVo. This cell line, which lacks hMSH2, a component of the human mismatch binding heterodimer hMutSalpha, is resistant to low doses of 6-TG. The level of activity of hypoxanthine-guanine phosphoribosyltransferase, the enzyme responsible for the phosphoribosylation of the thiopurine, was comparable to that expressed in the parental cells. No significant difference was found in the levels of enzyme activities involved in the conversion of 6-TG or its derivatives into non-toxic compounds. In contrast, a significant difference was found in the uptake kinetics of 6-TG in the 2 cell types. Net uptake of 6-TG ceased after 100-sec incubation in the 6-TG(r) cells, while it appeared to continue throughout the 10-min incubation in the wild-type cells. As a consequence, after 10-min incubation, the total amount of 6-TG taken up by the parental LoVo cells was approximately 3 times higher than that present in the 6-TG(r) cells.

Identification, separation and characterisation of two forms of cytosolic 5'-nucleotidase/nucleoside phosphotransferase in calf thymus.

Cytosolic 5'-nucleotidase, acting preferentially on IMP, GMP and their deoxyderivatives, endowed with phosphotransferase activity, is a widespread enzyme responsible for the regulation of intracellular IMP and GMP concentrations and the phosphorylation of purine nucleoside pro-drugs. The enzyme activity is stimulated by ATP, ADP and 2,3-bisphosphoglycerate (BPG), and is inhibited by phosphate. Calf thymus possesses two active proteins with a different electrophoretic mobility. In this report we show that the two forms can be separated by ADP-agarose affinity chromatography. Whereas form A binds weakly to the column, form B is tightly bound and is released by the addition of ADP into the elution buffer. The two enzyme forms differ in terms of electrophoretic, chromatographic behaviour and regulatory characteristics. Form B, as already described for the enzyme purified from the same source (Pesi et al., 1996, Biochim Biophys Acta 294, 191-194), exhibits three different sites for the three activators with a synergistic effect between ADP and BPG. Form A has a high affinity regulatory site for BPG, while ADP and ATP appear to share the same low affinity site and no synergistic effect is observed.

Deoxyadenosine metabolism in a human colon-carcinoma cell line (LoVo) in relation to its cytotoxic effect in combination with deoxycoformycin.

We have assessed the intracellular metabolism of 2'-deoxyadenosine in a human colon-carcinoma cell line (LoVo), both in the absence and in the presence of deoxycoformycin, the powerful inhibitor of adenosine deaminase. The combination of 2'-deoxyadenosine and deoxycoformycin has been reported to inhibit the growth of LoVo cells in culture. In this paper we demonstrate that the observed toxic effect is strictly dependent on cell density. In the absence of deoxycoformycin, 2'-deoxyadenosine is primarily deaminated to 2'-deoxyinosine and then converted into hypoxanthine. In the presence of the inhibitor, the deoxynucleoside, in addition to a phosphorylation process, undergoes phosphorolytic cleavage giving rise to adenine. The conversion of 2'-deoxyadenosine to adenine might represent a protective device, emerging when the activity of adenosine deaminase is reduced or inhibited. There is much evidence to indicate that the enzyme catalyzing this process may be distinct from methylthioadenosine phosphorylase and S-adenosyl homocysteine hydrolase, which are the enzymes reported to be responsible for the formation of adenine from 2'-deoxyadenosine in mammals.

Channelling of deoxyribose moiety of exogenous DNA into carbohydrate metabolism: role of deoxyriboaldolase.

In bacteria, the addition of (deoxy)nucleosides or (deoxy)ribose to the growth medium causes induction of enzymes involved in their catabolism, leading to the utilisation of the pentose moiety as carbon and energy source. In this respect, deoxyriboaldolase appears the key enzyme, allowing the utilisation of deoxyribose 5-P through glycolysis. We observed that not only deoxynucleosides, but also DNA added to the growth medium of Bacillus cereus induced deoxyriboaldolase; furthermore, the switch of the culture from aerobic to anaerobic conditions caused a further increase in enzyme activity, leading to a more efficient channelling of deoxyribose 5-P into glycolysis, probably as a response to the low energy yield of the sugar fermentation. In eukaryotes, the catabolism of (deoxy)nucleosides is well known. However, the research in this field has been mainly devoted to the salvage of the bases formed by the action of nucleoside phosphorylases, whereas the metabolic fate of the sugar moiety has been largely neglected. Our results indicate that the deoxyriboaldolase activity is present in the liver of several vertebrates and in a number of cell lines. We discuss our observations looking at the nucleic acids not only as informational molecules, but also as a not negligible source of readily usable phosphorylated sugar.

