Adenosine Phosphorylase Research Articles

Specific adenosine phosphorylase from hepatopancreas of gastropod Helix pomatia

1. 1. Specific adenosine phosphorylase from Helix pomatia hepatopancreas was separated from inosine-guanosine phosphorylase and purified 100–165 times; molecular weights were found to be 71,000 and 90,000, respectively. 2. 2. The enzyme is specific for deoxy- and adenosine; it is inactive for 5′-methylthioadenosine and 5-amino-4-imidazole-carboxyamide riboside. Its sensitivity to several inhibitors differs from that of eucaryotic and bacteriol purine nucleoside phosphorylases, and is not identical with the sensitivity of the S. mansoni adenosine-splitting enzyme. 3. 3. H. pomatia adenosine phosphorylase differs in its mol. wt and K m values for P i, ribose IP, Ado and Ade from adenosine phosphorylase of B. subtilis.

Targeting 5'-deoxy-5'-(methylthio)adenosine phosphorylase by 5'-haloalkyl analogs of 5'-deoxy-5'-(methylthio)adenosine

A series of 5'-haloalkyl-modified analogues of 5'-deoxy-5'-(methylthio)adenosine (MTA), a nucleoside byproduct of polyamine biosynthesis, has been synthesized: 5'-deoxy-5'-[(2-monofluoroethyl)thio]adenosine (10), 5'-deoxy-5'-[(2-chloroethyl)thio]adenosine (4), 5'-deoxy-5'-[(2-bromoethyl)thio] adenosine (5), and 5'-deoxy-5'-[(3-monofluoropropyl)thio]adenosine (13). On the basis of their abilities to serve as substrates of MTA phosphorylase prepared from mouse liver, several of these analogues were characterized for their growth inhibitory effects in MTA phosphorylase-containing (murine L5178Y and human MOLT-4) and MTA phosphorylase-deficient (murine L1210 and human CCRF-CEM) leukemia cell lines. The MTA phosphorylase-containing tumor cell lines, especially of human origin, were found to be more sensitive to treatment by these analogues. Of the analogue series, 10 was the most potent inhibitor of growth in each of the cell lines tested. The analogues, especially compound 10, displayed a reduced capacity to alter polyamine pools relative to MTA, mechanistically indicating a decreased potential for interactions at sites other than MTA phosphorylase. The results indicate that of the analogues tested, compound 10 displayed the best inhibitor/substrate interaction with MTA phosphorylase, which, in turn, correlated with more potent growth inhibition in tumor cell lines containing MTA phosphorylase. Overall, this supports the concept that MTA phosphorylase plays a role in the activation of such analogues.

Mycoplasma contamination greatly enhances the apparent transoport and concentrative accumulation of formycin B by mammalian cell culture

S49 mouse leukemia cells exhibit both equilibrative and Na +-dependent, concentrative formycin B transport. The latter represents only a minor nucleoside transport component and is detectable only when equilibrative nucleoside transport is inhibited by dipyridamole or another transport inhibitor. Thus in uncontaminated S49 cells formycin B accumulated only to slightly above the intracellular equilibrium level. In contrast, in suspensions of S49 cells contaminated with mycoplasma, formycin B accumulated in the intracellular water space in unmodified form to 40–50-times the extracellular concentration in a dipyridamole-independent manner during 90 min of incubation at 37°C. The mycoplasma active formycin B transport system was inhibited by all nucleosides tested, including thymidine and deoxycytidine, which are not substrates for the concentrative nucleosid transporter of S49 cells. Mycoplasma contamination was detected by the presence of cell-associated adenosine phosphorylase activity.

