Studies on the topography of the catalytic site of acetylcholinesterase using polyclonal and monoclonal antibodies.
Polyclonal and monoclonal antibodies were generated against a synthetic peptide (25 amino acid residues) corresponding to the amino acid sequence surrounding the active site serine of Torpedo californica acetylcholinesterase (AChE). Prior to immunization, the peptide was either coupled to bovine serum albumin or encapsulated into liposomes containing lipid A as an adjuvant. To determine whether this region of AChE is located on the surface of the enzyme and thus accessible for binding to antibodies, or located in a pocket and thus not accessible to antibodies, the immunoreactivity of the antibodies was determined using enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, Western blots, and competition ELISA. The polyclonal antibody and several of the monoclonal antibodies failed to react with either Torpedo or fetal bovine serum AChE in their native conformations, but showed significant cross-reactivity with the denatured enzymes. Human serum butyrylcholinesterase, which has a high degree of amino acid sequence homology with these AChEs, failed to react with the same antibodies in either native form or denatured form. Chymotrypsin also failed to react with the monoclonal antibodies in either form. Eighteen octapeptides spanning the entire sequence of this region were synthesized on polyethylene pins, and epitopes of representative monoclonal antibodies were determined by ELISA. The reactivity of peptides suggest that a portion of the 25 mer peptide in AChE containing the active site serine is the primary epitope. It is not exposed on the surface of the enzyme and is most likely sequestered in a pocket-like conformation in the native enzyme.
Ogert
R A
RA
Division of Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100.
Gentry
M K
MK
Richardson
E C
EC
Deal
C D
CD
Abramson
S N
SN
Alving
C R
CR
Taylor
P
P
Doctor
B P
BP
D000110:Acetylcholinesterase / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002091:Butyrylcholinesterase / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007118:Immunoassay; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D014101:Torpedo
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D000110:Acetylcholinesterase; D002091:Butyrylcholinesterase