Ligand-Binding in the Reaction Mechanism of DAO
DAO 反应机制中的配体结合
基本信息
- 批准号:6838246
- 负责人:
- 金额:$ 7.5万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2003
- 资助国家:美国
- 起止时间:2003-06-01 至 2006-05-31
- 项目状态:已结题
- 来源:
- 关键词:DNA damageamine oxidase (copper)animal tissueatomic absorption spectrometrycapillary electrophoresiscatalystchemical bindingenzyme activityenzyme substrategel filtration chromatographyheparinhigh performance liquid chromatographyion exchange chromatographyisozymesligandsmanganesemass spectrometrynucleoproteinsoxidative stressprotein purificationstop flow technique
项目摘要
DESCRIPTION (provided by applicant): Diamine oxidase (DAO) activities are elevated in boundaries separating quiescent and rapidly dividing cells where the transformation of molecular oxygen and biogenic amines to aminoaldehydes and hydrogen peroxide by these enzymes may play a significant role in the regulation of cell division in normal and cancerous tissues. Biochemical studies of this enzyme will help to elucidate the molecular mechanism of cellular response to changes in polyamine concentration in normal and disease states. Here we propose to 1) develop and optimize purification schemes for diamine oxidase from bovine liver and kidney and evaluate the substrate specificities of these isoforms 2) identify the role of manganese in ligand-binding and the catalytic mechanism, 3) characterize the heparin, and nucleic acid-binding interactions of DAO and the modulation of its catalytic activity associated with these biopolymers. The primary structure, glycosylation, cofactor content, specific activity, and substrate specificities of the purified isozymes will be compared. Catalytic reaction mechanisms of the purified enzymes will be studied through a combination of ligand binding, steady state, and pre-steady state kinetic measurements. DNA, RNA, heparin, and metal binding mechanisms, as well as the polynucleotide sequence binding specificity of these isozymes will be investigated by using a combination of titrametric, stopped-flow and continuous flow measurements. The role of polynucleotide, metal, and heparin-binding in catalysis will be established through the measurement of ligand-specific effects on steady-state kinetic parameters and reaction product distributions. The influence of nucleic acid binding on individual reaction steps will be established by observing changes in the kinetics of the oxidative and reductive half reactions by stopped-flow spectroscopy. The role of polyamines in the DAO-nucleic acid binding interaction and the kinetics of oxidation of DAO with polyamines will be investigated. Elevated concentrations of diamines and increased diamine oxidase activity generates significant concentrations of hydrogen peroxide and aminoaldehydes, which may lead to oxidative damage and the formation of inter- and intramolecular cross-links between reactive groups of susceptible proteins and nucleic acids. Products recovered from in vitro reactions of defined composition will be screened for DAO-induced molecular modifications by capillary electrophoresis and mass spectrometry.
描述(由申请人提供):二胺氧化酶(DAO)活性在分隔静止和快速分裂细胞的边界中升高,其中这些酶将分子氧和生物胺转化为氨基醛和过氧化氢,可能在细胞的调节中发挥重要作用正常组织和癌组织的分裂。这种酶的生化研究将有助于阐明正常和疾病状态下细胞对多胺浓度变化做出反应的分子机制。在此,我们建议 1) 开发和优化牛肝和肾二胺氧化酶的纯化方案,并评估这些亚型的底物特异性 2) 确定锰在配体结合中的作用和催化机制,3) 表征肝素,以及DAO 的核酸结合相互作用及其与这些生物聚合物相关的催化活性的调节。将比较纯化同工酶的一级结构、糖基化、辅因子含量、比活性和底物特异性。 将通过配体结合、稳态和预稳态动力学测量的组合来研究纯化酶的催化反应机制。 DNA、RNA、肝素和金属结合机制以及这些同工酶的多核苷酸序列结合特异性将通过滴定、停流和连续流测量的组合进行研究。通过测量配体特异性对稳态动力学参数和反应产物分布的影响,将确定多核苷酸、金属和肝素结合在催化中的作用。通过停流光谱法观察氧化和还原半反应动力学的变化,可以确定核酸结合对各个反应步骤的影响。将研究多胺在 DAO-核酸结合相互作用中的作用以及多胺氧化 DAO 的动力学。 二胺浓度升高和二胺氧化酶活性增加会产生显着浓度的过氧化氢和氨基醛,这可能导致氧化损伤以及易受影响的蛋白质和核酸的反应基团之间形成分子间和分子内交联。从确定成分的体外反应中回收的产品将通过毛细管电泳和质谱法筛选 DAO 诱导的分子修饰。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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George T. Gassner其他文献
George T. Gassner的其他文献
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{{ truncateString('George T. Gassner', 18)}}的其他基金
Repurposing Styrene Catabolic Enzymes for the Synthesis of Penicillins
重新利用苯乙烯分解代谢酶来合成青霉素
- 批准号:
10411114 - 财政年份:2022
- 资助金额:
$ 7.5万 - 项目类别:
Repurposing Styrene Catabolic Enzymes for the Synthesis of Penicillins
重新利用苯乙烯分解代谢酶来合成青霉素
- 批准号:
10686815 - 财政年份:2022
- 资助金额:
$ 7.5万 - 项目类别:
Structure and Mechanisms of Styrene Monooxygenase
苯乙烯单加氧酶的结构和机制
- 批准号:
7678363 - 财政年份:2007
- 资助金额:
$ 7.5万 - 项目类别:
Structure and Mechanisms of Styrene Monooxygenase
苯乙烯单加氧酶的结构和机制
- 批准号:
7488409 - 财政年份:2007
- 资助金额:
$ 7.5万 - 项目类别:
Structure and Mechanisms of Styrene Monooxygenase
苯乙烯单加氧酶的结构和机制
- 批准号:
7910560 - 财政年份:2007
- 资助金额:
$ 7.5万 - 项目类别:
Structure and Mechanisms of Styrene Monooxygenase
苯乙烯单加氧酶的结构和机制
- 批准号:
7289486 - 财政年份:2007
- 资助金额:
$ 7.5万 - 项目类别:
NMR SOLUTION STRUCTURE OF THE MMOB COMPONENT
MMOB 组件的 NMR 解决方案结构
- 批准号:
2910033 - 财政年份:1999
- 资助金额:
$ 7.5万 - 项目类别:
NMR SOLUTION STRUCTURE OF THE MMOB COMPONENT
MMOB 组件的 NMR 解决方案结构
- 批准号:
2521187 - 财政年份:1998
- 资助金额:
$ 7.5万 - 项目类别:
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