Mycoplasma and Epidemic Group A Meningococcal Meningitis-Reply

In Reply. — As Drs Kenny and Foy point out, mycoplasma contamination of cell cultures is a common and insidious problem. We are confident that the M hominis isolated in our study came from the specimens and not the cell cultures due to the precautions taken to avoid and detect contamination. Ten uninoculated control cell cultures of each cell type were passaged in parallel with each batch of inoculated cultures; none of these controls became positive. As part of our quality assurance policies, each cell line is also tested monthly for mycoplasma at the Centers for Disease Control using an adenosine phosphorylase test (Mycotect, Gibco) and at the American Type Culture Collection (Rockville, Md) using both direct culture on Hayflick's medium and a DNA fluorescent staining test. 1,2 All three tests were consistently negative throughout the study period. Furthermore, when study specimens were positive for mycoplasma, all four cell types

Mycoplasma contamination alters 2′‐deoxyadenosine metabolism in deoxycoformycin‐treated mouse leukemia cells

Deoxycoformycin-treated P388 and L1210 mouse leukemia cells salvage 2'-deoxyadenosine from the medium only inefficiently, because deoxyadenosine deamination is blocked and its phosphorylation is limited by feedback controls. Mycoplasma contamination at a level that had no significant effect on the growth of the cells increased the salvage of deoxyadenosine greater than 10 fold over a 90 min period of incubation at 37 degrees C, but in this case deoxyadenosine was mainly incorporated into ribonucleotides and RNA via adenine formed from deoxyadenosine by mycoplasma adenosine phosphorylase. Deoxyadenosine was an efficient substrate for this enzyme, in contrast to 2',3'-dideoxyadenosine which was not phosphorolyzed. Mycoplasma infection was confirmed by the presence of uracil phosphoribosyltransferase activity and by culture isolation. The contaminant has been identified as Mycoplasma orale. Mycoplasma infection had no effect on the deamination and phosphorylation of deoxyadenosine and adenosine, on the salvage of hypoxanthine and adenine, or on the degradation of dAMP and dATP by the cells or on their acid and alkaline phosphatase activities.

Adenine salvage enzymes and intracellular nucleotide triphosphate content in Physarum flavicomum amoebae during growth and development

The specific activity of adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) and adenosine phosphorylase (EC 2.4.2.-), two enzymes involved in the utilization of exogenous adenine, was measured in extracts of myxamoebae-swarm cells of Physarum flavicomum undergoing growth, microcyst formation (control), and during adenine inhibition of encystment. Both enzymes showed a higher specific activity in adenine-inhibited cells (AIC) compared to normal control (NC) or growing cells (GC). These experiments revealed that the specific activity of APRT was 7.1-, 5.3-, and 1.7-fold higher than that of adenosine phosphorylase in AIC, GC, and NC, respectively. This suggests a predominant role for the enzyme APRT in the salvage of adenine in this organism. The major route for the utilization of adenine thus seems to be by its direct conversion to AMP rather than via its riboside adenosine. HPLC analysis of the ribonucleotide triphosphates in cell extracts of GC, NC, and AIC revealed a 2.6- and a 3.3-fold increase in the ATP and GTP content, respectively, in the AIC compared with the NC cells. The ATP content in the GC was higher by a factor of 2.2 compared with the NC cells, while the GTP content in the GC was only 0.6 times that in the NC cells. UTP levels in AIC and GC were 1.3- and 1.4-fold higher than in the NC cells. In contrast, the CTP level in AIC was lower than in NC cells and was not detectable in the growing cells.

Indicator cell lines for the detection of hidden mycoplasma contamination, using an adenosine phosphorylase screening test

Mycoplasmas are a major cause of cell culture contamination and are especially troublesome during HAT selection. The enzyme adenosine phosphorylase (adoP) is present in all common mycoplasma species but is considered to have a low activity in mammalian cells. However, using an adoP screening test, we have observed that some cell cultures do possess an intrinsic adoP activity leading to false positive results. Moreover, as a false negative result, we encountered a variant of Mycoplasma orale (identified after cultivation on agar and immunostaining) which was not detectable with the adoP screening in cell culture supernatants and only at low levels in cell lysates. To increase the low signal/noise adoP ratio found there, we used an indicator cell line with low intrinsic activity. Indicator cells were inoculated with the test supernatant and the adoP activity of these infected cells were measured after lysis. The procedure diminished the effect of biological variation in intrinsic enzyme activity between the several cell lines tested. Furthermore, in another mycoplasma infected cell line (with M. fermentans), this infection was only reliably detected using these indicator cells. With this procedure we obtained rapid results which were concordant with those obtained using the time consuming cultivation on agar.

