PTERIDINE DEPENDENT HYDROXYLASE FROM LIVER AND BRAIN

来自肝脏和大脑的蝶啶依赖性羟化酶

• 批准号: 3270945
• 负责人: JOHN M WHITELEY
• 金额: $ 0.64万
• 依托单位: SCRIPPS CLINIC AND RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-04-01 至 1987-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3270945
• 关键词: affinity chromatography; aminoacid analog; aminoacid biosynthesis; brain; brain metabolism; chemical models; chemical substitution; circular dichroism; conformation; dimer; enzyme structure; enzyme substrate analog; fluorescent dye /probe; gel electrophoresis; gel filtration chromatography; liver; liver metabolism; molecular site; nucleotide analog; phenylalanine 4 monooxygenase; pteridines; radiotracer; spectrometry; tritium; tyrosine 3 monooxygenase; unspecific monooxygenase

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.

该提案继续探索机制 四氢蝶啶与蝶啶还原酶结合， 苯丙氨酸或酪氨酸羟化酶导致插入一个原子 分子氧作为羟基取代基进入芳香核 后者酶的底物。 该项目的一个组成部分需要 三种酶的有效分离和表征。 按小说 蝶啶还原酶和亲和层析技术 已从大鼠肝脏中分离出苯丙氨酸羟化酶 均质性和部分特征化。 的高比活性 恢复的羟化酶使得许多先前报道的 重新检查酶的特性。 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、特定氨基酸标记、动力学 参数等，蛋白质的铁和磷酸盐含量均应 被确定。 两者活性位点的进一步表征 将尝试使用氨基酸的类似物和 含有光亲和或荧光标记的核苷酸底物。 酪氨酸羟化酶，难以大规模纯化 常规技术，将从牛肾上腺髓质中分离出来 含有兔抗羟化酶配体的亲和基质，由 注射 PAG 电泳纯化的酶。 这种酶会 那么也可以通过上述程序来表征。 的可达性 毫克量的还原酶和羟化酶将允许广泛 底物和酶相互作用的光谱和动力学分析， 结合模型氧化途径的检查 四氢蝶啶，将有助于进一步解释氧合作用 过程。