DE NOVO PURINE NUCLEOTIDE BIOSYNTHESIS

这项研究的长期目标是定义 高等生物体内从头合成嘌呤核苷酸的代谢调节 真核生物。这将需要分离和表征 催化从头合成嘌呤核苷酸关键步骤的酶 生物合成。分离相应的cDNA和基因也将 是必要的。这现在是可能的，因为最近开发的 编码嘌呤生物合成酶的cDNA克隆策略 来自高等真核生物。这项建议侧重于主要的监管 从头途径中的酶，谷氨酰胺PRPP氨基转移酶。 描述了克隆人谷氨酰胺PRPP的实验 氨基转移酶cD4A。此cDNA将用于研究反馈 高尿酸血症患者细胞内酶的调节及与酶的比较 病人。两个关键的结构元件，一个11个氨基酸的前肽和 最近将对一种未表征的铁成分进行研究 克隆的禽氨基转移酶。工作假设是，这些 细菌和脊椎动物中保守的成分可能有 在催化和/或调节中的重要作用。鸟类的基因 将克隆氨基转移酶，以完成其结构 MRNA5‘端和评估富含GC的5’端未翻译的mRNA 调节酶合成的功能。

The long term goal of this research is to define the mechanisms for metabolic regulation of de novo purine nucleotide synthesis in higher eukaryotes. This will require the isolation and characterization of enzymes which catalyze key steps in de novo purine nucleotide biosynthesis. Isolation of the corresponding cDNAs and genes will also be necessary. This is now possible because of recently developed strategies to clone cDNAs which encode the purine biosynthetic enzymes from higher eukaryotes. This proposal focuses on the primary regulatory enzyme in the de novo pathway, glutamine PRPP amidotransferase. Experiments are described to clone the human glutamine PRPP amidotransferase cD4A. This cDNA will be used to investigate feedback regulation and for comparison with the enzyme in cells from hyperuricemic patients. Two key structural elements, an 11 amino acid propeptide and an uncharacterized Fe component will be investigated in the recently cloned avian amidotransferase. The working hypothesis is that these components which are conserved in bacteria and vertebrates may have important roles in catalysis and/or regulation. The gene for the avian amidotransferase will be cloned in order to complete the structure of the mRNA 5' end and to evaluate whether the GC-rich 5' untranslated mRNA functions to modulate enzyme synthesis.