Comparison of Biochemical and Pharmacokinetic Properties of Native and Recombinant Bovine Acetylcholinesterase

摘要

The successful demonstration of plasma-derived cholinesterases (ChEs) as organophosphorus compound (OP) scavengers is attributed to their ability to rapidly sequester a wide variety of OPs and to their long residence time in circulation. The production of large quantities of these enzymes is limited by high cost. However, the utilization of recombinant enzymes with similar biochemical and pharmacokinetic properties to those of the plasma- derived enzymes would provide a therapeutic solution. To further understand the biochemical and pharmacokinetic properties of different molecular forms of acetylcholinesterase (AChE), we cloned the mature T-subunit of bovine (Bo) brain AChE (1851 base pairs) including the bovine signal peptide sequence in exon2. The full-length cDNA clone was truncated at the C-terminus to obtain a 1733 base pair cDNA clone for the monomeric subunit of bovine brain AChE. Functional expression and secretion of the Bo AChE enzyme was achieved by transfecting CHOK1 cells. The secreted recombinant Bo AChE proteins, tetramer and truncated monomer forms, were purified, characterized and compared with fetal bovine serum (FBS) AChE. The catalytic and inhibitory properties of the two recombinant forms were very similar to those of FBS AChE. However, differences were observed in the reactivation of OP-inhibited recombinant forms by different oximes compared to FBS AChE. As expected, the two recombinant forms displayed different pharmacokinetic profiles, which were different from the plasma-derived enzyme. These results confirm our previous suggestion that the subunit assembly makes a significant contribution to the circulatory stability of ChEs.