Solubilization and partial characterization of acetylcholinesterase from the sarcotubular system of skeletal muscle.
Attempts were made to solubilize acetylcholinesterase (AChE) from microsomal membranes isolated from rabbit white muscle. The preparative procedure included a step in which the microsomes were incubated in a solution containing high salt concentration (0.6 M KCl). About 15% of the total enzyme activity could be solubilized with dilute buffer. Addition of EDTA (1 mM), EGTA (1 mM) or NaCl (0.5 and 1 M) to the extraction buffer did not improve the solubilization yield. Several non-ionic detergents and biliary salts were then used to bring the enzyme into solution. Triton X-100, C12E9 (dodecylnonaethylenglycol monoether) and biliary salt, above their critical micellar concentration, proved to be very effective as solubilizing agents. The occurrence of multiple molecular forms in detergent-soluble AChE was investigated by means of molecular sieving, centrifugation analysis, and slab gel electrophoresis. Experiments on gel filtration showed that, during the process, half of the enzyme was transformed into aggregates, the rest of the activity appearing as peaks with Stokes radii ranging from 3.7 to 7.9 nm. Both ionic strength and detergent nature modify the number and relative proportion of these peaks. Centrifugation analysis of Triton-saline-soluble AChE yielded molecular forms of 4.8S, 10-11S, and 13.5S, whereas deoxycholate extracts revealed species of 4.8S, 10S, and 15S, providing that gradients were prepared with 0.5 M NaCl. In the absence of salt, forms of 6.5-7.5S, 10S, and 15S were measured. The lightest species was always the predominant form. Slab gel electrophoresis showed several bands (68,000-445,000). The 4.8S component only yielded bands of 65,000-70,000.(ABSTRACT TRUNCATED AT 250 WORDS)
D000110:Acetylcholinesterase / Q000302:isolation & purification*; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D007425:Intracellular Membranes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008870:Microtubules / Q000201:enzymology*; D009132:Muscles / Q000201:enzymology*; D011817:Rabbits; D012995:Solubility; D013329:Structure-Activity Relationship
D000110:Acetylcholinesterase