BINDING-PROTEIN-DEPENDENT ARGININE TRANSPORT IN PASTEURELLA-HAEMOLYTICA

A periplasmic arginine transport system that is a member of the ATP-de pendent transport superfamily was identified in Pasteurella haemolytic a. The gene encoding the periplasmic binding protein (lapT) was cloned and the protein overexpressed in Escherichia coli. LapT was purified to homogeneity using a modified osmotic shock procedure and anion-exch ange column chromatography. Filter-binding assays established that Lap T is an L-arginine-binding protein. Various amino acids were tested fo r their ability to inhibit L-arginine binding to LapT. When present in 100-fold excess, only L-arginine, D-arginine and citrulline competed with L-arginine for binding. Arginine transport in P. haemolytica whol e cells was competitively inhibited by the same amino acids, suggestin g that the LapT permease specifically transports L-arginine. The disso ciation constant for the L-arginine-LapT complex was 170 nM and the st oichiometry of binding was approximately 0.8 mol L-arginine (mol LapT) (-1). A polyclonal antibody raised against the purified protein permit ted detection of LapT in P. haemolytica periplasmic fractions.