CHEMISTRY OF FOLATE AND PTERIDINE COENZYMES

作为蝶呤生物化学既定计划的一部分，二氢叶酸 还原酶(DHFR)和二氢蝶啶还原酶(DHPtR)，这两种酶 将7，8-二氢叶酸和喹诺酮类二氢生物蝶呤转化为其 各自的四氢类似物和苯丙氨酸羟基酶(PAH)，它 将苯丙氨酸转化为酪氨酸，每个都被提纯到均一 大鼠肝脏中的镁含量；三种酶在其中起作用的组织 通力合作。在目前的提案中，纯化的还原酶将是 在各种条件下(例如，不同的pH、离子 强度、激活剂、抑制剂等)对于交叉专一性， 底物。这些结果可能表明两者的功能重叠。 体内的酶，这一观察结果将特别令人感兴趣，因为 DHPtR广泛分布于细胞培养和含有NO的组织中。 辅助羟基酶。需要DHPtR的二聚体、NADH(MR约. 51,000)将进一步通过与荧光的相互作用来表征 放射性耐光底物类似物探针，单克隆体的产生 抗体，通过初步的高分辨率核磁共振分析，测序和 用X-射线技术进行晶化处理。无论在哪里进行结构比较 可能是信息性的，DHFR(MR约22,500)将来自同一来源 也要接受检查。各种荧光叶酸类似物将被 合成了作为上述酶特性的辅助和AS的 蝶啶和叶酸细胞摄取途径的探针。此外，还有 这个实验室长期以来一直对不同的氧化反应感兴趣 因此，四氢蝶呤的途径是补充酶 经调查，多种甲基取代四氢蝶呤类化合物均有 已综合，并将在模型系统中进行检查，以定义 控制其退化路径的因素。 作为该项目的必然结果，甲氨蝶呤将与一系列 用于检测其体内性质的合成肽，定位于 携带者，化疗潜力得到改善。

As part of an established program in pteridine biochemistry, dilhydrofolate reductase (DHFR), and dihydropteridine reductase (DHPtR), two enzymes which convert 7,8-dihydrofolate and 'quinonoid' dihydrobiopterin to their respective tetrahydro analogs, and phenylalanine hydroxylase (PAH), which converts phenylalanine to tyrosine, have each been purified to homogeneity in mg quantities from rat liver; a tissue in which the three enzymes work cooperatively. In the current proposal the purified reductases are to be examined under a variety of conditions (e.g., differing pH, ionic strengths, activators, inhibitors, etc.) for cross-specificity of substrates. These results might suggest overlapping functions for the two enzymes in vivo, an observation that would be of particular interest as DHPtR is widely distributed in cell cultures and tissues which contain no ancillary hydroxylases. The dimeric, NADH requiring DHPtR (Mr approx. 51,000) will be further characterized by interaction with fluorescent probes, radioactive photolabile substrate analogs, generation of monoclonal antibodies, by preliminary high resolution NMR analysis, sequencing, and after crystallization by X-ray techniques. Wherever structural comparisons could be informative, DHFR (Mr approx. 22,500) from the same source will also be examined. A variety of fluorescent folate analogs are to be synthesised both as aids to the above enzyme characterizations and as probes for pteridine and folate cellular uptake pathways. Additionally, of long standing interest to this laboratory have been the diverse oxidation path ways of tetrahydropteridines, therefore, to complement the enzymatic investigations, a variety of methyl-substituted tetrahydropteridines have been synthesized and are to be examined in model systems to define the factors which control their paths of degradation. As a corollary to the project methotrexate will be bound to a series of synthetic peptides to examine their properties as in vivo, site-directed carriers, with improved chemotherapeutic potential.