Kinetic mechanism and specificity of the arginine-ornithine antiporter of Lactococcus lactis.
The kinetic mechanism and specificity of the arginine-ornithine antiporter was investigated in membrane vesicles derived from Lactococcus lactis. Membrane vesicles loaded with ornithine, and diluted into an arginine-free medium, rapidly released a limited amount of ornithine during the first seconds of incubation. The amount of ornithine released was independent of the amount initially present on the inside and roughly matched the number of ornithine-binding sites in the membrane. Net flow of ornithine was only observed in membrane vesicles derived from induced cells and blocked by p-chloromercuribenzene sulfonic acid. These results suggest that net flow of ornithine is caused by a single turnover of the antiporter. With saturating concentrations of arginine in the external medium, efflux of ornithine was stoichiometrically coupled to uptake of arginine. Arginine-ornithine exchange and net flow of ornithine are electrically silent and not regulated by the electrical potential. The kinetics of the homologous exchange reactions indicate that the Vmax values for arginine and ornithine uptake are comparable, whereas the apparent Kt values differ. No major sidedness of the apparent Kt values are observed for both surfaces of the cytoplasmic membrane. Various basic amino acid analogues, including optical isomers, are transported as well, albeit with different efficiencies (Vmax/Kt). Evidence for a competitive character of arginine and ornithine interactions for binding sites on the antiporter are provided by transport and binding measurements. The Vmax and apparent Kt for arginine uptake increases with increasing internal ornithine, with little effect on the ratio of Vmax to apparent Kt. These results are discussed in terms of a simple carrier model in which the substrate-binding site is presented alternately to the two surfaces of the membrane as in a Ping Pong mechanism for enzyme kinetics.
Driessen
A J
AJ
Department of Microbiology, University of Groningen, Haren, The Netherlands.
Molenaar
D
D
Konings
W N
WN
D026905:Amino Acid Transport Systems; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D007700:Kinetics; D013294:Lactococcus lactis / Q000378:metabolism*; D009952:Ornithine / Q000378:metabolism*; D033902:Periplasmic Binding Proteins
D026905:Amino Acid Transport Systems; D002352:Carrier Proteins; D033902:Periplasmic Binding Proteins; C035727:arginine-ornithine periplasmic transport protein, bacteria; D001120:Arginine; D009952:Ornithine