Adenosine metabolism in wild‐type and enzyme‐deficient variants of Chinese hamster ovary and Novikoff rat hepatoma cells

Abstract

Variants of Chinese hamster ovary and Novikoff rat hepatoma cells resistant to tubercidin and 2,5-diaminopurine, or to both drugs, were isolated, and their ability to convert adenosine and various adenosine analogs to nucleotides was compared to that of wild-type cells, both in intact cells and cell-free extracts. Adenosine deamination, and thus its conversion to nucleotides via inosine-hypoxanthine-inosine monophosphate, was inhibited by pretreatment of the cells or cell extracts with 2-deoxycoformycin. Cell-free extracts of the tubercidin-resistant variants, as well as of two adenosine-resistant mutants of Chinese hamster ovary cells, phosphorylated adenosine, tubercidin, pyrazofurin, or tricyclic nucleoside in the presence of ATP at less than 1% of the rate of extracts of wild-type cells. However, addition of phosphoribosyl pyrophosphate stimulated the conversion of adenosine to nucleotides 40-fold. Similarly, intact adenosine kinase-deficient cells failed to phosphorylate the adenosine analogs, but still converted adenosine to nucleotides at 5-10% the rate observed with wild-type cells. Phosphorylation of adenosine and tubercidin in wild-type cells was inhibited by substrate at concentration above 5-10 microM. In contrast, the rate of conversion of adenosine to nucleotides by adenosine kinase-deficient cells increased linearly up to a concentration of 400 microM adenosine, with the consequence that, at this concentration, these cells took up adenosine almost as rapidly as wild-type cells. Adenosine uptake by these kinase-deficient cells was inhibited by adenine and 5'-deoxyadenosine, and was largely abolished in mutants devoid also of adenine phosphoribosyltransferase. We conclude that adenosine is converted to nucleotides in adenosine kinase-deficient cells via adenine. Indirect evidence implicates 5'-methylthioadenosine phosphorylase as the enzyme responsible for the degradation of adenosine to adenine.

Transport and metabolism of adenosine in human erthrocytes: Effect of transport inhibitors and regulation by phosphate

“…Vice versa, Ado transport occurred independently of either deamination or phosphorylation. This conclusion is in agreement with earlier work by us and other investigators Gum et al, 1979;Plagemann and Wohlhueter, 1983a;Plagemann et al, 1985a;Fazio et al, 1980;Fernandez-Rivero-Rio and Gonzalez-Garzia, 1985). Only when transport was inhibited about 95% ( Fig.…”

Adenosine uptake, transport, and metabolism in human erythrocytes

“…In both experiments, the rate of phosphorylation reached a maximum at about 1 p M and then decreased at higher concentrations. The results indicate that Ado phosphorylation in human red cells is subject to substrate inhibition similarly as has been observed for cultured mammalian cells (Plagemann and Wohlhueter, 1983a).…”

Residual nitrobenzylthioinosine-resistant nucleoside transport in a transport mutant (AE1) of S49 murine T-lymphoma cells.

“…For fractionating the acid-soluble pool of labeled cells, samples of cells were centrifuged through an oil layer directly into 100 of a solution composed of 0.5 N trichloroacetic acid and 10% (wt/vol) sucrose, which rapidly quenches all metabolism (18,25). The acid layer was further processed and analyzed by ascending paper chromatography with a solvent composed of 30 ml of 1 M ammonium acetate, pH 5, and 70 ml of 95% ethanol (solvent 28) as described previously (18).…”

“…The acid layer was further processed and analyzed by ascending paper chromatography with a solvent composed of 30 ml of 1 M ammonium acetate, pH 5, and 70 ml of 95% ethanol (solvent 28) as described previously (18).…”