Two-site immunoradiometric assay of chicken acetylcholinesterase: active and inactive molecular forms in brain and muscle.
Several monoclonal antibodies were raised against chicken acetylcholinesterase (AChE; EC 3.1.1.7). Some of these antibodies react with quail AChE but not with AChEs from nonavian vertebrates or invertebrates and not with butyrylcholinesterase. They may be classified in several mutually compatible groups, i.e., that can bind simultaneously to the monomeric form of AChE. Most antibodies recognize a peptidic domain that does not exist in mammalian AChE and that may be digested by trypsin without loss of activity or dissociation of quaternary structure. The only exception is the antibody C-131, which is conformation dependent and preferentially recognizes active AChE. We have set up two-site immunoradiometric assays, using an immobilized capture antibody, C-6 or C-131, and a radiolabeled antibody, 125I-C-54. The C-6/C-54 assay quantifies the totality of inactive and active AChE subunits: It detects 10(-3) Ellman unit (approximately 40 pg of protein) and yields a linear response up to at least 25 10(-3) Ellman units. An analysis of gradient fractions, using C-6/C-54 and C-131/C-54 assays as well as activity determination, shows that the A12 and G4 forms are exclusively composed of active subunits, whereas inactive molecules cosediment with the active G2 and G1 forms. Both active and inactive G2 and G1 forms are amphiphilic, as indicated by the influence of detergents on their sedimentation coefficients and Stokes radii. In brain, the proportion of inactive forms decreases from 40% at embryonic day 11 (E11) to 20% at birth [day 1 (D1)]. In muscle, we observed no inactive AChE at E11 and a small proportion of inactive G1 at D1.(ABSTRACT TRUNCATED AT 250 WORDS)
Chatel
J M
JM
Laboratoire de Neurobiologie, CNRS URA 295, Ecole Normale Supérieure, Paris, France.
Eichler
J
J
Vallette
F M
FM
Bon
S
S
Massoulié
J
J
Grassi
J
J
D000110:Acetylcholinesterase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001921:Brain / Q000196:embryology / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D004789:Enzyme Activation; D015592:Immunoradiometric Assay / Q000379:methods*; D008823:Micelles; D009132:Muscles / Q000196:embryology / Q000201:enzymology*; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D008823:Micelles; D000110:Acetylcholinesterase