Lipid protein interactions in bovine erythrocyte acetylcholinestrase
Creators: name: Sekar, Chandra
Publisher: name: University of British Columbia
Year: 2010

description: Involvement of lipid in the activity of mammalian erythrocyte acetylcholinesterase (AChE) has been proposed by various workers. In bovine erythrocyte AChE a tightly bound fraction of cardiolipin (CL) was proposed to be involved in the modulation of AChE catalytic activity. Methods previously used for the isolation of CL resulted in enzyme denaturation. In the present study various methods for the separation of CL under non-denaturing conditions have been investigated. It was reported earlier that all the lipoprotein forms of the enzyme containing CL showed a biphasic Arrhenius plot with a break around 20°C. It was suggested that treatment of the enzyme with 1.8M sodium chloride, 2mM sodium phosphate, pH 7.4 or IM sodium bicarbonate pH 8-10, caused dissociation of cardiolipin and it was accompanied by abolition of the Arrhenius plot break. Methods used for the separation of CL from AChE were based on the difference in size and density between the two components. Enzyme was treated with "high salt" conditions which were postulated to cause dissociation of CL. The resulting mixture was passed through a Sephadex gel column so that CL can be separated from the enzyme because of its size difference. The enzyme obtained from the Sephadex gel column gave a partial specific volume of 0.81 ml/g, which is higher than that expected from the amino acid composition of the protein, indicating that CL is still bound to the enzyme. In another experiment ("flotation experiment") an attempt was made to separate the dissociated CL from the enzyme on a sucrose gradient, based on the density difference between the phopholipid and protein. Arrhenius plots were obtained at different time intervals on the enzyme recovered from the sucrose gradient. A linear Arrhenius plot was observed after 24 h. Storage of the enzyme for 5 to 8 days gave rise to a distinct break in the Arrhenius plot. The reapperarance of the break was observed even when the centrifugation was done in the presence of 0.09% Triton X-100. This was interpreted to indicate that endogeneous CL was bound to the enzyme through ionic and hydrophobic interaction. "High salt" treatment may abolish the ionic interaction, causing "functional dissociation" of CL (as shown by disappearance of the Arrhenius plot break) but simultaneous strengthening of the hydrophobic interactions may account for the reappearance of the break. The next method attempted for the separation of CL was based on the principle that, if enzyme could be bound to a solid support, then washing of the enzyme with chaotropic agents, detergents and "high salt" may result in the release of CL. The suitability of the N-methylacridinium (MAC) affinity column as a solid support for this enzyme was investigated. The choice of MAC as an affinity ligand was based on the recent reports regarding its suitability as an affinity ligand for purification of eel and pig brain AChE. The elution profile of the enzyme in 0.IM NaCl, 20mM sodium phosphate, pH 7.4 at different ligand (MAC) concentrations indicated that a minimum of 2.8 umole/ml gel required for sufficient retention of the enzyme. As the affinity of the ligand for the enzyme will further decrease with increasing ionic strength, the MAC affinity column, is unsuitable as a solid support for bovine erythrocyte AChE. It was found that the lower retention of the bovine erythrocyte AChE compared to the eel enzyme on the MAC affinity columns was due to the lower affinity of the former for the ligand, rather than to any structural difference or a different mode of binding. Preliminary findings suggest that CL dissociation did not alter the affinity of the enzyme for the ligand. Finally, as a primary requirement for the preparation of large quantities', of pure AChE, so that CL can be exchanged by a detergent exchange method, various steps for the purification of the native forms of AChE by a detergent free method have been characterized. The following findings were made. Butanol treatment enhanced the enzyme release .from the membrane, from 40 to 80 percent, by extracting the "mobile" phospholipids. The extraction of AChE can be increased by increasing the ionic strength of the medium and by calcium chelation. Purification of the above enzyme can be acheived by affinity purification but optimum conditions required for the above purification are still under investigation. Characterization of the molecular forms of the enzyme on sucrose density gradient indicates extensive aggregation at low ionic strength, while a lower degree of aggregation with a prominant 11S peak was observed in the presence of 0.IM sodium chloride, 20mM sodium phosphate.