Transport of Sugars and Amino Acids in Bacteria: I. Purification and Specificity of the Galactose- and Leucine-Binding Proteins

Abstract

Abstract Two proteins, a galactose-binding protein and a leucine-binding protein, have been isolated from shock fluid of a mutant of Escherichia coli K12 that lacks galactokinase. They were purified by conventional methods to a homogeneous state as judged by electrophoresis on acrylamide columns and other physicochemical criteria. The leucine-binding protein was crystallized by treatment with ammonium sulfate. Binding activity was measured by means of equilibrium dialysis. The osmotic shock procedure was an essential part of the scheme of purification. The purified galactose-binding protein was substantially free of galactokinase, acid hexose phosphatase, and uridine diphosphoglucose pyrophosphatase activities. Of a number of carbohydrates tested, only galactose and glucose were bound. A number of related sugars, including mannose, fucose, lactose, galactosamine, and galactose 1- and 6-phosphate did not interfere with the binding of galactose. The leucine-binding protein showed specificity for leucine, isoleucine, and valine but not for other amino acids tested. Dissociation constants were measured for the galactose-binding protein with galactose and glucose. The values were in the range of 10-6 m. The binding proteins carried out a reversible binding of galactose or leucine in the absence of any chemical alteration of the substrates.