THE INHIBITION OF MALTOSE TRANSPORT BY THE UNLIGANDED FORM OF THE MALTOSE-BINDING PROTEIN OF ESCHERICHIA-COLI - EXPERIMENTAL FINDINGS AND MATHEMATICAL TREATMENT

Binding protein-dependent transport systems in Gram-negative enteric bacteria are multicomponent systems in which a soluble periplasmic binding protein of high substrate binding affinity establishes the major substrate recognition site. Usually, there are two integral membrane proteins which are thought to interact with the substrate loaded form of the binding protein to allow transport of substrate to occur. Transport is against the concentration gradient and needs energization by an ATP hydrolizing polypeptide. Overall transport is considered mainly unidirectional due to the high energy of ATP hydrolysis coupled to transport. In the study reported here, maltose transport in membrane vesicles in the presence of varying concentrations of unliganded maltose-binding protein but with constant amounts of maltose was measured. The conditions were chosen such that the concentration of maltose was always smaller than that of the binding protein and the initial concentration of the liganded binding protein was essentially constant. It was found that the initial rate of transport went through a maximum with increasing amounts of binding protein and declined thereafter. This finding strongly supports the conclusion that both the liganded and the unliganded forms of the binding protein interact with the membrane components of the transport system. The mathematical treatment of the experimental data allowed the ratio of the affinities for the membrane components of the substrate loaded and unloaded binding protein to be estimated. Published data on the binding protein-dependent transport of histidine in membrane vesicles of Salmonella typhimurium were also used. The data allowed the ratio of the binding affinity of the membrane components to the substrate-loaded and free binding protein to be determined. In addition, the K-M of transport to the K-D of binding protein was approximated. (C) 1995 Academic Press Limited