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.

Deoxyribose 5-phosphate aldolase of Bacillus cereus: purification and properties.

Deoxyribose 5-phosphate aldolase was purified 41 times from Bacillus cereus induced by growth on deoxyribonucleosides. The purification procedure includes ammonium sulphate fractionation, gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and preparative electrophoresis on 10% polyacrylamide gel. The enzyme is stable above pH 6.5, but is rapidly inactivated by sulfhydryl reagents. Being insensitive to EDTA, it may be considered as a Class I aldolase. Among a number of compounds tested (including some carboxylic acids, free and phosphorylated pentoses, nucleotides and nucleosides), none has been found to affect the enzyme activity. The enzyme appears to be dimeric, with a subunit Mr of 23,600. A Km of 4.4 x 10(-4) M was calculated for dRib 5-P.

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.

Purification and characterization of the larvicidal toxin of Bacillus sphaericus 1593M.

We present here a procedure for purifying the larvicidal toxin from sporulating cells of Bacillus sphaericus 1593M and describe some of the biochemical and biophysical properties of this toxin. The procedure involves solubilization of the cell-wall/membrane bound toxin by sonication of cells followed by repeated rounds of freezing and thawing at 50 degrees C. Further purification involved Sephadex G-100 and DEAE Sephacel chromatography. We show by Sephadex G-100 chromatography that at pH 7.5 the smallest active form of the toxin has an Mr of 38,000 and that this toxin can reversibly aggregate to molecular forms of a size higher than 2 X 10(5) Mr. By shifting the pH from 7.5 to 8.5 only the aggregated forms can be observed.

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.

Induction of deoxyribose-5-phosphate aldolase of Bacillus cereus by deoxyribonucleosides.

In Bacillus cereus purine ribonucleosides and deoxyribonucleosides share a common inducible catabolic pathway, leading to the formation of ribose-5-P or deoxyribose-5-P respectively inside the cell, while the purine ring remains in the external medium. Both ribo- and deoxyribonucleosides are inducers of adenosine deaminase, inosine-guanosine phosphorylase and phosphopentomutase, the enzymes of the catabolic pathway. We now show that deoxyribonucleosides, but not ribonucleosides, induce the aldolase specific for deoxyribose-5-P (2-deoxy-D-ribose-5-phosphate acetaldehyde lyase, EC 4.1.2.4), thus allowing the sugar moiety of exogenous deoxyribonucleosides to be utilized as an energy source.

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).

Preliminary characterization of adenosine deaminase from Bacillus cereus.

Adenosine deaminase from Bacillus cereus is quite unstable, similarly to other bacterial deaminases, but it shows a peculiar stabilizing effect by some monovalent cations. These include K+, Li+, NH4+ and to a lesser extent Cs+. Maximal stabilization of the deaminase is exerted by K+ at concentrations higher than 20 mM. The enzyme can be rapidly inactivated by sulphydryl reagents such as p-hydroxymercuribenzoate. Since adenosine deaminase from B. cereus, in addition to monovalent cations, is stabilized also by dithiothreitol, a possible influence of monovalent cations on the reactivity of some sulphydryl groups on the enzyme has been suggested.

Induction and repression of enzymes involved in exogenous purine compound utilization of 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.

Utilization of exogenous purine compounds in Bacillus cereus. Translocation of the ribose moiety of inosine.

Intact cells of Bacillus cereus catalyze the breakdown of exogenous AMP to hypoxanthine and ribose 1-phosphate through the successive action of 5'-nucleotidase, adenosine deaminase, and inosine phosphorylase. Inosine hydrolase was not detectable, even in crude extracts. Inosine phosphorylase causes a "translocation" of the ribose moiety (as ribose 1-phosphate) inside the cell, while hypoxanthine remains external. Even though the equilibrium of the phosphorolytic reaction favors nucleoside synthesis, exogenous inosine (as well as adenosine and AMP) is almost quantitatively transformed into external hypoxanthine, since ribose 1-phosphate is readily metabolized inside the cell. Most likely, the translocated ribose 1-phosphate enters the sugar phosphate shunt, via its prior conversion into ribose 5-phosphate, thus supplying the energy required for the subsequent uptake of hypoxanthine in B. cereus.