CHARACTERIZATION OF CITRATE SYNTHASE PURIFIED FROM DROSOPHILA-MELANOGASTER

Citrate synthase which condenses acetyl-CoA and oxaloacetate to citrat e was purified from Drosophila melanogaster. Some physicochemical as w ell as enzymatical properties were investigated. The optimum pH and te mperature were pH 8.0-9.0 and 45 degrees C, respectively. The molecula r weight of the enzyme was determined as 81,000 Da by gel filtration a nd the purified active enzyme consisted of two identical subunits whic h had a molecular mass of 48,700 on SDS-PAGE. Homogeneity of the purif ied enzyme was confirmed by SDS-PAGE and also by N-terminal amino acid sequence analysis. The Michaelis constants (K-m) of the enzyme for ac etyl-CoA and oxaloacetate were 6.7 mu M and 3.1 mu M, respectively. Ki netic studies showed that citrate synthase follows the concerted mecha nism which forms a ternary complex. Propionyl-CoA, ATP, and intermedia tes of the TCA cycle, succinyl-CoA and alpha-ketoglutarate, behaved as inhibitors in vitro. Using pig and chicken heart enzymes for comparis on, we found similarities at the N-terminal region. However, in the Ou chterlony immunodiffusion test, the polyclonal antibody raised against Drosophila citrate synthase did not show any crossreaction with pig, chicken or pigeon enzymes.