Activation and inactivation of bovine caudate acetylcholinesterase by trivalent cations

Abstract

Kinetic analysis of the interaction of trivalent cations with mammalian brain acetyl-cholinesterase revealed at least three distinct concentration-dependent effects on enzyme activity. Acetylcholinesterase was purified from bovine caudate nucleus by affinity chromatography to a specific activity of 1.1 mmoles acetylthiocholine · hr −1 · (mg protein) −1. The cations studied included the chloride salts of lanthanum, terbium, yttrium and scandium in low and high ionic strength buffers (2mM Pipes ± 0.1 M NaCl). At low ionic strength, high affinity noncompetitive or allosteric activation was observed at very low cation concentrations (1–10 μm); at higher concentrations (50–200 μM) these cations were noncompetitive inhibitors; and at 200–500 μM they exerted a mixed competitive-noncompetitive inhibition. Activation by low cation concentrations was not evident in high ionic strength buffers, while enzyme inhibition by all the trivalent cations was similar at low and high ionic strength. Inhibition by all of the multivalent cations was fully reversed by a 10-fold excess of EDTA or by a 100-fold dilution of the inhibited enzyme. The water-soluble carboxyl group affinity reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, was shown to specifically block the activating effect of the multivalent cations supporting the suggestion that the β- or “activator” peripheral anionic sites (P 1) involve a carboxyl group outside the enzyme active site.