A simple procedure was developed for preparing two species of branched chain amino acid-binding proteins of Escherichia coli. The procedure involved osmotic shock, fractionation with ammonium sulfate and chromatography on DEAE-Sephadex and DEAE-cellulose columns. The major binding protein binds leucine, isoleucine, and valine and is referred to as LIV-BP, while the minor binding protein binds only leucine and is referred to as LS-BP.
Antibody against purified LIV-BP was prepared in rabbits. The antibody gave a single precipitin line with LIV-BP in an Ouchterlony double diffusion plate. The antibody also cross-reacted with LS-BP, but spur formation was observed, indicating a difference in the antigenicities of the two proteins.
A protein released by osmotic shock having binding activity for threonine was studied. The protein was co-purified with the LIV-BP activity, and purified, homogeneous LIV-BP was competitively inhibited by threonine. Based on these findings we propose that LIV-BP should be referred to as LIVT-BP, which specifically binds leucine, isoleucine, valine, and threonine.
The structure-binding activity relationship of the two binding proteins was investigated using various compounds which are structurally related to branched chain amino acids.