PTERIDINE DEPENDENT HYDROXYLASE FROM LIVER AND BRAIN

本提案将继续探讨下列机制 四氢蝶呤类药物与蝶啶还原酶联合应用 苯丙氨酸或酪氨酸羟基酶导致一个原子的插入 分子氧作为羟基取代基进入芳香族原子核 后者是酶的底物。该项目的一个组成部分需要 这三种酶的高效分离和鉴定。通过小说 蝶啶还原酶亲和层析技术和 大鼠肝脏中的苯丙氨酸羟基酶已经被分离出来 同质性和部分特征化。酶的高比活度 恢复的羟基酶使许多先前报道的 重新检查该酶的性质。因此，除了典型的 蛋白质表征程序，如氨基酸、N-末端和 序列分析，PAG电泳，凝胶过滤，光谱和 圆二色性能、特定氨基酸标记、动力学 参数等，蛋白质的铁和磷含量都是 要下定决心。两者活性中心的进一步表征 将尝试使用氨基酸和氨基酸的类似物来尝试酶 含有光亲和标记或荧光标记的核苷酸底物。 酪氨酸羟基酶，对大规模纯化的顽固 常规技术将从牛肾上腺髓质中分离出 含有兔抗羟基酶配体的亲和矩阵，由 注射PAG-电泳纯化的酶。这种酶会 然后还以上述程序为特征进行说明。的可访问性 还原酶和羟基酶的镁含量将允许广泛的 底物和酶相互作用的光谱和动力学分析， 再加上对模型氧化途径的研究 四氢蝶呤，将允许进一步解释氧合作用 进程。

This proposal continues to explore the mechanisms whereby tetrahydropteridines in conjunction with pteridine reductase and phenylalanine or tyrosine hydroxylase cause the insertion of one atom of molecular oxygen as a hydroxyl substituent into the aromatic nucleus of the latter enzymes' substrates. An integral part of the project requires the efficient isolation and characterization of the three enzymes. By novel affinity chromatographic techniques both pteridine reductase and phenylalanine hydroxylase from rat liver have already been isolated to homogeneity and partially characterized. The high specific activity of the recovered hydroxylase has necessitated that many of the previously reported properties of the enzyme be re-examined. Therefore, in addition to typical protein characterization procedures such as amino acid, N-terminal and sequence analyses, PAG electrophoresis, gel filtration, spectral and circular dichroic properties, specific amino-acid labelling, kinetic parameters, etc., both the iron and phosphate content of the protein are to be determined. Further characterization of the active sites of both enzymes will be attempted by employing analogs of the amino acid and nucleotide substrates containing photoaffinity or fluorescent labels. Tyrosine hydroxylase, recalcitrant to large scale purification by conventional techniques, will be isolated from bovine adrenal medulla using affinity matrices containing a rabbit anti-hydroxylase ligand, generated by injection of PAG-electrophoretically purified enzyme. This enzyme will then also be characterized by the above procedures. The accessibility of the reductase and hydroxylases in mg quantities will allow extensive spectral and kinetic analyses of substrate and enzyme interactions, which coupled with an examination of the oxidation pathways of model tetrahydropteridines, will allow further interpretation of the oxygenation process.