PTERIDINE DEPENDENT HYDROXYLASE FROM LIVER AND BRAIN

这项建议继续探讨各种机制， 四氢蝶啶与蝶啶还原酶结合， 苯丙氨酸或酪氨酸羟化酶引起的插入一个原子的 分子氧作为羟基取代基进入芳核的 后者是酶的底物。 该项目的一个组成部分需要 三种酶的有效分离和表征。 采用新型 亲和层析技术蝶啶还原酶和 来自大鼠肝脏的苯丙氨酸羟化酶已经被分离出来， 同质性和部分特征。 高比活度的 回收的羟化酶需要许多以前报道的 酶的性质需要重新研究。 因此，除了典型的 蛋白质表征程序，例如氨基酸、N-末端和 序列分析，PAG电泳，凝胶过滤，光谱和 圆二色性，特异性氨基酸标记，动力学 参数等，蛋白质中的铁和磷酸盐含量 被确定。 进一步表征两者的活性位点 酶将通过使用氨基酸的类似物来尝试， 含有光亲和性或荧光标记的核苷酸底物。 酪氨酸羟化酶，可用于大规模纯化， 将使用常规技术从牛肾上腺髓质中分离 含有兔抗羟化酶配体的亲和基质，通过 注射PAG-经层析纯化的酶。 这种酶将 然后也可以通过上述程序进行表征。 的可及性 mg量的还原酶和羟化酶将允许广泛的 底物和酶相互作用的光谱和动力学分析， 结合模型的氧化途径的检查 四氢蝶啶，将允许进一步解释氧化 过程

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.