Open AccessMultiple Molecular Forms of Purified Human Erythrocyte Acetylcholinesterase
Author(s) -
OTT Peter,
JENNY Bruno,
BRODBECK Urs
Publication year - 1975
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1975.tb02322.x
Subject(s) - isoelectric point , chemistry , isoelectric focusing , chromatography , ionic strength , size exclusion chromatography , polyacrylamide gel electrophoresis , enzyme , enzyme assay , centrifugation , molecular mass , gel electrophoresis , biochemistry , organic chemistry , aqueous solution
1 Human erythrocyte acetylcholinesterase was solubilized by Triton X‐100 and purified by affinity chromatography to a specific activity of 3800 IU/mg of protein. The yield of the purified enzyme was 25–45%. 2 Gel filtration on Sepharose 4‐B in the presence of Triton X‐100 revealed one peak of enzyme activity with a Stokes' radius of 8.7 nm. Density gradient centrifugation in 0.1% Triton X‐100 showed one peak of enzyme activity with an S 4 value of 6.3S. 3 Isoelectric focusing in Triton X‐100 resolved the enzyme into five molecular forms with isoelectric points of 4.55, 4.68, 4.81, 4.98 and 5.18. Upon incubation with neuraminidase the enzyme activity in the first four forms was decreased with a concommitant increase in activity in the form with the higher isoelectric point. 4 After removal of excess Triton X‐100 on Bio‐Gel HTP, polyacrylamide gel electrophoresis showed seven bands of protein and corresponding bands of enzyme activity. Density gradient centrifugation of the detergent‐depleted enzyme at high ionic strength revealed five multiple molecular forms with S 4 values of 6.3 S, 10.2 S, 12.2 S, 14.2 S and 16.3 S. At low ionic strength, higher aggregates were observed in addition to the other forms. Dodecylsulfate‐polyacrylamide gel electrophoresis gave one subunit only with an apparent molecular weight of 80000. 5 These results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X‐100, exists in various forms differing in net charge but of apparently similar molecular dimensions. After removal of the detergent, forms with different molecular sizes are observed.