The Crystallization of Acetylcholinesterase (AChE) from Torpedo Electric Organ
Abstract
The objective of this project is the crystallization of the enzyme acetylcholinesterase (AChE), with the long-term objective of determining its three-dimensional structure and, thereby, the detailed topography of its active site. Torpedo electric organ was selected since it is a rich source of AChE and possesses an amino acid sequence very similar to that of mammalian AChE. A dimeric form of this enzyme was purified by a procedure which involved selective solubilization with a phosphatidyl-inositol-specific phospholipase C of bacterial origin, followed by affinity chromatography employing a Sepharose conjugate of a suitable quaternary affinity ligand. A highly purified AChE preparation was obtained in amounts which permitted a systematic attempt to crystallize the enzyme. We were able to obtain two different crystal forms which diffract to better than 3 resolution. One of these forms, which was obtained from ammonium sulfate-phosphate buffer, is capable of being 'shock-cooled' to liquid nitrogen temperatures, which permitted X-ray data to be collected at this temperature. This 'shock-cooling' procedure prolongs the lifetime of the crystal in the X-ray beam almost indefinitely and has already permitted us to collect a preliminary set of three-dimensional X-ray data. We are thus in a position to proceed with the determination of the three-dimensional structure of AChE.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 25, 1988
- Accession Number
- ADA203411
Entities
People
- I. Silman
- J. L. Sussman
Organizations
- Weizmann Institute of Science