PURIFICATION AND PROPERTIES OF ACETYLCHOLINESTERASE FROM HUMAN BRAIN

Acetylcholinesterase from human caudate nucleus and partial thalamus w as purified by using Con A-Sepharose, short-arm and long-arm ligand Se pharose affinity chromatographies. SDS-PAGE of the purified AChE under the reduced condition showed one main band, corresponding to a molecu lar weight of 66 kD. The purified AChE with a specific activity of 338 4 U/mg protein represented 20% activity of the homogenate supernatant. Analysis of purified AChE by gradient slab PAGE and DISC-PAGE with ac tivity staining revealed the existence of monomer, dimer, tetramer, he xamer and octomer of the enzyme. The isoelectric point of AChE ranged between pH 5.6 and 6.0. Con A-Sepharose affinity chromatography retain ed most of the applied AChE activity implying that the enzyme is a kin d of ,glycoprotein. The isolated human brain AChE had no cross-immunor eactivity with 3F3 and weak cross-immunoreactivity with 2G8 and 1H11 a nti-Torpedo AChE antibodies. Balb/c mice were immunized with human cer ebellum AChE purified with Con A and short-arm ligand affinity chromat ographies. The antiserum produced showed strong cross-immunoreactivity with Torpedo AChE but weak cross-immunoreactivity with human RBC memb rane AChE. The purified human brain striatum AChE was reduced and alky lated, and then hydrolyzed by immobilized TPCK-treated trypsin. Trypti c peptides in the hydrolysate was separated by RP-HPLC. Several large peptide peaks and numbers of small peaks were observed. The large peak s showed obvious immunoreactivity with the mouse anti-human cerebellum AChE anti-serum.