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KMID : 0578319970070050599
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Molecules and Cells
1997 Volume.7 No. 5 p.599 ~ p.604
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Characterization of Citrate synthase Purified from Drosophila melanogaster
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Lee, Soojin
Park, Chaehwa/Yim, Jeongbin
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Abstract
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Citrate synthase which condenses acetyl-CoA and oxaloacetate to citrate was purified from Drosophila melanogaster. Some physicochemical as well as enzymatical properties were investigated. The optimum pH and temperature were pH 8.0-9.0 and 45℃, respectively. The molecular weight of the enzyme was determined as 81,000 Da by gel filtration and the purified active enzyme consisted of two identical subunits which had a molecular mass of 48,700 on SDS-PAGE. Homogeneity of the purified emzyme was confirmed by SDS-PAGE and also by N-terminal amino acid sequence analysis. The Michaelis constants (K_(m)) of the enzyme for acetyl-CoA and oxaloacetate were 6.7㎛ and 3.1㎛, respectively. Kinetic studies showed that citrate synthase follows the concerted mechanism which forms a ternary complex. Propionyl-CoA, ATP, and intermediates of the TCA cycle, succinyl-CoA and intermediates of the TCA cycle, succinyl-CoA and α-ketoglutarate, behaved as inhibitors in vitro. Using pig and chicken heart enzymes for comparison, we found similarities at the N-terminal region. However, in the Ouchterlony immunodiffusion test, the polyclonal antibody raised against Drosophila citrate synthase did not show any crossreaction with pig, chicken or pigeon enzymes.
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