Effect of larval trematode infection on AMP catabolism in the freshwater pulmonate snail Lymnaea luteola

Activities of the AMP-catabolizing enzymes adenosine phosphorylase, adenosine deaminase, and adenine deaminase and their alterations on larval trematode infection were observed in all the major tissues of Lymnaea luteola. No significant change was seen in the activity of adenosine phosphorylase. Though the activities of adenosine deaminase and adenine deaminase were elevated on infection, the former was significant only in mantle tissue and the latter was significant only in digestive gland tissue, and not in other tissues. Based on the enzyme activity ratios, it was found that there was a tendency for adenosine levels to rise in the infected digestive gland, while its levels decrease in other tissues. It is suggested that adenosine may be one of the larval trematode requirements met by the host snail.

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.

Radioenzymatic determination of adenosine

Adenosine has been measured at the nanomolar level by an enzymatic radioactive assay. The nucleoside is converted into [U- 14C]ribose-labeled inosine via the following reactions: adenosine + H 2O → adenine + ribose (adenosine nucleosidase); adenine + [U- 14C]ribose 1-phosphate noneth [U- 14C]ribose-adenosine + P i (adenosine phosphorylase); [U- 14C]ribose-adenosine + H 2O → [U- 14C]ribose-inosine + NH 3 (adenosine deaminase). The radioactivity of inosine, separated by thin-layer chromatography, is a measure of the adenosine initially present.

Genetic and physiological characterization of Bacillus subtilis mutants resistant to purine analogs.

Bacillus subtilis mutants defective in purine metabolism have been isolated by selecting for resistance to purine analogs. Mutants resistant to 2-fluoroadenine were found to be defective in adenine phosphoribosyltransferase (apt) activity and slightly impaired in adenine uptake. By making use of apt mutants and mutants defective in adenosine phosphorylase activity, it was shown that adenine deamination is an essential step in the conversion of both adenine and adenosine to guanine nucleotides. Mutants resistant to 8-azaguanine, pbuG mutants, appeared to be defective in hypoxanthine and guanine transport and normal in hypoxanthine-guanine phosphoribosyltransferase activity. Purine auxotrophic pbuG mutants grew in a concentration-dependent way on hypoxanthine, while normal growth was observed on inosine as the purine source. Inosine was taken up by a different transport system and utilized after conversion to hypoxanthine. Two mutants resistant to 8-azaxanthine were isolated: one was defective in xanthine phosphoribosyltransferase (xpt) activity and xanthine transport, and another had reduced GMP synthetase activity. The results obtained with the various mutants provide evidence for the existence of specific purine base transport systems. The genetic lesions causing the mutant phenotypes, apt, pbuG, and xpt, have been located on the B. subtilis linkage map at 243, 55, and 198 degrees, respectively.

Mechanism of adenosine production by xanthine-requiring mutants derived from a Bacillus strain

The xanthine-requiring mutants defective in adenine deaminase (adenase) derived from a Bacillus strain accumulate much adenosine. The mechanism of adenosine production was investigated. Limitation of the guanine-related substances in the fermentation medium facilitated the adenosine accumulation, but the excess of those suppressed it. Metabolic regulation of the purine nucleotide biosynthesis was supposed to be released from both feedback inhibition and repression by limiting the concentration of guanine-related substances in the cells caused by xanthine-requirement. Deficiency in the deaminase activities of adenine, adenosine and AMP and the weak adenosine phosphorylase activity contributed to adenosine accumulation. No apparent changes were observed in the adenylosuccinate synthetase activity and the dephosphorylation activity of AMP compared with the wild strain.

Adenosine accumulation in Saccharomyces cerevisiae cultured in medium containing low levels of adenine.

By monitoring the in vivo incorporation of low concentrations of radiolabeled adenine into acid-soluble compounds, we observed the unusual accumulation of two nucleosides in Saccharomyces cerevisiae that were previously considered products of nucleotide degradation. Under the culture conditions used in the present study, radiolabeled adenosine was the major acid-soluble intracellular derivative, and radiolabeled inosine was initially detected as the second most prevalent derivative in a mutant lacking adenine aminohydrolase. The use of yeast mutants defective in the conversion of adenine to hypoxanthine or to AMP renders very unlikely the possibility that the presence of adenosine and inosine is attributable to nucleotide degradation. These data can be explained by postulating the existence of two enzyme activities not previously reported in S. cerevisiae. The first of these activities transfers ribose to the purine ring and may be attributable to purine nucleoside phosphorylase (EC 2.4.2.1) or adenosine phosphorylase (EC 2.4.2.-). The second enzyme converts adenosine to inosine and in all likelihood is adenosine aminohydrolase (EC 3.5.4.4).

