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 depolymerization procedure. Previous studies in our laboratory showed that its primary structure shared a high sequence identity with citrate synthases known so far and that the 49K protein possessed citrate synthase activity. To ascertain whether or not Tetrahymena's mitochondrial citrate synthase is identical to the 49K protein, citrate synthase was purified from Tetrahymena mitochondria using ammonium sulfate fractionation, Butyl-Toyopearl and SP-Toyopearl column chromatographies, based on monitoring of the enzymatic activity. The molecular weight of the purified citrate synthase was estimated to be 49 kDa, as was that of the 49K protein and the enzyme cross-reacted with an anti-49K protein antiserum. The purified citrate synthase showed much the same optimum pH, optimum KCl concentration, effects of substrate concentrations (acetyl-CoA and oxaloacetate), and inhibitory effect by ATP as those of purified 49K protein. Furthermore, an anti-49K protein monoclonal antibody strongly suppressed the enzymatic activity of the purified citrate synthase. 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 synthase, in mitochondria.
Institute of Biological Sciences, University of Tsukuba, Ibaraki.
H
H
Kojima
J
J
Chiba
Y
Y
Watanabe
O
O
Numata
D000818:Animals; D000953:Antigens, Protozoan / Q000032:analysis*; D002478:Cells, Cultured; D002950:Citrate (si)-Synthase / Q000276:immunology / Q000302:isolation & purification*; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D015800:Protozoan Proteins / Q000032:analysis* / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D013768:Tetrahymena / Q000201:enzymology* / Q000648:ultrastructure
D000953:Antigens, Protozoan; D015800:Protozoan Proteins; C067125:49K protein, Tetrahymena; D002950:Citrate (si)-Synthase