Adenosine phosphorylase

The lethal effect of mycoplasmas on cell cultures can be accentuated by culture in the presence of 6-methylpurine deoxyribonucleoside (6-MPDR) and this forms the basis of the MycoTect kit available from Gibco. All mycoplasmas possess high levels of adenosine phosphorylase which converts the non-toxic 6-MPDR into 6-methylpurine and 6-methylpurine ribonucleoside, both of which are toxic to mammalian cells (McGarrity and Carson, 1982). 

Add 0.2 ml test sample to a well and incubate for 24 h. It is suggested that some wells are left as negative controls and to others 0.2 ml of a solution of adenosine phosphorylase is added to act as a positive control. This still leaves room to test five different cell lines using the single 24-well plate. 

Few detailed studies have been done on the purine salvage enzymes of procyclic African trypanosomes. Tb. gambiense has high levels of guanine deaminase and lacks adenine and adenosine deaminase activities (8). Tb. brucei, T.b. gambiense and T.b. rhodesiense convert allopurinol into aminopyrazolopyrimidine nucleotides and incorporates these into RNA (49). This indicates that HPRTase, succino-AMP synthetase, and succino-AMP lyase are present. At least three nucleoside cleavage activities are present (Berens, unpublished results) two are hydrolases, of which one is specific for purine ribonucleosides and the other is specific for purine deoxyribonucleosides. The third nucleoside cleavage activity is a methylthioadenosine/adenosine phosphorylase. The adenosine kinase is similar to that of L. donovani (Berens, unpublished results). 

There is a relatively high activity of adenosine phosphorylase and low levels of adenosine kinase, deaminase and phosphotransferase (66). The adenosine kinase is responsible for incorporation of adenosine analogs such as tubercidin (67). 

Nucleotidase [15] followed by adenosine nucleosidase [16] are expected to be the enzymes responsible for the step-by-step conversion of the cytokinin nucleotide to the base iPA. Both of these reactions may proceed also in the opposite direction, and in this case they are catalysed by adenosine phosphorylase (ribosylation of iPA, [17]) and adenosine kinase (phosphorylation of iPAR, [18-20]). These enzymes are common in the mutual conversions of adenine and purine metabolites (reviewed in [21]) and their properties have been summarised by [22]. These enzyme activities seem to be the key for understanding the fate of C-labelled adenine (Ade) and adenosine (Ado) in feeding experiments [summarised by 23]. 

Generally, all conversions in the biosynthetic direction, i.e. iPARMP— iPAR— iPA (catalysed by 5 -nucleotidase, (EC 3.1.3.5), and adenosine nucleosidase, (EC 3.2.2.7), respectively, c/. Fig. 2) may also proceed in the opposite direction, i.e. base-— nucleoside — nucleotide (catalysed by adenosine phosphorylase and adenosine kinase, respectively). All these enzymes require both Ade and iPA or Ado and iPAR, respectively, as substrates. They were characterised in wheat germ [15-18] and lupin seeds [19]. Interestingly, no K, -constants were reported for Z-type cytokinins (see summary in [22]). However, as seen in H-labelled Z-derivatives feeding experiments, Z-type cytokinins are also interconverted in a similar way [82,121,122]. Moreover, the specificity of these enzymes is not too strict with respect to the side chain configuration and one may speculate that this complex may function for most if not all native cytokinins [21,81]. 

Brewerton, L.J. et al., Polyethylene glycol-conjugated adenosine phosphorylase Development of alternative enzyme therapy for adenosine deaminase deficiency, Biochim. Biophys. Acta, 1637, 171, 2003. 

Fig. 20.3 Pathway of methionine metabolism. The numbers represent the following enzymes or sequences (1) methionine adenosyltransferase (2) S-adenosylmethionine-dependent transmethylation reactions (3) glycine methyltransferase (4) S-adenosylhomocysteine hydrolase (5) betaine-homocysteine methyltransferase (6) 5-methyltetrahydrofolate homocysteine methyltransferase (7) serine hydroxymethyltransferase (8) 5,10-methylenetetrahydrofolate reductase (9) S-adenosylmethionine decarboxylase (10) spermidine and spermine synthases (11) methylthio-adenosine phosphorylase (12) conversion of methylthioribose to methionine (13) cystathionine P-synthase (14) cystathionine y-lyase (15) cysteine dioxygenase (16) cysteine suplhinate decarboxylase (17) hypotaurine NAD oxidoreductase (18) cysteine sulphintite a-oxoglutarate aminotransferase (19) sulfine oxidase. MeCbl = methylcobalamin PLP = pyridoxal phosphate...

