Research Article 3: On the Stoichiometry and Kinetics of ω Complementation of E. coli β-galactosidase

Abstract

We have reported elsewhere on observations which demonstrate that certain complementary fragments of the normally single polypeptide chain which forms the protomer of E. coli β-galactosidase, may reassemble both in vivo and in vitro into an enzymatically active structure [For references and map positions of the mutants see Ullmann and Perrin, this volume.] The remarkably high and specific affinity exhibited by these polypeptide fragments, and their capacity to reconstruct a structure presumably close to the native one, albeit via a very different pathway, poses some interesting questions concerning the mechanism of formation of tertiary and quaternary structures. In the present paper we report some, as yet in-complete, experiments aimed at analyzing this mechanism. These experiments concern the kinetics and Stoichiometry of the so called "ω complementation". Let us recall that the ω peptide, formed by certain mutants of β-galactosidase corresponds to the last (operator distal) quarter, approximately, of the normal polypeptide. A complementary fragment or "ω acceptor" may be furnished by any mutant (including deletions and nonsense mutations) within the ω segment. Most of the experiments reported here were done using crude extracts of mutants containing respectively, the ω peptide and the ω acceptor peptide. The relative amounts of each of the two peptides, in crude extracts can be estimated by titration against an excess of its complement under standardized conditions.

The ω peptide has been purified and its molecular weight determined as 39,000. The ω acceptor has not, as yet, been purified in a state where it is still...