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C

基本信息

批准号：
3287957
负责人：
EARL S SHRAGO
金额：
$ 7.27万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-07-01 至 1989-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3287957
关键词：
acyl coA adenosine diphosphate adenosine triphosphate affinity labeling azides chemical binding chemical synthesis covalent bond cow gel electrophoresis heart mitochondrial membrane palmitates photochemistry transport proteins

项目摘要

The specific aims of this proposal are to synthesize a photoaffinity labeled radioactive acyl CoA to study the mechanism of interaction of long chain acyl CoA esters with the adenine nucleotide translocase (ANT) in the inner mitochondrial membrane after covalent binding of the ligand to specific receptor sites on the carrier. Since acyl CoAs are known effectors of a number of enzymes in cell metabolism, the availability of photoaffinity labeled palmitoyl CoA would also be a very useful probe for investigations of a number of these enzymes. The compound, 8-Azido (2-3H) palmitoyl CoA will be prepared from the critical intermediates including 8N3-3',5'ADP(2-3H) and S-palmitoyl 4' phosphopantetheine. Experiments will be conducted to establish: Inhibition of ANT by the photoaffinity labeled acyl CoA; the binding characteristics, approximate binding and inhibition constants; and the specificity of the binding of the photolabel to the carrier prior to photoirradiation. The photoaffinity labeled radioactive ANT will then be purified by solubilization in Triton X-100 followed by hydroxylapatite chromatography, and the homogeneous 30,000 dalton subunit acyl CoA-protein complex identified by SDS gel electrophoresis. The critical experiments will be to obtain identical gel patterns starting with either isolated mitochondria or inverted submitochondrial particles. Attempts will then be made to identify the sequence of the peptide fragment which binds the photoaffinity labeled palmitoyl CoA. The results obtained from these investigations would provide further evidence to support the specificity of interactions of acyl CoAs with the ADP/ATP carrier, and indicate that they may be the physiological ligands for the carrier and a number of other enzymes.

这项提议的具体目的是合成一种亲光性标记放射性酰基辅酶A研究Long的相互作用机理含腺嘌呤核苷酸转位酶(ANT)的乙酰辅酶A链酯配体共价结合后的线粒体膜内层载体上的特定受体位置。由于已知酰基COA 一些酶在细胞新陈代谢中的效应器，光亲和标记棕榈酰辅酶A也将是一种非常有用的探针对这些酶中的一些进行研究。化合物8-叠氮(2-~3H) 棕榈酰辅酶A将由关键中间体制备，包括 8N3-3‘，5’-腺苷二磷酸(2-3H)和S-棕榈酰基4‘磷酸丙氨酸。将进行实验以确定：通过抑制蚂蚁光亲和标记的酰基辅酶A；结合特性，近似结合常数和抑制常数；以及结合的特异性在光照射之前将光标记到载体上。光亲和力然后通过在Triton中的增溶来提纯标记的放射性蚂蚁 X-100后经羟基磷灰石层析，均相 3万道尔顿亚基酰辅酶A-蛋白复合体的十二烷基硫酸钠凝胶鉴定电泳法。关键的实验将是获得相同的凝胶从孤立的线粒体或倒置开始的图案亚线粒体颗粒。然后，将尝试识别结合光亲和标记的多肽片段的序列棕榈酰辅酶A。从这些调查中获得的结果将为支持酰基相互作用的特异性提供进一步的证据与ADP/ATP承运商的COAS，并表明他们可能是载体的生理配体和许多其他酶。