Repression and derepression of purine biosynthesis in mammalian hepatoma cells in culture.

The rates of biosynthesis of purines from glycine have been examined in an established line of rat hepatoma cells in continuous culture. The presence of low4 M adenine in the culture medium causes a prompt 4-fold to 5-fold decline in the rate of [14C]glycine incorporation into purines, and the capacity for de novo puke biosynthesis decays with a halftime of approximately 4 hours. Upon removal of adenine, the rate promptly increases, but the increase can be prevented by cycloheximide or actinomycin D. Neither cycloheximide nor actinomycin D prevents the decline in purine synthesis caused by adenine. Cycloheximide or actinomycin D alone causes a fall in the rate of purine formation, and the rate decays with a half-time of about 2 hours. The presence of 5-azacytidine, a pyrimidine analog which does not significantly inhibit RNA synthesis but is probably incorporated into RNA molecules, also causes the rate to decay with a similar half-time. The intracellular accumulation of a-N-formylglycinamide ribonucleotide (in the presence of 6.4 x low6 M azaserine in the culture medium) is repressed and derepressed by the addition and removal, respectively, of exogenous adenine. The kinetics of derepression and the inhibition by cycloheximide and 5-azacytidine of the rate of formylglycinamide ribonucleotide accumulation suggest that the regulation in the first third of the pathway is responsible for the control of the entire pathway by adenine. Under the conditions employed, the intracellular and extracellular concentrations of the precursor, glycine, do not change. The first three enzymes committed to de novo puke synthesis, phosphoribosylpyrophosphate glutamyl amidotransferase, glycinamide ribonucleotide synthetase, and glycinamide ribonucleotide transformylase, are not repressed or derepressed by the presence or absence of adenine in the culture medium. The identity of the repressible macromolecule(s) is unknown at present but it appears not to be specifically committed to purine biosynthesis.