Chick acetylcholinesterase : purification, molecular properties and monoclonal antibodies
Acetylcholinesterase (AChE, E.C. 126.96.36.199) from 1-day chick brain was enriched over 2,000-fold by N-methylacridinium affinity column. Using this preparation as immunogen, two monoclonal antibodies (mAbs), 1E2 and 3D10, were isolated. Both mAbs react with all molecular forms of AChE from chick but not with butyrylcholinesterases (BuChE, E.C. 188.8.131.52). The mAb, 1E2, was used to immunopurify chick brain globular and muscle asymmetric AChE to homogeneity. The purified brain AChE showed a specific activity of 2,200 U/mg of protein, and it appeared to be a hydrophobic tetramer with a subunit mass of 105 kDa. The purified muscle asymmetric AChE has a specific activity of 1,100 U/mg of protein, it exhibits catalytic and inhibition properties characteristic of both AChE and BuChE and contains three distinct subunits with an apparent size of 110 kDa, 72 kDa and 58 kDa in the ratio 2:2:1. The discovery of an AChE/BuChE hybrid asymmetric form has been further supported by: (1) the identification of active site properties of AChE in the 110kDa subunit and of BuChE in the 72-kDa subunit, (2) the purification and precipitation of both activities by a BuChE-specific mAb (7D11), and (3) the evidence that all subunits are bound in the asymmetric form by disulphide bonds. The 58-kDa subunit is the only one that is sensitive to digestion with purified collagenase; it carries the collagenous 'tail'.