CITRATE SYNTHASE PURIFIED FROM TETRAHYMENA MITOCHONDRIA IS IDENTICAL WITH TETRAHYMENA 14-NM FILAMENT PROTEIN

A 14-nm filament protein (designated as 49K protein) was purified from a ciliated protozoan, Tetrahymena, using the polymerization and depol ymerization procedure, Previous studies in our laboratory showed that its primary structure shared a high sequence identity with citrate syn thases known so far and that the 49K protein possessed citrate synthas e activity, To ascertain whether or not Tetrahymena's mitochondrial ci trate synthase is identical to the 49K protein, citrate synthase was p urified from Tetrahymena mitochondria using ammonium sulfate fractiona tion, Butyl-Toyopearl and SP-Toyopearl column chromatographies, based on monitoring of the enzymatic activity, The molecular weight of the p urified citrate synthase was estimated to be 49 kDa, as was that of th e 49K protein and the enzyme cross-reacted with an anti-49K protein an tiserum, The purified citrate synthase showed much the same optimum pH , optimum KCI concentration, effects of substrate concentrations (acet yl-CoA and oxaloacetate), and inhibitory effect by ATP as those of pur ified 49K protein, Furthermore, an anti-49K protein monoclonal antibod y strongly suppressed the enzymatic activity of the purified citrate s ynthase, Thus, we suggest that mitochondrial citrate synthase and the 49K protein are identical and that the 49K protein has dual functions in the cytoskeleton in cytoplasm and as a TCA cycle enzyme, citrate sy nthase, in mitochondria.