Becker M A, Kim M
Department of Medicine, University of Chicago Pritzker School of Medicine, Illinois 60637.
J Biol Chem. 1987 Oct 25;262(30):14531-7.
Previous studies of purine nucleotide synthesis de novo have suggested that major regulation of the rate of the pathway is affected at either the phosphoribosylpyrophosphate (PP-Rib-P) synthetase reaction or the amidophosphoribosyltransferase (amido PRT) reaction, or both. We studied control of purine synthesis de novo in cultured normal, hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient, and PP-Rib-P synthetase-superactive human fibroblasts by measuring concentrations and rates of synthesis of PP-Rib-P and purine nucleotide end products, proposed effectors of regulation, during inhibition of the pathway. Incubation of cells for 90 min with 0.1 mM azaserine, a glutamine antagonist which specifically blocked the pathway at the level of conversion of formylglycinamide ribotide, resulted in a 5-16% decrease in purine nucleoside triphosphate concentrations but no consistent alteration in generation of PP-Rib-P. During this treatment, however, rates of the early steps of the pathway were increased slightly (9-15%) in normal and HGPRT-deficient strains, more markedly (32-60%) in cells with catalytically superactive PP-Rib-P synthetases, and not at all in fibroblasts with purine nucleotide feedback-resistant PP-Rib-P synthetases. In contrast, glutamine deprivation, which inhibited the pathway at the amido PRT reaction, resulted in time-dependent nucleoside triphosphate pool depletion (26-43% decrease at 24 h) accompanied by increased rates of PP-Rib-P generation and, upon readdition of glutamine, substantial increments in rates of purine synthesis de novo. Enhanced PP-Rib-P generation during glutamine deprivation was greatest in cells with regulatory defects in PP-Rib-P synthetase (2-fold), but purine synthesis in these cells was stimulated only 1.4-fold control rates by glutamine readdition. Stimulation of these processes in normal and HGPRT-deficient cells and in cells with PP-Rib-P synthetase catalytic defects was, respectively: 1.5 and 2.0-fold; 1.5 and 1.7-fold; and 1.6 and 4.1-fold. These studies support the following concepts. 1) Rates of purine synthesis de novo are regulated at both the PP-Rib-P synthetase and amido PRT reactions by end products, with the latter reaction more sensitive to small changes in purine nucleotide inhibitor concentrations. 2) PP-Rib-P exerts its role as a major regulator of purine synthetic rate by virtue of its interaction with nucleotide inhibitors to determine the activity of amido PRT. 3) Activation of amido PRT by PP-Rib-P is nearly maximal at base line in fibroblasts with regulatory defects in PP-Rib-P synthetase.
先前关于嘌呤核苷酸从头合成的研究表明，该途径速率的主要调节作用于磷酸核糖焦磷酸（PP-Rib-P）合成酶反应或酰胺磷酸核糖转移酶（amido PRT）反应，或两者均有影响。我们通过测量PP-Rib-P和嘌呤核苷酸终产物（推测的调节效应物）的浓度及合成速率，研究了培养的正常、次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HGPRT）缺陷型和PP-Rib-P合成酶超活性人成纤维细胞中嘌呤从头合成的调控情况。用0.1 mM重氮丝氨酸（一种谷氨酰胺拮抗剂，它在甲酰甘氨酰胺核苷酸转化水平特异性阻断该途径）孵育细胞90分钟，导致嘌呤核苷三磷酸浓度下降5 - 16%，但PP-Rib-P的生成没有一致的改变。然而，在此处理过程中，该途径早期步骤的速率在正常和HGPRT缺陷型细胞系中略有增加（9 - 15%），在具有催化超活性PP-Rib-P合成酶的细胞中增加更显著（32 - 60%），而在具有嘌呤核苷酸反馈抗性PP-Rib-P合成酶的成纤维细胞中则完全没有增加。相反，谷氨酰胺缺乏在amido PRT反应处抑制该途径，导致核苷三磷酸池随时间减少（24小时时减少26 - 43%），同时PP-Rib-P生成速率增加，并且在重新添加谷氨酰胺后，嘌呤从头合成速率大幅增加。谷氨酰胺缺乏期间PP-Rib-P生成的增强在PP-Rib-P合成酶具有调节缺陷的细胞中最大（增加2倍），但这些细胞中的嘌呤合成仅被谷氨酰胺重新添加刺激至对照速率的1.4倍。在正常和HGPRT缺陷型细胞以及具有PP-Rib-P合成酶催化缺陷的细胞中，这些过程的刺激倍数分别为：1.5和2.0倍；1.5和1.7倍；1.6和4.1倍。这些研究支持以下概念。1）嘌呤从头合成速率在PP-Rib-P合成酶和amido PRT反应处均受到终产物的调节，后者反应对嘌呤核苷酸抑制剂浓度的微小变化更敏感。2）PP-Rib-P通过与核苷酸抑制剂相互作用来决定amido PRT的活性，从而发挥其作为嘌呤合成速率主要调节因子的作用。3）在PP-Rib-P合成酶具有调节缺陷的成纤维细胞中，PP-Rib-P对amido PRT的激活在基线时几乎达到最大值。
