Research Article 6: Antibody Mediated Activation of a Defective β-galactosidase (AMEF). Characteristics of Binding and Activation Processes

Abstract

INTRODUCTION
Recently Rotman and Celada (1968) described the activating effect of anti-β-galactosidase serum on AMEF, the gene product of a lac⁻ point mutant E. coli (lac 201, W6101). The magnitude of the activation is between 500 and 1000 times over the low native enzyme activity of AMEF. The specificity of the site(s) of attachment of the activating antibodies on the enzyme molecule is demonstrated by the fact that antisera obtained by injecting mutant enzyme, and undistinguishable from anti-z by immunodiffusion, are not activating (Celada, Ellis, Bodlund, and Rotman, in prep.). The molecular mechanism of activation is not known; it probably involves a conformational change of the AMEF tetramer. In view of the interest of such a phenomenon both from a physico-chemical and an immunological point of view, we have done experiments to determine (a) whether divalent antibodies are required to bring about activation, (b) the effect of varying antibody concentration on the enzyme activity reached by a given amount of AMEF, and (c) the kinetics of activation of antibody-AMEF mixtures. To interpret the results of (b)-type experiments, we also present a theoretical treatment of the data following lines suggested to us by Jeffries Wyman.

MATERIALS AND METHODS
1. Buffers and solutions. Buffer B contained 10 mM tris (hydroxymethyl) aminomethane, 10 mM MgCl₂,0.1 M Na Cl and 0.05M 2-mercaptoethanol and its pH was adjusted to 7.05 (23°C) with acetic acid. The complete buffer was prepared daily by adding mercaptoethanol to a stock salt solution. For papain cleavage of antibodies, the following...