DE NOVO PURINE NUCLEOTIDE BIOSYNTHESIS

这项研究的长期目标是确定 嘌呤核苷酸从头合成的代谢调控 真核生物 这将需要分离和表征 催化嘌呤核苷酸从头合成关键步骤的酶 生物合成 相应cDNA和基因的分离也将 是必要的。 这是因为最近开发的 克隆编码嘌呤生物合成酶的cDNA的策略 从高等真核生物。 该提案侧重于主要监管 从头途径中的酶，谷氨酰胺PRPP酰胺转移酶。 本实验克隆了人谷氨酰胺PRPP 酰胺转移酶cD 4A。 该cDNA将用于研究反馈 调节，并与高尿酸血症患者细胞中的酶进行比较 患者 两个关键的结构元件，一个11个氨基酸的前肽和 一个未表征的铁组分将在最近的研究 克隆的禽类酰胺转移酶。 工作假设是，这些 在细菌和脊椎动物中保守的成分可能具有 在催化和/或调节中的重要作用。 鸟类的基因 酰胺转移酶将被克隆，以完成结构的 mRNA 5'末端，并评估富含GC的5'非翻译mRNA是否 功能是调节酶的合成。

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.