辅酶A修饰的线粒体基质蛋白的免疫化学检测
Immunochemical detection of CoA-modified mitochondrial matrix proteins.
作者信息
Huth W, Pauli C, Möller U
机构信息
Georg-August-Universität Göttingen, Institut für Biochemie und Molekulare Zellbiologie, Germany.
出版信息
Biochem J. 1996 Dec 1;320 ( Pt 2)(Pt 2):451-7. doi: 10.1042/bj3200451.
An in vitro assay mixture consisting of mitochondrial matrix proteins from rat liver and CoA resulted in the formation of CoA-modified proteins. CoA-modified proteins were demonstrated by detection of CoA. CoA was released from the proteins by dithioerythritol treatment under denaturing conditions and was identified by (a) its retention time on HPLC, (b) its absorption spectrum and (c) its activity in a CoA-specific assay. This CoA represents protein-bound CoA and, in addition, protein-bound palmitoyl-CoA when MgATP was also present in the in vitro assay. The detection of immunoreactive proteins using mono-specific anti-CoA antibodies exclusively identifies CoA-modified proteins. The specificity of these antibodies can be used to identify both endogenously occurring CoA-modified proteins and proteins that have been modified in the in vitro assay. An intact thiol group of CoA is an essential precondition for the modification to occur.
由大鼠肝脏线粒体基质蛋白和辅酶A组成的体外分析混合物导致了辅酶A修饰蛋白的形成。通过检测辅酶A证实了辅酶A修饰蛋白的存在。在变性条件下用二硫苏糖醇处理可从蛋白中释放出辅酶A，并通过以下方法进行鉴定：(a) 其在高效液相色谱上的保留时间；(b) 其吸收光谱；(c) 其在辅酶A特异性分析中的活性。这种辅酶A代表与蛋白结合的辅酶A，此外，当体外分析中也存在MgATP时，还代表与蛋白结合的棕榈酰辅酶A。使用单特异性抗辅酶A抗体检测免疫反应性蛋白可专门鉴定辅酶A修饰蛋白。这些抗体的特异性可用于鉴定内源性存在的辅酶A修饰蛋白以及在体外分析中被修饰的蛋白。辅酶A完整的巯基是发生修饰的必要前提条件。
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