Purification and properties of a periplasmic D-xylose-binding protein from Escherichia coli K-12.

Escherichia coli contains a cold osmotic shock-releasable binding protein for D-XylOSe which has been purified and characterized.The D-xylose-binding protein has an apparent molecular weight of 37,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid analysis.D-Xylose-binding protein has an apparent absolute specificity for D-XY- lose and binds one molecule of D-XylOSe per molecule of protein with a Kd of 0.6 PM.The protein exhibits native tryptophan fluorescence (hex 284 nm, he, 345 nm) and a fluorescence enhancement (32%) associated with D-xylose binding which saturates with a Kd of 0.5 PM.D-Xylose-binding protein does not bind to a Cibacron blue 3FGA affinity column but does interact with a tetraiodofluorescein-agarose affinity column.D-XYlose-binding protein interaction with tetraiodofluorescein, if present, does not produce a red shift in the absorption spectrum of the dye in contrast to the L- arabinose-and D-galactose-binding proteins.Tryptophan oxidation with N-bromosuccinimide results in a concomitant drop in xylose binding.D-Xylose-binding protein contains no cysteine and possesses a PI of 7.4.In recent years, considerable evidence has bee? obtained implicating a transport and, in some cases, a chemotactic role for a group of periplasmic, osmotic shock-releasable proteins which are generally termed binding proteins (1, 2).These proteins are capable of specific, high affinity binding of certain metal ions, carbohydrates, amino acids, and other metabolites (2).Carbohydrate-binding proteins for L-arabinose (3-5), Dribose (6, 7), D-gahCtOSe (3, 4, 8, 9), and maltose (10) have been previously purified and characterized.D-Xylose transport in Escherichia coli has been reported previously (11-15), and it has been suggested that the genes coding for transport, xylose isomerase and xylulokinase, are probably regulated as an operon (11-14).L-Arabinose and D- ribose are transported via two transport systems, a "low affinity" and a "high affinity" transport system; in both cases, binding proteins have been found to be associated with the high affinity transport system (3, 7, 15, 16).Studies in this laboratory and others have indicated the presence of at least two xylose uptake systems with varying affinities (14, 15).Preliminary studies of xylose transport in E. coli K-12 have indicated the release of a periplasmic binding protein subsequent to osmotic shock and a reduction of the high affinity