Mechanism of Na+/proline symport in Escherichia coli: reappraisal of the effect of cation binding to the Na+/proline symport carrier.
The proton and sodium ion dependences of the proline binding and transport activities of the proline carrier in Escherichia coli were investigated in detail. The binding activity in cytoplasmic membrane vesicles from a carrier over-producing strain (PT21/pTMP5) was absolutely dependent on the presence of Na+, but did not necessarily require protonation of the carrier, in contrast to the model previously reported (Mogi, T., Anraku, Y. 1984. J. Biol. Chem. 259:7797-7801). Based on this and previous observations, we propose a modified model of the proline binding reaction of the proline carrier, in which a proton is supposed to be a regulatory factor for the binding activity. The apparent Michaelis constant of proline (Kt) of the transport activity of cytoplasmic membrane vesicles from the wild type E. coli strain driven by a respiratory substrate, ascorbate, showed dependence on a low concentration of sodium ion. The Michaelis constant of sodium ion for transport (KtNa) was estimated to be 25 microM. The proline transport activities in membrane vesicles and intact cells were modulated by H+ concentration, the inhibitory effect of protons (pKa approximately equal to 6) being similar to that observed previously (Mogi, T., Anraku, Y. 1984. J. Biol. Chem. 259:7802-7806). Based on these observations and the modified model of substrate binding to the proline carrier, a model of the proline/Na+ symport mechanism is proposed, in which a proton is postulated to be a regulatory factor of the transport activity.
Yamato
I
I
Department of Biology, Faculty of Science, University of Tokyo, Japan.
Anraku
Y
Y
D026921:Amino Acid Transport Systems, Neutral; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002412:Cations / Q000378:metabolism* / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D018021:Lithium Chloride; D008956:Models, Chemical; D011392:Proline / Q000378:metabolism*; D011522:Protons; D012964:Sodium / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D027981:Symporters
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D002412:Cations; D002712:Chlorides; D029968:Escherichia coli Proteins; D011522:Protons; D027981:Symporters; C060609:PutP protein, E coli; D012965:Sodium Chloride; D011392:Proline; D008094:Lithium; D012964:Sodium; D018021:Lithium Chloride