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Aggregation Properties of the Acetylcholinesterase from the Central Nervous System of Manduca sexta
Author(s) -
Silver Lewis H.,
Prescott David J.
Publication year - 1982
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1982.tb06653.x
Subject(s) - manduca sexta , sedimentation coefficient , chromatography , chemistry , acetylcholinesterase , ionic strength , size exclusion chromatography , enzyme , sedimentation equilibrium , sedimentation , stokes radius , biochemistry , ultracentrifuge , biology , botany , sediment , paleontology , larva , aqueous solution
The CNS of the tobacco hornworm, Manduca sexta , provides a rich source of true acetylcholinesterase (AChE, acetylcholine hydrolase, EC 3.1.1.7.). Optimal extraction of the enzyme was obtained with a nonionic detergent at high ionic strength (1% Triton X‐100, 0.5 M NaCl). Velocity sedimentation of the Triton + salt‐extracted enzyme demonstrated a single peak whose sedimentation coefficient was dependent upon the enzyme concentration layered on top of the gradient. When more than 20 units were applied to the gradient, a sedimentation coefficient of 8.6 S (205,000) was obtained, and extrapolation to zero units yielded a 5.7 S (110,500) species. Sedimentation in the absence of detergents (1.0 M NaCl or 10 mM phosphate buffer, pH 7.4) yielded pelleted enzyme and species with mean values of 18.6 S (650,000) and 17.5 S (600,000), respectively. The detergent‐extracted enzyme also demonstrated a concentration‐dependent size in gel filtration experiments. When less than 300 units were applied to the column, a single species was recovered, with a molecular radius of 40.15 ± 2.08 Å, (108,000) or 43.4 ± 2.38 Å, (117,000) calculated by different methods. If the sample contained 300 to 1300 units, two species were observed, with molecular radii of 40.15 ± 2.08 Å or 43.4 ± 2.38 Å and 78.4 ± 3.94 Å, (319,000) or 80.25 ± 3.01 Å (326,000). Velocity sedimentation and gel filtration of AChE have demonstrated that the enzyme has a minimum molecular weight of approximately 110,000 and also exists as higher‐molecular‐weight aggregates of this value.