Solubilization, purification, and salt activation of acyl-acyl carrier protein synthetase from Escherichia coli.
Acyl-acyl carrier protein (acyl-ACP) synthetase is an inner membrane enzyme from Escherichia coli that ligates free fatty acids to acyl carrier protein (ACP) in the presence of ATP and M&+. Acyl-ACP synthetase is neither a component of the /I oxidation (fad) regulon nor catabolite repressed by g...
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| Main Authors: | , |
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| 格式: | Artigo |
| 语言: | 英语 |
| 出版: |
1979
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| 在线阅读: | https://doi.org/10.1016/s0021-9258(18)50292-1 |
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| 总结: | Acyl-acyl carrier protein (acyl-ACP) synthetase is an inner membrane enzyme from Escherichia coli that ligates free fatty acids to acyl carrier protein (ACP) in the presence of ATP and M&+. Acyl-ACP synthetase is neither a component of the /I oxidation (fad) regulon nor catabolite repressed by growth on glucose. The enzyme has been solubilized by Triton X-100 and purified 3000-fold over the crude extract. Acyl-ACP synthetase has a half-life of 2 min at 55°C; however, in the presence of ATP the half-life increases to >32 min at 55°C. M&’ is not required for ATP to protect the enzyme from heat denaturation. We found no evidence for a phospholipid requirement for the solubilized protein. Kinetic constants were determined for ATP (0.3 DIM), ACP (5 PM), palmitate (5.3 PM), and oleate (30 PM). Acyl-ACP synthetase activity is also dependent on the presence of salt in the assay system. With the exception of M&+, no specific ion requirements were found and all salts tested are capable of activating the enzyme to comparable extents. The salt effect is not related to ionic strength per se since the more chaotropic the ion, the lower the concentration required to maximally activate acyl-ACP synthetase. The ATP-induced heat stability of the protein is abolished by salt concentrations that maximally activate acyl-ACP synthetase. We conclude that the salts exert a destabilizing influence on the acyl-ACP synthetase protein that is required for the production of acyl-ACP in vitro. |
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