Reconstitution of the L-leucine-H+ cotransporter of the plasma membrane from Chang liver cells into proteoliposomes.
L-Leucine is cotransported with H+ in the plasma membrane of Chang liver cells (Mitsumoto, Y. et al. (1986) J. Biol. Chem. 261, 4549). The leucine transport system was solubilized from the plasma membrane of the cells with ocytl glucoside and reconstituted in proteoliposomes prepared by a rapid dilution of a mixture of the solubilized proteins, octyl glucoside and liposomes. The proteoliposomes exhibited H(+)-gradient and electrical potential-stimulated leucine uptake. The H(+)-gradient-stimulated leucine uptake could be completely inhibited by carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (FCCP) and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH). The stimulatory effect of H+ gradient on leucine uptake was shown to be mainly due to decrease of the Km, but not to change of the Vmax, of the transport kinetics. These results suggest that the leucine-H+ cotransporter is solubilized and reconstituted into proteoliposomes.
Mitsumoto
Y
Y
Second Division, School of Pharmacy, Hokuriku University, Kanazawa, Japan.
Mohri
T
T
D000596:Amino Acids / Q000494:pharmacology; D000598:Amino Acids, Cyclic; D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002352:Carrier Proteins / Q000302:isolation & purification*; D002462:Cell Membrane / Q000737:chemistry*; D002478:Cells, Cultured; D029968:Escherichia coli Proteins; D005960:Glucosides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000737:chemistry; D033902:Periplasmic Binding Proteins; D012995:Solubility; D014634:Valinomycin / Q000494:pharmacology
D000596:Amino Acids; D000598:Amino Acids, Cyclic; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005960:Glucosides; D008081:Liposomes; C024913:LivK protein, E coli; D033902:Periplasmic Binding Proteins; D014634:Valinomycin; C006504:2-aminobicyclo(2,2,1)heptane-2-carboxylic acid; C018619:octyl-beta-D-glucoside; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D007930:Leucine