Identification of the CoA-modified forms of mitochondrial acetyl-CoA acetyltransferase and of glutamate dehydrogenase as nearest-neighbour proteins

Abstract

A 52 kDa protein could only be co-purified with the CoA-modified forms of acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase) (EC 2.3.1.9) from rat liver mitochondria. Immunoprecipitations of these modified forms with anti-(acetyl-CoA acetyltransferase) IgG or anti-(52 kDa protein) IgG yielded, in addition to the appropriate proteins, the 52 kDa protein or the CoA-modified form of acetyl-CoA acetyltransferase (41 kDa) respectively. This was demonstrated by SDS/PAGE and immunoblots. The modified forms containing the 52 kDa protein could be cross-linked by 1,5-difluoro-2,4-dinitrobenzene to a high-molecular-mass complex containing both the 41 kDa and 52 kDa proteins. The 52 kDa protein was identified as mitochondrial glutamate dehydrogenase (EC 1.4.1.3) by amino acid sequence analysis. The results of co-immunoprecipitation and cross-linking characterize the CoA-modified forms of acetyl-CoA acetyltransferase and the glutamate dehydrogenase as nearest-neighbour proteins.