Immunochemical detection of CoA-modified mitochondrial matrix proteins

Abstract

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 monospecific 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.