Formes moleculaires multiples de la butyrylcholinesterase du plasma humain: II. electrophorèse d'affinité des formes C 1, C 3 ET C 4

Abstract

Multiple molecular forms of human plasma butyrylcholinesterase II.-Study of the C 1, C 3 and C 4 components by means of affinity electrophoresis Affinity electrophoresis has been applied to the analysis of the multiple molecular forms of human plasma cholinesterase allozyme U. A water-soluble p-amino-substituted-phenyltrimethylammoniumpolyacrylamide was synthetized by copolymerization of an unsaturated derivative of the ligand with acrylamide, and entrapped at various concentrations within the matrix of separating gels. Electrophoresis was carried out in these gels, and the relative mobility of the molecular forms of the enzyme was decreased. From the variation of mobility ( R m) as a function of immobilized ligand concentrations, the apparent dissociation constants of monomer (C 1), dimer (C 3) and tetramer (C 4) of phenotype U were calculated. The decrease in mobility was reversed by addition of non-immobilized competitive ligands ( N-methylpyridinium and N-methylacridinium). The appearance of the slopes of R mi -1 vs. concentration does not give sufficient information for determination of the number of anionic binding sites of C 4, but the slight curvature of the plots suggests that bivalent or higher interactions occur when the concentration is sufficiently high. For all three size isomers from a critical ligand concentration, a second zone, named B, appears and intensifies rapidly at the expense of the first zone (A) as the immobilized ligand concentration increases. Among several possible explanations of this phenomenon, it is proposed that the ligand induces a conformational isomerization of the enzymes with a change in affinity ( K D,B < K D,A), and that the interconversion process between the two states B⇆A is slow compared with the ligand-association⇄dissociation steps.