MONOCLONAL ANTIBODIES AS PROBES OF CYTOCHROME OXIDASE
MONOCLONAL ANTIBODIES AS PROBES OF CYTOCHROME OXIDASE
批准号:
3280897
负责人:
SAMUEL H CHAN
金额:
$14.91万
依托单位国家:
美国
项目类别:
财政年份:
1985
资助国家:
美国
项目状态:
已结题
起止时间:
1985-09-05 至 1988-08-31
中文摘要
细胞色素氧化酶,细胞中的终极电子传递酶
真核生物的线粒体内膜,以某种形式由
都是好氧生物。因为它能立刻排出氧气,是一处
对于生命存在的可能性,它是至关重要的
地球。然而,人们对分子的实际构象迁移率知之甚少
TOTO中的酶,因为它在电子传递和
能量转导。拟议的研究将结合新出现的
用传统工艺生产单抗的技术
与线粒体生物化学领域相关。我们会做好准备
抗酶及其亚基的单抗。然后我们会放映
对于决定因素识别敏感的克隆
活性状态(使用新开发的酶联免疫吸附剂
检测)抗原检测)或识别与其结合的决定因素
影响氧化还原功能(通过分析细胞色素C氧化酶活性)
已与克隆抗体预先孵育的酶)。决定因素
它们显示了有趣的结合-活性关系(以及其
抗原性对洗涤剂变性不敏感)将定位于
用Western blotting技术测定该酶的亚基。一个
二维SDS-PAGE将解析亚基并展示多肽
由有限的蛋白质分解引起的。这些多肽的蛋白质印迹图可以
揭示亚基的相关部分,以确定初级
结构。通过使用这些技术来探测体内或
隔离(溶解的或人工重组的),休眠或
活性,有或没有配体,以及有或没有亚基
似乎与能量传递有最直接的联系,我们将
获得一系列地形变异性的结构-功能图
最终,导致了对酶的全面概述
这种酶的结构和功能之间的动态相互作用
对氧化磷酸化至关重要--对生命本身来说!
英文摘要
Cytochrome oxidase, the ultimate electron transport enzyme in the
mitochondrial inner membrane of eukaryotes, is possessed in some form by
all aerobic organisms. Because it at once detoxifies oxygen and is a site
of energy transduction, it is essential to the very possibility of life on
earth. Yet little is known of the actual conformational mobility of the
enzyme in toto as it performs its functions on electron transport and
energy transduction. The proposed study will combine the newly emerging
technology of monoclonal antibody production with techniques traditionally
associated with the field of mitochondrial biochemistry. We will prepare
monoclonal antibodies to the enzyme and its subunits. We will then screen
for clones for which the recognition of determinants is sensitive to
activity state (using a newly developed ELISA (enzyme linked immuno-sorbant
assay) antigen assay) or which recognize determinants to which binding
affects redox function (by assaying the cytochrome c oxidase activity of
enzyme which has been preincubated with the cloned antibody). Determinants
which show interesting binding-activity relationships (and whose
antigenicity is insensitive to detergent denaturation) will be localized on
the subunits of the enzyme using the technique of Western blotting. A
two-dimensional SDS-PAGE will resolve the subunits and display the peptides
resulting from limited proteolysis. Western blots of these peptides can
reveal the relevant portions of the subunit for determination of primary
structure. By using these techniques to probe the enzyme in vivo or
isolated (either solubilized or artificially reconstituted), resting or
active, with and without ligands, and with or without the subunit which
appears to be most directly associated with energy transduction, we will
obtain a series of structure-function maps of the topographical variability
of the oxidase, ultimately, leading to a comprehensive overview of the
dynamic interaction between structure and function of this enzyme which is
so vital to oxidative phosphorylation-to life itself!
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MONOCLONAL ANTIBODIES AS PROBES OF CYTOCHROME OXIDASE
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批准号:3280901
-
项目类别:
-
资助金额:$11.92万
-
财政年份:1985
-
负责人:SAMUEL H CHAN
-
依托单位:
MONOCLONAL ANTIBODIES AS PROBES OF CYTOCHROME OXIDASE
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批准号:3280900
-
项目类别:
-
资助金额:$13.54万
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财政年份:1985
-
负责人:SAMUEL H CHAN
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依托单位:
海外基金