Molecular forms of acetylcholinesterase present in the white and grey matter of pig brain.

Extraction of the white matter of pig brain with EDTA, lysolecithin or Triton X-100 gave poor yields of soluble acetylcholinesterase although these agents had proved effective at solubilizing the enzyme in the grey matter. This finding, together with the observation that the strong detergent sodium deoxycholate, was needed to solubilize the enzyme, shows that it is more difficult to remove acetylcholinesterase from the white matter of brain than from the grey. This could mean that the enzyme in the white matter is more firmly bound to the membrane than the enzyme in the grey matter. The difference in binding of the enzyme from the two regions of the brain is also reflected in the affinity chromatography experiments which showed a lower recovery for the acetylcholinesterase of white matter compared with the enzyme from grey matter. Starch-block electrophoresis of acetylcholinesterase showed a single negatively charged peak of activity for both the naturally soluble and the deoxycholate solubilized preparations. The presence of only one form on electrophoresis suggests that the molecular species of acetylcholinesterase do not arise from differences in charge. Sucrose density gradient centrifugation of the two preparations from white matter gave a single peak of activity with a sedimentation constant of about 10 S. This corresponds closely to the major species of molecular weight 260,000 detected by gradient gel electrophoresis. Other forms detected in both enzyme preparations by gradient gel electrophoresis were species with molecular weights of 660,000, 180,000, 130,000 and 115,000. The significance of these species in terms of the formation of oligomers is discussed. A comparison was made with the corresponding preparations of acetylcholinesterase from the grey matter and the results showed that acetylcholinesterase from the white and grey matter of pig brain were very similar. The exception to this was the species with a molecular weight of 68,000 which was present in the grey but not the white matter of pig brain.

The interaction of purified acetylcholinesterase from pig brain with liposomes.

The binding of pig brain acetylcholinesterase to artificial phospholipid membranes was investigated at different temperatures. Calculation of the thermodynamic parameters revealed a small negative enthalpy change, but a large negative change in the free energy and a large positive change in the entropy on binding. The large entropy change might be interpreted as being responsible for forming the enzyme-membrane complex and was indicative of hydrophobic interactions between lipid and protein. This conclusion would also favour the hypothesis that the enzyme was an integral protein. Further support for this theory was provided by the study of acetylcholinesterase binding to liposomes containing the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Lowering the temperature below the transition temperature or incorporating cholesterol into the liposomes decreased enzyme binding. Both factors could be interpreted as decreasing the fluidity of the hydrocarbon side chains of the phospholipids, causing an increase in bilayer thickness due to closer packing of side chains. This membrane condensation would certainly not favour the binding of integral protein molecules.

Purification of acetylcholinesterase from pig cerebral cortex by affinity chromatography.

Acetylcholinesterase from pig cerebral cortex was solubilised with 1% (w/v) Triton X-100 and purified by affinity chromatography. Three different ligands were investigated and details are given for their preparation. The elution profile depended on the presence of Triton X-100, the ionic strength and the inhibitor used to remove the enzyme from the column as well as the nature of the affinity material. The most efficient purification was obtained when the enzyme was eluted from a column containing the acetylcholinesterase inhibitor [1-methyl-9-(Nbeta-epsilon-amino-caproyl)-beta-aminopropylamino] acridinium bromide hydrobromide covalently linked to Sepharose 4B. A recovery of 44% of the applied enzyme was eluted from the column with a specific activity of 148 mumoles min-1 mg-1 and a purification of 900-fold.