Kinetic analysis of lactose exchange in proteoliposomes reconstituted with purified lac permease.
Lactose exchange catalyzed by purified lac permease reconstituted into proteoliposomes was analyzed with unequal concentrations of lactose on either side of the membrane and at low pH so as to prevent equilibration of the two pools. Exchange with external concentrations below 1.0 mM is a single-exponential process, and the apparent affinity constants for external and internal substrate are close to the apparent KMs reported for active transport and efflux, respectively [Viitanen, P.V., Garcia, M. L., & Kaback, H. R. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1629]. At external lactose concentrations above 1.0 mM, a second kinetic pathway becomes evident with an apparent affinity constant of about 6 mM which is similar to the apparent KM for facilitated influx. A second pathway is not observed with respect to internal lactose even when the concentration is increased up to 80 mM. Furthermore, high internal or external lactose concentrations do not inhibit the exchange reaction. Biphasic kinetics with respect to external lactose are retained in a mutant permease that catalyzes exchange but is defective in H(+)-coupled lactose transport. It is suggested that lac permease has more than one binding site and that this may be the underlying reason for the biphasic kinetics observed for both exchange and H(+)-coupled lactose transport.
Lolkema
J S
JS
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Carrasco
N
N
Kaback
H R
HR
D001692:Biological Transport; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D066298:In Vitro Techniques; D007700:Kinetics; D007785:Lactose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D011510:Proteolipids; D013329:Structure-Activity Relationship; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; C021499:lactose permease; D007785:Lactose