Elucidation of aberrant purine metabolism. Application to hypoxanthine-guanine phosphoribosyltransferase- and adenosine kinase-deficient mutants, and IMP dehydrogenase- and adenosine deaminase-inhibited human lymphoblasts

Abstract

We propose that the ratio of [ 14C]formate-labelled purine nucleosides and bases (both intra and extracellular) to nucleic acid purines provides, in exponentially growing cultures, a sensitive index for comparative studies of purine metabolism. This ratio was 4-fold greater for an HGPRT − mutant than for the parental HGPRT + human lymphoblast line. The major components of the labelled nucleoside and base fraction were hypoxanthine and inosine. By blocking adenosine deaminase activity with coformycin we found that approx. 90% of inosine was formed directly from IMP rather than the route IMP → AMP → adenosine → inosine. The ratio of labelled base + nucleosides to nucleic acids was essentially unchanged for an AK − lymphoblast line and 2-fold greater than control for an HGPRT −-AK − line, demonstrating that a deficiency of adenosine kinase alone has little effect on the accumulation of purine nucleosides and bases. Although adenosine was a minor component of the nucleoside and base fraction, the adenosine fraction increased from 3 to 13% with the addition of coformycin to the HGPRT −-AK − line. In the parental and HGPRT − lines, adenosine was shown to be primarily phosphorylated rather than deaminated at concentrations less than 5 μM. Inhibition of IMP dehydrogenase activity by mycophenolic acid caused a 12- and 3-fold increase in the rate of production of labelled base and nucleoside in the parent and HGPRT − cells respectively. These results suggest that a mutationally induced partial deficiency in the activities converting IMP to guanine nucleotides may result in an increased catabolism of IMP.