This thesis describes the affinity purification of the enzyme acetylcholinesterase from the electric organ tissue of the electric eel (Electrophorus electricus) and the characterization of the enzyme by selective proteolytic cleavage monitored by sucrose gradient sedimentation,
sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel chromatography. It describes conditions, using N-nmethylacri-dinium-Sepharose 2B, for the purification of the asymmetric forms of the enzyme from high salt extracts of electric tissue and for the purification of the globular form of the enzyme subsequent to treatment with the enzyme trypsin. In addition it describes for the first time the selective purification of either asymmetric or globular acetylcholinesterase
from mixtures containing both forms of the enzyme. A distinction between autolytic and tryptic degradation of asymmetric acetylcholinesterase is described for the first time and two new forms of the enzyme generated by collagenase proteolysis of the asymmetric
18S and 14S forms are described. The species derived from the 18S form of acetylcholinesterase has a sedimentation coefficient of 21.IS and a Stokes radius of 12.9 nm while the 14S form gives rise to a 17.3S species with a Stokes radius of 11.1 nm. The changes in the sodium dodecyl sulphate-polyacrylamide gel electrophoresis migration pattern of acetylcholinesterase fragments following trypsin or collagenase
proteolysis and the changes in sedimentation coefficient and Stokes radius with collagenase proteolysis are compared to identify a component with a molecular weight of 45,000 daltons on electrophoresis gels, that contributes greatly to the asymmetry but only minimally to
the mass of the 18S and 14S forms of acetylcholinesterase. An appendix discusses some efforts at the purification of the individual subunits of the 18S and 14S forms of acetylcholinesterase and describes several observations made on the proteolytic instability of even highly purified asymmetric acetylcholinesterase. / Science, Faculty of / Chemistry, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/21299 |
| Date | January 1978 |
| Creators | Webb, Geoffrey |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
Page generated in 0.0018 seconds