Acetylcholinesterase from Koi Carp Body Muscle: Tissue Distribution, Purification and Characterization
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概要
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The activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BtChE) were examined in various tissue homogenates of Koi carp (<I>Cyprinus carpio</I>). The AChE activity was highest in brain followed by body muscle, kidney, liver and spleen, and the BtChE activity was also highest in the brain. On the other hand, the activity ratio of AChE to BtChE was high (11 - 17) in the body muscle and plasma (or serum), and lower in the brain, kidney, liver and spleen (1 - 4). The AChE activity in the body muscle was mostly localized in the insoluble fraction when the homogenate was prepared with a buffer of low salt concentration, but more than 85 % of the activity in the fraction was extracted with another buffer containing 1 M NaCl. This suggests that most of AChE in the muscle is in the membrane-bound, collagen-tailed asymmetric forms. We purified AChE 79700-fold from the muscle homogenates and the final preparation had a specific activity of 14900 μmol?min<SUP>-1</SUP>?mg<SUP>-1</SUP> protein. Analysis of the purified AChE by SDS-polyacrylamide slab gel electrophoresis under the reducing condition also suggests that the AChE is in the collagen-tailed asymmetric forms and the molecular mass of the catalytic subunit was 76.8 kDa. The enzyme is subject to substrate inhibition, at high substrate concentrations, and showed a markedly higher V<SUB>max</SUB>/K<SUB>m</SUB> and a lower K<SUB>i</SUB> with acetylthiocholine as a substrate than those with propionyl- and butyrylthiocholine as substrates. The enzyme is highly sensitive to the known inhibitors for AChE and far less sensitive to those for BtChE. Those results confirm that the enzyme thus purified from Koi carp muscle is certainly an AChE.
- 日本環境毒性学会の論文
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