De novo synthesis of purine nucleotides in human peripheral blood leukocytes. Excessive activity of the pathway in hypoxanthine-guanine phosphoribosyltransferase deficiency.

Abstract

A B S T R A C T Human peripheral blood leukocytes were studied for the presence and the regulatory properties of the pathway of de novo synthesis of purine nucleotides. The cells were found to incorporate the labeled precursors formate and glycine into purines. The rate of [14C]-formate incorporation was decreased by several compounds known to inhibit purine synthesis by affecting the activity of glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase, the first committed enzyme in the pathway, either through decreasing the availability of PRPP, a substrate for this enzyme, or through exerting inhibition on this enzyme.PRPP availability in the leukocyte was found to be limiting for purine synthesis. Increased PRPP availability resulting from activation of PRPP synthetase by increasing inorganic phosphate (Pi) concentration caused acceleration of purine synthesis. On the other hand, no clear-cut evidence was obtained for the availability of ribose-5-phosphate in the leukocyte being rate limiting at physiological extracellular Pi concentration for PRPP generation, and thus for purine synthesis. However, the addition of methylene blue, which accelerates the oxidative pentose shunt that produces ribose-5-phosphate, resulted in acceleration of PRPP gen- eration and of purine synthesis only when PRPP synthetase was largely activated at high Pi concentration. These results may be taken to suggest that ribose-5-phosphate availability is indeed not limiting for PRPP generation under physiological conditions. Purine synthesis de novo was accelerated more than 13-fold in the leukocytes of two gouty patients affected with partial deficiency of hypoxanthine-guanine phosphoribosyltransferase, but was normal in the leukocytes of an obligate heterozygote for this enzyme abnormality.The results demonstrate in peripheral human leukocytes the presence of the complete pathway of de novo synthesis of purine nucleotides and the manifestation in these cells of the biochemical consequences of hypoxanthine-guanine phosphoribosyltransferase deficiency, i.e., increased availability of PRPP and acceleration of purine synthesis de novo. The results indicate the usefulness of leukocytes as a model tissue for the study of purine metabolism in man.

<i>De novo</i> Synthesis of Purine Nucleotides in Human Blood Platelets

“…The finding that despite increased consumption of R-5-P for PRPP formation the cellular content of R-5-P remained constant, may reflect the fact that R-5-P is an intermediate in several active pathways, and that its cellular content does not relate to its turnover. The finding in the present study that the increase in PRPP concentration was not associated with an increase in the rate of de novo purine synthesis, is not compatible with the findings in human blood leukocytes (3), and may be taken to suggest that in the platelet, PRPP concen tration is saturating for the PRPP-amidotransferase, catalyzing the first com mitted and rate-limiting step in the pathway of purine synthesis de novo. Moreover, the above difference between platelets and leukocytes may also be taken as an additional proof for the fact that the de novo purine nucleotide synthesis in our system was only due to platelet activity.…”

“…This low rate of activity of de novo purine synthesis in normal human blood platelets, which may have been the reason for the inability to detect its presence up to now could reflect residual activity from the parent megakaryocyte cell and may have no quantita tive physiological role in the platelets. PRPP and R-5-P contents in human blood platelets were found to be of a similar order as that found previously for human blood erythrocytes (22) and leukocytes (3). As with these cells, also in the platelets, incubation at high Pi concentrations caused a marked increase in cellular PRPP content.…”

“…It was recently calculated that only 0.6% of the adenine nucleotides of platelets is produced from hypoxanthine (20). The rate of purine synthesis de novo in the platelets, as gauged by the rate of (14C)formate incorporation into purines was found to be slow relatively to other tissues studied in our laboratory (2,3,25). This low rate of activity of de novo purine synthesis in normal human blood platelets, which may have been the reason for the inability to detect its presence up to now could reflect residual activity from the parent megakaryocyte cell and may have no quantita tive physiological role in the platelets.…”

Amidophosphoribosyltransferase Limits the Rate of Cell Growth-linked de Novo Purine Biosynthesis in the Presence of Constant Capacity of Salvage Purine Biosynthesis

“…Also, the proliferation of T lymphocytes from the patients in response to mitogenic and antigenic stimulation was normal (33). In the leukocytes from two gouty patients affected with a partial deficiency of HPRT, de novo synthesis was accelerated to more than 13 times that of normal controls (34).…”

Feedback Inhibition of Amidophosphoribosyltransferase Regulates the Rate of Cell Growth via Purine Nucleotide, DNA, and Protein Syntheses

“…Even in patients with Lesch-Nyhan syndrome, the proliferation of T lymphocytes in response to mitogenic and antigenic stimulation was normal (17). Moreover, in leukocytes from two gouty patients affected with a partial deficiency of HPRT, de novo synthesis was accelerated to more than 13 times that of normal controls (18). These reports suggest that the large capacity of purine de novo synthesis can compensate for the defect in the salvage pathway.…”