Purine ribonucleoside kinases

J. A., Purine ribonucleoside kinase activity and resistance to some analogs of adenosine. Mol. Pharmacol, i, 432 (1966). 

It is particularly interesting that 6-mercaptopurine ribonucleoside (XXI) [158] is an inhibitor of, but not a substrate for, the kinase [47] but 6-(methylthio)purine ribonucleoside (XXII) [159, 160], resulting from the... 

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 At least three nucleoside cleavage activities are present (Berens, unpublished results) two are hydrolases, of which one is specific for purine ribonucleosides and the other is specific for purine deoxyribonucleosides.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 The adenosine kinase is similar to that of L. donovani (Berens, unpublished results). 

The possibility has been considered that purine bases can be converted to ribonucleosides by purine nucleoside phosphorylase, and thence to ribonucleotides by the purine nucleoside kinases. 

Fluoroadenine, 2-chloroadenine, 2-aminoadenine, 2- and 8-aza-adenines, 4-aminopyra7.olo[3, 4-d] pyrimidine and 6-methylpurine are converted to their ribonucleosidesby adenosine phosphoribosyltransferase,and their ribonucleosides are converted to the ribonucleotides by adenosine kinase most of the ribonucleotides are then converted to the di- and triphosphates. A -Aminoadenine, A -hydroxyadenine, A -methyladenine, purine, 7-deaza-adenine, and 7-amino-pyrazolo[4, 3-d] pyrimidine are either not substrates or are very poor substrates for the phosphoribosyltransferase, but their ribonucleosides are excellent substrates for the kinase. The ribonucleotides of purine, 7-deaza-adenine and... 

Purine bases can be converted to ribonucleotides via phosphoribosyl-transferases PP-ribose-P provides the ribosyl phosphate moiety. Purine nucleosides can be phosphorylated by ATP-requiring nucleoside kinases to form the same ribonucleotides. Finally, the possibility also exists that purine bases are first converted to ribonucleosides via nucleoside phos-phorylase, and then to ribonucleotides by the above-mentioned kinases. These routes of ribonucleotide synthesis are summarized as follows ... 

Pyrimidine ribonucleotides, like those of purines, may be synthesized de novo from amino acids and other small molecules (Chapter 11). Preformed pyrimidine bases and their ribonucleoside derivatives, derived from the diet of animals or found in the environment of cells, may be converted to ribonucleotides via nucleoside phosphorylases and nucleoside kinases. In some cells a more direct pyrimidine phosphoribosyltransferase pathway has also been recognized (Chapter 12). Ribonucleotides are catabolized by dephosphorylation, deamination, and cleavage of the glycosidic bond, to uracil. Uracil may be either oxidatively or reductively cleaved, depending on the organism involved, and can be converted to CO and NH (Chapter 13). 

The inhibitory effect of allopurinol on the first three steps of purine biosynthesis in Neurospora crassa cultures could be due 1. to the formation of allopurin-ol-l-ribonucleotide (by HGPRTase 1,19 nMol/mg protein/ hr in our Neurospora crassa cultures) 2. due to an oxipurinol-7-ribonucleotide effect, namely the formation of oxipurinol (by xanthine oxidase) and then oxipurinol-7-ribonucleotide (by OPRTase) and 3. by formation of allopurinol-1-ribotide through allopurin-ol-1-riboside (by a purine nucleoside phosphorylase) to allopurinol-1-ribonucleotide (by a nucleoside kinase). This latter pathway seems rather improbable because we could not demonstrate an effect on purine biosynthesis by direct addition of allopurinol-1-ribonucleoside to the incubation medium. Allopurinol-1-ribonucleotide inhibits significantly at higher concentrations. This is to explain if there is free access of the ribonucleotide form through the cell membrane (at least in these high concentrations) or if there is conversion to the ribonucleoside (which then enters the cell) by a nucleotidase located on the cell membrane. This latter mechanism however seems more improbable bcause there was no effect by allopurinol-1-ribonucleoside (which excludes the possibility for subsequent formation of allopurinol-1 -ribonucleotide by a nucleoside kinase, at least... 

The inhibitory effects of allopurinol and oxipurinol on purine and pyrimidine biosynthesis in human fibroblasts could be explained by formation of their respective ribonucleotides either by HGPRTase (allopurinol) or OPRTase (oxipurinol). The effects of allopurinol-1-ribonucleoside are hardly to be explained. Theoretically the allopurinol-1-ribonucleoside can be converted either to the free base (catalyzed by a purine nucleoside phosphorylase) or to allopurinol-1-ribonucleotide (catalyzed by a nucleoside kinase). According to Utter et al. (4) there seems however to be a lack of kinases in mammals which effectively phosphorylate inosine. Furthermore, indirect experiments of Elion et al. (5), where no detectable nucleotide formation or incorporation into nucleic acids was observed in vivo with Hc-allopurinol would support this. Nevertheless, our results would claim for a direot conversion of allopurin-ol-1-ribonucleoside to allopurinol-1-ribonucleotide in human fibroblast, at least at doses from 10 5 -to 10-3 M. Otherwise we would have to explain the inhibitory effects of allopurinol-1-ribonucleoside by direct influences on purine synthesis, for the other possibility theoretical-... 

Our results could not conclusively explain the mechanisms for allopurinol- and oxipurinol-mediated inhibition of purine and pyrimidine biosynthesis. Most of our results are in accordance with the view that allopurinol-1-ribonucleotide and oxipurinol-7-ribonucleo-tide are to be accounted for the inhibitory effects on both purine and pyrimidine biosynthesis. Relatively high concentrations of allopurinol-1-ribonucleoside and oxipurinol-7-ribonucleoside have Inhibitory effects which are difficult to explain with the view that there exist no inosinic or uridine specific kinases in fibroblasts, catalyzing to the respective ribonucleotides. 

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