The susceptibilities of some of these fluorinated purine nucleosides to the action of enzymes are now described. In contrast to the inertness of the 2 -deoxy-2 -fluoro- and 3 -deoxy-3 -fluorocytidine analogs 739, 744, and 821 towards cytidine deaminase, the adenosine compounds 867, 883, and 906 are readily deaminated - by the adenosine deaminase in erythrocytes and calf intestine, but the resulting (deaminated) inosine compounds (from 867 and 883), as well as 888, are highly resistant - to cleavage by purine nucleoside phosphorylase (to give hypoxanthine base for the first two). The reason was discussed. Both 867 and 883 can form the 5 -triphosphates, without deamination, in human erythrocytes or murine sarcoma cells in the presence of 2 -deoxycoformycin, an adenosine deaminase inhibitor, but... 

The biosynthesis of purines and pyrimidines is stringently regulated and coordinated by feedback mechanisms that ensure their production in quantities and at times appropriate to varying physiologic demand. Genetic diseases of purine metabolism include gout, Lesch-Nyhan syndrome, adenosine deaminase deficiency, and purine nucleoside phosphorylase deficiency. By contrast, apart from the orotic acidurias, there are few clinically significant disorders of pyrimidine catabolism. 

Adenosine Deaminase Purine Nucleoside Phosphorylase Deficiency... 

Adenosine deaminase deficiency is associated with an immunodeficiency disease in which both thymus-derived lymphocytes (T cells) and bone marrow-derived lymphocytes (B cells) are sparse and dysfunctional. Purine nucleoside phosphorylase deficiency is associated with a severe deficiency of T cells but apparently normal B cell function. Immune dysfunctions appear to result from accumulation of dGTP and dATP, which inhibit ribonucleotide reductase and thereby deplete cells of DNA precursors. 

H4. Hershfield, M. S., and Mitchell, B. S., Immunodeficiency diseases caused by adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency. In Metabolic and Molecular Bases of Inherited Disease, 7th ed. (C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, eds.), pp. 1725-1768. McGraw-Hill, New York, 1995. 

Low-density protein receptor Adenosine deaminase Purine nucleoside phosphorylase Sphingomylinase Glucocerebrosidase... 

ATP diphosphohydrolase Diadenosine polyphosphatase 5 nucleotidase Nucleoside transporter Adenosine deaminase Adenosine kinase Xanthine oxidase Nucleoside phosphorylase... 

Nucleoside phosphorylases that catalyse the reversible cleavage of purine nucleosides to the free bases and ribose-1-phosphate are found in most cells, although a phosphorylase that will cleave adenosine has so far been identified only in bacteria. Recent studies have shown that ribo- and 2 -deoxyribofurano-syltransferase activity is associated with phosphorylase activity [19, 23., 222] and that both activities reside in one enzyme, which can be converted from one form to the other by substrate or product binding [20]. Upon crystallization of the enzyme from human erythrocytes a marked decrease in the ribosyl transfer reaction was observed [21b]. 

Severe combined immunodeficiency Adenosine deaminase, purine nucleoside phosphorylase... 

Table 7.1.4 Concentration range of purine and pyrimidine metabolites in urine (pmol/mmol creatinine) from patients. ADA Adenosine deaminase, APRT adenine phosphoribosyltransferase, ASA adenylosuccinate lyase, DHP dihydropyrimidinase, DPD dihydropyrimidine dehydrogenase, HGPRT hypoxanthine-guanine phosphoribosyltransferase, PNP purine nucleoside phosphorylase, TP thymidine phosphorylase, UMPS uridine monophosphate synthase, / -UP fi-ureidopropionase... 

The activity of hypoxanthine-guanine phosphoribosyltransferase, adenine phos-phoribosyltransferase, adenosine deaminase, and purine nucleoside phosphorylase can be determined in dried blood spots using an HPLC-linked assay [3]. 

A guanosine aminohydrolase preparation from Pseudomonas convexa No. 149 free from guanine, adenine and adenosine aminohydrolase and nucleoside phosphorylase activities has been described (63). Of the 22... 

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