Action of tubercidin and other adenosine analogs on Schistosoma mansoni schistosomules

The incorporation of the radiolabeled adenosine analogs tubercidin, formycin A, 9-deaza-adenosine, and adenine arabinoside into nucleotides of Schistosoma mansoni schistosomules was studied in vitro. Of the four analogs, only tubercidin and formycin A were incorporated into the nucleotide pool, at rates respectively one-tenth and one-fiftieth the rate of adenosine incorporation. Tubercidin inhibited schistosomule motility in vitro with an approximate IC 50 value of 1 μM, whereas formycin A exerted no visible effect even when more of it than of tubercidin was incorporated into the nucleotides and nucleic acids. Formycin A thus acts like a nontoxic adenosine analog. 7-Deaza-adenine, the purine base of tubercidin, was not incorporated into nucleotides. 7-Deaza-adenine, 9-deaza-adenosine, and adenine arabinoside all had no effect on schistosomule motility at concentrations up to 100 μM. Formycin A blocked the incorporation of tubercidin and of adenosine with equal effectiveness, as did p-nitrobenzyl-6-mercaptopurine ribonucleoside, a specific inhibitor of nucleoside transport in many mammalian cells. Thus, formycin A, tubercidin, and adenosine appear to have a common mechanism of cellular uptake. The significant levels of adenosine phosphorylase and adenine phosphoribosyl transferase activity found in schistosomule extracts suggests that most of the transported adenosine is converted to adenine before conversion to AMP. The levels of adenosine kinase and tubercidin kinase, while low, can more than account for the rate of tubercidin incorporated into intact schistosomules. The kinase(s) may also represent a minor pathway for direct adenosine incorporation. It may have a rather unusual substrate specificity because it is able to recognize adenosine, tubercidin, and formycin A as substrates, but not 9-deaza-adenosine or adenine arabinoside.

Spectrophotometric and radioenzymatic determination of ribose 5-phosphate

The present work describes an assay which is higly specific or fibose 5-phosphate. The method is based on the following three-stage enzymatic conversion: (1) ribose 5-phosphate ⇌ ribose 1-phosphate associated with the conversion of adenine to inosine, or radioenzymatically by measuring the radioactivity (phosphopentomutase); (2) ribose 1-phosphate + adenine ⇌ adenosine + P i (adenosine phosphorylase); (3) adenosine + H 2O → inosine + NH 3 (adenosine deaminase). Ribose 5-phosphate may be detemined 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 inosing formed form [8- 14C]adenine, after chromatrographic separation on polyethy-eneimine-cellulose. The spectrophotometric assay was used to follow ribose 5-phosphate formation and ribose 1-phosphagte consumption catalyzed by phosphopentomutase. Further, the ability of alkaline phospatase, 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.

Deoxyribose 1-phosphate: radioenzymatic and spectrophotometric assays

A method has been developed to measure deoxyribose 1-phosphate in the presence of ribose 1-phosphate and other sugar phosphates. The specificity of the method is based on the observation that only deoxyribose 1-phosphate is hydrolyzed by heating at pH 7.4, while both deoxyribose 1-phosphate and ribose 1-phosphate remain unchanged when heated at pH 10. A tissue extract is heated at pH 10. The amount of deoxyribose 1-phosphate plus ribose 1-phosphate is determined from that of deoxyinosine plus inosine formed in a coupled enzymatic reaction, based on the following two-stage transformation: deoxyribose 1-phosphate (ribose 1-phosphate) + adenine ⇋ deoxyadenosine (adenosine)+inorganic phosphate, catalyzed by adenosine phosphorylase; deoxyadenosine (adenosine)+H 2O → deoxyinosine (inosine), catalyzed by adenosine deaminase. By taking advantage of its unique heat lability, deoxyribose 1-phosphate is eliminated by heating the tissue extract at pH 7.4, and ribose 1-phosphate is determined as above. The amount of deoxyribose 1-phosphate stems from the difference between the amount of deoxyinosine plus inosine plus inosine measured in the tissue extract heated at pH 10 and that of inosine measured in the tissue extract heated at pH 7.4. Free deoxyribose 1-phosphate has been found in rat tissues, as well as in Bacillus cereas during stationary phase of growth.

