Biological Synthesis of a Protein Analogue of Acetylcholinesterase: Monoclonal Anti-Idiotype Antibody Analogue of the Esteratic Site

摘要

This study was designed to characterize the molecular structure of the active site of human acetylcholinesterase (AChE). Human erythrocyte acetylcholinesterase was purified to >98% Homogeneity by monoclonal antibody affinity chromatography and homogeneity by monoclonal antibody affinity chromatography and size-exclusion HPLC, and amino acid sequence data for >100 residues were obtained from tryptic, chymotryptic, and V-8 peptide fragments of the enzyme. These sequence data were used to synthesize oligonucleotide probes with which to screen four human cDNA libraries for the gene encoding human acetylcholinesterase. Putative positive clones were detected in one of these libraries, a muscle cDNA library in pBR322. In addition, a number of monoclonal antibodies that bind to acetycholinesterase were produced; however, only one of these antibodies, C1B7, inhibited the activity of the enzyme. C1B7 was extensively characterized and compared to the inhibitory antibody AE-2. A kinetic analysis of inhibition by C1B7 and AE-2 suggested that their inhibition of acetylholinesterase is non-competitive and allosteric. Thus, these two antibodies were not suitable for studies of the active site of AChE; nevertheless, they identify two new ly described sites on the enzyme that represent potential points of enzyme regulation. (aw)