PHOTOAFFINITY LABELED ACYL COA FOR ADP/ATP CARRIER

用于 ADP/ATP 载体的光亲和标记酰基 COA

• 批准号: 3287952
• 负责人: EARL S SHRAGO
• 金额: $ 4.85万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3287952
• 关键词: acyl coA; adenosine diphosphate; adenosine triphosphate; affinity labeling; azides; chemical binding; chemical synthesis; covalent bond; gel electrophoresis; heart; mitochondrial membrane; palmitates; photochemistry

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.

这个建议的具体目标是合成一种光亲和性 标记的放射性酰基辅酶A，以研究长 链酰基辅酶A酯与腺嘌呤核苷酸转位酶（ANT）在 配体与线粒体内膜共价结合后， 载体上的特定受体位点。 由于已知酰基辅酶A 细胞代谢中许多酶的效应物， 光亲和标记的棕榈酰辅酶A也将是一种非常有用的探针， 对这些酶的研究。 化合物8-叠氮基（2- 3 H） 棕榈酰CoA将从关键中间体制备，包括 8 N3 - 3 '，5' ADP（2- 3 H）和S-棕榈酰基4'磷酸泛酰巯基乙胺。 将进行实验以确定： 光亲和标记的酰基辅酶A;结合特性，近似 结合和抑制常数;和结合的特异性， 在光照射之前将其光标记到载体上。 光亲和性 然后通过在Triton中溶解来纯化标记的放射性ANT X-100，然后通过羟基磷灰石色谱法， SDS凝胶鉴定的30，000道尔顿亚基酰基CoA-蛋白复合物 电泳 关键的实验将是获得相同的凝胶 从分离的线粒体开始， 亚线粒体颗粒 然后将尝试识别 结合光亲和标记的肽片段的序列 棕榈酰辅酶A 这些调查的结果将 提供了进一步的证据，以支持特定的相互作用的酰基 CoA与ADP/ATP载体，并表明它们可能是 载体的生理配体和许多其他酶。