Purification and properties of acetylcholinesterase from human brain.
Acetylcholinesterase from human caudate nucleus and partial thalamus was purified by using Con A-Sepharose, short-arm and long-arm ligand Sepharose affinity chromatographies. SDS-PAGE of the purified AChE under the reduced condition showed one main band, corresponding to a molecular weight of 66 kD. The purified AChE with a specific activity of 3384 U/mg protein represented 20% activity of the homogenate supernatant. Analysis of purified AChE by gradient slab PAGE and DISC-PAGE with activity staining revealed the existence of monomer, dimer, tetramer, hexamer and octomer of the enzyme. The isoelectric point of AChE ranged between pH 5.6 and 6.0. Con A-Sepharose affinity chromatography retained most of the applied AChE activity implying that the enzyme is a kind of glycoprotein. The isolated human brain AChE had no cross-immunoreactivity with 3F3 and weak cross-immunoreactivity with 2G8 and 1H11 anti-Torpedo AChE antibodies. Balb/c mice were immunized with human cerebellum AChE purified with Con A and short-arm ligand affinity chromatographies. The antiserum produced showed strong cross-immunoreactivity with Torpedo AChE but weak cross-immunoreactivity with human RBC membrane AChE. The purified human brain striatum AChE was reduced and alkylated, and then hydrolyzed by immobilized TPCK-treated trypsin. Trypsin peptides in the hydrolysate was separated by RP-HPLC. Several large peptide peaks and numbers of small peaks were observed. The large peaks showed obvious immunoreactivity with the mouse anti human cerebellum AChE antiserum.
Zhu
M C
MC
Institute of Pharmacology and Toxicology, Beijing, PRC.
Xin
Y B
YB
Sun
M J
MJ
Fang
Y Z
YZ
D000110:Acetylcholinesterase / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D000911:Antibodies, Monoclonal; D001923:Brain Chemistry; D002421:Caudate Nucleus / Q000737:chemistry; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004910:Erythrocyte Membrane / Q000737:chemistry; D006801:Humans; D051379:Mice; D013788:Thalamus / Q000737:chemistry; D014101:Torpedo
D000911:Antibodies, Monoclonal; D000110:Acetylcholinesterase