CHEMISTRY OF FOLATE AND PTERIDINE COENZYMES

作为蝶啶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“喹喔啉”二氢生物蝶呤转化为它们的 各自的四氢类似物和苯丙氨酸羟化酶（PAH）， 将苯丙氨酸转化为酪氨酸， 从老鼠肝脏中提取的毫克数量;这三种酶起作用的组织 合作。 在当前的提议中，纯化的还原酶将被纯化。 在各种条件下检查（例如，不同pH值，离子 规格、活化剂、抑制剂等）交叉特异性 印刷受体. 这些结果可能表明这两个功能重叠 酶在体内，观察，这将是特别感兴趣的， DHPtR广泛分布于不含DHPtR的细胞培养物和组织中。 辅助羟化酶。 二聚体，需要DHPtR的NADH（Mr约为 51，000）将进一步通过与荧光 探针，放射性光不稳定底物类似物，单克隆抗体的产生 抗体，通过初步的高分辨率NMR分析，测序， 通过X射线技术结晶后。 无论在何处，结构比较 可能是信息，DHFR（先生约。22,500）从同一来源将 也要检查。 各种荧光叶酸盐类似物将被用于治疗癌症。 作为上述酶表征的辅助物和作为 用于蝶啶和叶酸细胞摄取途径的探针。 此外， 这个实验室长期以来的兴趣是各种氧化 因此，四氢蝶啶的途径，以补充酶促 研究表明，各种甲基取代的四氢蝶啶 将在模型系统中进行审查，以确定 控制其降解路径的因素。 作为该项目的必然结果，甲氨蝶呤将与一系列 合成肽以检查其体内定点性质 载体，具有改善的化疗潜力。

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.