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Protein chemistry of acetylcholinesterase Morrod, Peter John


The protein acetylcholinesterase (AChE) has been isolated from the electroplax tissue of the electric eel (Electrophorus electricus) and purified by affinity chromatography. Working with fresh tissue, the structural stability of this kind of preparation towards proteolysis and/or autolysis has been investigated. Gel electrophoresis of the purified enzyme, in the presence of sodium dodecylsulphate and dithiothreitol, shows predominantly one component at 80,000 molecular weight. However, gels run at various times after purification demonstrate that the 80,000 polypeptide is susceptible to cleavage generating peptides of 55,000, 28,000 and 25,000 molecular weight. Evidence is presented to show that AChE is composed of four identical subunits arranged as a dimer of dimers (α₂)₂. Incubation of the freshly affinity purified AChE with trypsinis shown to mimic the cleavage of the 80,000 subunit by endogeneous protease. Sucrose gradient centrifugation of purified AChE shows it to be composed of two forms characterised by their sedimentation coefficients of 18S and 14S which upon proteolysis convert to a globular 11S form. Furthermore conversion of the 'native' molecular forms to the globular form occurs faster than proteolytic cleavage of the catalytic subunit. Some chemical modification of the protein is described in the last section of the thesis. The enzyme has been labelled, by two different and complementary methods, so as to incorporate radioactive iodide¹²⁵, I. Of particular interest is the result observed with an enzymatic, lacto-peroxidase iodination of the 11S form of the enzyme which shows that greater than 90% of the label is incorporated into the low molecular weight components of the subunit. The other results of the two iodination methods are described and discussed. Finally, an appendix describing the characterisation of AChE via isokinetic sucrose gradients in included.

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