Purification and Properties of a Component of Histidine Transport in Salmonella typhimurium

Abstract

Abstract The purification of the J histidine-binding protein, a component of high affinity histidine transport in Salmonella typhimurium, is described. A mutant strain producing elevated levels of this protein is used as the optimal source of this protein. The purification involves a modified osmotic shock procedure, chromatography on DEAE-cellulose, and isoelectric focusing. The final preparation is homogeneous by several criteria. The J protein has a molecular weight of 25,000 by gel filtration and 26,000 by sedimentation equilibrium. Histidine binding is reversible, with a dissociation constant for the J protein-histidine complex of 0.15 µm. The J protein at saturating substrate (10-6 m histidine) maximally binds 1 molecule of histidine per molecule protein. Histidine binding decreases slightly at high ionic strength, and decreases with increase in pH. Binding activity is relatively stable to boiling. The protein has an isoelectric pH of 5.5 and an amino acid composition representative of S. typhimurium protein. The J protein is located near the cell surface, as indicated by its release by osmotic shock and during spheroplast formation. The J protein has no detectable enzymatic activity. In addition to the J protein, osmotic shock treatment releases three separable components which bind histidine.