Lactose transport system of Streptococcus thermophilus. Functional reconstitution of the protein and characterization of the kinetic mechanism of transport.
The kinetic mechanism of the lactose transport system of Streptococcus thermophilus was studied in membrane vesicles fused with cytochrome c oxidase containing liposomes and in proteoliposomes in which cytochrome c oxidase was coreconstituted with the lactose transport protein. Selective manipulation of the components of the proton (and sodium) motive force indicated that both a membrane potential and a pH gradient could drive transport. The galactoside/proton stoichiometry was close to unity. Experiments which discriminate between the effects of internal pH and delta pH as driving force on galactoside/proton symport showed that the carrier is highly activated at alkaline internal pH values, which biases the transport system kinetically toward the pH component of the proton motive force. Galactoside efflux increased with increasing pH with a pKa of about 8, whereas galactoside exchange (and counterflow) exhibited a pH optimum around 7 with pKa values of 6 and 8, respectively. Imposition of delta pH (interior alkaline) retarded the rate of efflux at any pH value tested, whereas the rate of exchange was stimulated by an imposed delta pH at pH 5.8, not affected at pH 7.0, and inhibited at pH 8.0 and 9.0. The results have been evaluated in terms of random and ordered association/dissociation of galactoside and proton on the inner surface of the membrane. Imposition of delta psi (interior negative) decreased the rate of efflux but had no effect on the rate of exchange, indicating that the unloaded transport protein carries a net negative charge and that during exchange and counterflow the carrier recycles in the protonated form.
Foucaud
C
C
Department of Microbiology, University of Groningen, Haren, The Netherlands.
Poolman
B
B
D001426:Bacterial Proteins / Q000378:metabolism*; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism; D029968:Escherichia coli Proteins; D005697:Galactosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D007785:Lactose / Q000378:metabolism*; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D013291:Streptococcus / Q000378:metabolism*; D027981:Symporters
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005697:Galactosides; D007476:Ionophores; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D007785:Lactose