Adenosine phosphorylase activity as a technique for detection of mycoplasmas in biological media

The importance of cell culture contamination by mycoplasmas is well recognized, but the means used to detect such contamination still need improvement. Most mycoplasmas possess an enzyme, adenosine phosphorylase, which is not found in cell lines. We used the ultramicromethod of Uitendaal et al. to detect the presence of mycoplasmas in sera and in tissue culture medium. The absence of adenosine phosphorylase activity seems to be the best guarantee that a serum is not contaminated by mycoplasmas. This test is also most efficient for the detection of mycoplasmas in tissue or cell cultures in vitro.

Adenosine phosphorylase activity in mycoplasma-free growth media for mammalian cells

Mammalian cells have enzymes that deaminate adenosine to inosine, which can readily be phosphorolysed to hypoxanthine. They do not, however, possess enzymes to form adenine by the cleavage of adenosine. For this reason, the release of adenine from adenosine by mammalian cell cultures has usually been interpreted as indicating the presence of mycoplasma, a frequent microbial contaminant that contains high levels of adenosine phosphorylase. We found that some human lymphoblast cultures free of mycoplasma showed high levels of adenosine cleavage and that this activity resulted from adenosine phosphorylase in the bovine serum used as the culture growth supplement. A survey of 13 serum supplements disclosed that fetal bovine serum (six lots) contains the highest adenosine phosphorylase activity, ranging from 9 to 648 nmol adenine produced per hour per ml serum; newborn calf serum (four lots) has much less activity, ranging from 0 to 5 nmol adenine produced per hour per ml serum; and donor horse serum (three lots) contains no detectable activity. These results suggest that mycoplasma tests dependent on the presence of adenosine phosphorylase or other enzyme activities may give false-positives with cultures containing fetal bovine serum supplements.

A coupled optical enzyme assay for phosphopentomutase

Published assays for phosphopentomutase activity are based on acid lability differences between ribose 1-phosphate and ribose 5-phosphate. The present work describes a new method in which the isomerization of ribose 5-phosphate to ribose 1-phosphate is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: ribose 1-phosphate + adenine ⇌ phosphate + adenosine (adenosine phosphorylase); adenosine + H 2O → inosine + NH 3 (adenosine deaminase). The method has been used to show some properties of Escherichia coli phosphopentomutase.

Adenosine phosphorylase-mediated nucleoside toxicity: Application towards the detection of mycoplasmal infection in mammalian cell cultures

Adenosine phosphorylase (adenosine: orthophosphate ribosyltransferase) activity is low in mammalian cells, but is abundant in certain prokaryotes. It is an appropriate target enzyme for the development of diagnostic and chemotherapeutic agents. Adenosine phosphorylase from Bacillus subtilis and mycoplasmas that commonly infect cell cultures converted the non-toxic deoxyadenosine analog, 6-methylpurine deoxyriboside (6MPDR) into the potent anti-metabolites 6-methylpurine and 6-methylpurine riboside. Consequently 6MPDR selectively killed mammalian cell cultures infected with mycoplasmas. 6MPDR was cytotoxic to 87 of 90 mycoplasma-infected cultures (96.6%). No toxicity was observed in nine different types of mycoplasma-free cell cultures. Use of a 3T6 mouse embryo fibroblast indicator cell culture improved standardization. Cytotoxicity was apparent 3–4 days after inoculation of 10 μM of 6MPDR to 3T6 cultures infected with the following mycoplasmas: M. hyorhinis, M. orale, M. arginini, M. salivarium, A, laidlawii, M. hominis, M. fermentons, M. sp. 70–159 and M. buccale. Cytotoxicity was produced in 28/28 3T6 indicator cultures inoculated with mycoplasma-infected cell cultures in the presence of 10 μM 6MPDR. The analog is apparently non-toxic to the mycoplasmas.