Purification and Properties of a Leucine-binding Protein from Escherichia coli

Abstract

Abstract A leucine-binding protein isolated from Escherichia coli K12 has been purified by DEAE-cellulose and hydroxylapatite cellulose chromatography and its chemical and physical properties were determined. The purified protein was crystallized by adding 2-methyl-2,4-pentanediol. The crystalline protein was homogeneous as judged by polyacrylamide electrophoresis, ultracentrifugation, and immunodiffusion. A molecular weight of 24,000 (ultracentrifugation), 36,000 (gel filtration), and 34,000 (amino acid analysis) was obtained. Amino acid analyses showed a low content of sulfur-containing amino acids. This protein proved to be heat-stable and could be reversibly denatured by urea. A large number of analogues were tested and showed that the binding was highly specific for the l isomers of the branched chain amino acids. The constants for the dissociation of the amino acid-protein complexes were less than 1.0 µm. The binding activity was not greatly affected either by changes in pH or ionic strength. Antiserum inhibited binding activity of the protein but had no effect on transport in whole cells. The synthesis of leucine-binding protein and the transport of branched chain amino acids were repressed in cells grown on leucine.