Regulation of de novo purine biosynthesis in human lymphoblasts. Coordinate control of proximal (rate-determining) steps and the inosinic acid branch point

Abstract

Purine nucleotide synthesis de novo has been studied in a permanent tissue culture line of human splenic lymphoblasts with particular attention to coordination of control of the proximal (rate-determining) steps with the distal branch point of the pathway. An assay was used which permits simultaneous determination of the overall rate of labeling of all intracellular purines with sodium [14C]formate, as well as the distribution of isotope into all intracellular guanine- and adenine-containing compounds. The guanine to adenine labeling ratio was used as an index of IMP branch point regulation. It was found that exogenous adenine and guanine produce feedback-controlling effects not only on the first step in the de novo pathway, but also on the IMP branch point. Concentrations of adenine which produce less than 40% inhibition of the overall rate of de novo purine synthesis do so by selectively inhibiting adenine nucleotide synthesis de novo by 50 to 70% while stimulating guanine nucleotide synthesis de novo by up to 20%. A reciprocal effect is seen with exogenous guanine. The adenosine analog 6-methylmercaptopurine ribonucleoside selectivity inhibits adenine nucleotide synthesis via the de novo pathway but not from exogenous hypoxanthine. Thus, the reactions of purine nucleotide interconversion, in particular adenylosuccinate synthetase, may be regulated differently in cells deriving their purine nucleotides solely from de novo synthesis than when deriving them via "salvage" of preformed hypoxanthine.