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

Abstract

To clarify the contributions of amidophosphoribosyltransferase (ATase) and its feedback regulation to the rates of purine de novo synthesis, DNA synthesis, protein synthesis, and cell growth, mutated human ATase (mhATase) resistant to feedback inhibition by purine ribonucleotides was engineered by site-directed mutagenesis and expressed in CHO ade ؊ A cells (an ATasedeficient cell line of Chinese hamster ovary fibroblasts) and in transgenic mice (mhATase-Tg mice). In Chinese hamster ovary transfectants with mhATase, the following parameters were examined: ATase activity and its subunit structure, the metabolic rates of de novo and salvage pathways, DNA and protein synthesis rates, and the rate of cell growth. In mhATase-Tg mice, ATase activity in the liver and spleen, the metabolic rate of the de novo pathway in the liver, serum uric acid concentration, urinary excretion of purine derivatives, and T lymphocyte proliferation by phytohemagglutinin were examined. We concluded the following. 1) ATase and its feedback inhibition regulate not only the rate of purine de novo synthesis but also DNA and protein synthesis rates and the rate of cell growth in cultured fibroblasts. 2) Suppression of the de novo pathway by the salvage pathway is mainly due to the feedback inhibition of ATase by purine ribonucleotides produced via the salvage pathway, whereas the suppression of the salvage pathway by the de novo pathway is due to consumption of 5-phosphoribosyl 1-pyrophosphate by the de novo pathway. 3) The feedback inhibition of ATase is more important for the regulation of the de novo pathway than that of 5-phosphoribosyl 1-pyrophosphate synthetase. 4) ATase superactivity leads to hyperuricemia and an increased bromodeoxyuridine incorporation in T lymphocytes stimulated by phytohemagglutinin.Purine nucleotides are synthesized both via the de novo pathway and via the salvage pathway and are vital for cell functions and cell proliferation through DNA and RNA syntheses and ATP energy supply. Amidophosphoribosyltransferase (ATase) 1 is the rate-limiting enzyme in the de novo pathway of purine ribonucleotide synthesis (1) and is regulated by feedback inhibition by AMP and GMP. Hypoxanthine (Hx) and hypoxanthine guanine phosphoribosyltransferase (HPRT) are the most important substrate and enzyme, respectively, of the salvage pathway (1). The Lesch-Nyhan syndrome is caused by a complete deficiency of HPRT. Although patients with this syndrome show hyperuricemia with accelerated de novo purine synthesis, the mechanism of activation of the de novo pathway is not fully understood.In a previous study, we demonstrated that the expression level of ATase limits the growth rate of cultured fibroblasts, and purine salvage strongly inhibits purine de novo synthesis (1). Two mechanisms by which the salvage pathway inhibits the de novo pathway were presumed. 1) Consumption of 5-phosphoribosyl 1-pyrophosphate (PRPP) by the salvage pathway decreases the activity of the de novo pathway because PRPP is the common source of both pathways. 2) Purin...

The complete genome sequence ofMycobacterium aviumsubspeciesparatuberculosis

“…Of particular interest is a gene (MAP0638) with the highest dN͞dS ratio of 7.3 that has homology to an amidophosphoribosyltransferase (ATase). This gene is primarily responsible for regulation and production of purine synthesis and has also been shown to play a role in the rate of DNA and protein synthesis and cell growth (33). It is tempting to speculate that the increased level of substitution in this gene may play a role in a decreased growth rate in Map in comparison to Mtb, a hypothesis that remains to be tested.…”

Microtubule-assisted mechanism for functional metabolic macromolecular complex formation

“…This work was funded by National Institutes of Health Grant GM24129 (to S.J.B.). Incorporation was found to be linear with time up to 4 h, and ratio of de novo purine biosynthesis rates in purine-depleted to purine-rich media was 1.42 by fitting data with the leastsquares line method (10,11). However, data could alternatively be fit with a single exponential function, resulting in larger difference between the two data sets (i.e., 1.60).…”

Purine and Pyrimidine Nucleotide Synthesis and Metabolism

“…growth (Yamaoka et al, 2001). While it is known that plant ATase is sensitive to feedback regulation (Ito et al, 1994) the impact of this inhibition on plant cellular metabolism has not been examined thoroughly.…”