Functional reconstitution of ATP-dependent transporters from the solubilized hepatocyte canalicular membrane.
The hepatocyte canalicular membrane contains several primary-active ATP-dependent export carriers including one for bile salts and one for leukotriene C4 and related conjugates. The molecular identity of both transporters has not been fully elucidated. To establish a transport assay that allows the purification and identification of the proteins involved in ATP-dependent bile salt transport and in leukotriene C4 transport, we reconstituted solubilized hepatocyte canalicular membranes into phospholipid bilayers using a rapid dilution method. The proteoliposomes formed exhibited both [3H]taurocholate and [3H]leukotriene C4 uptake, which was much higher in the presence of ATP than in the presence of the non-hydrolyzable ATP-analog AdoPP[CH2]P or in the absence of nucleotides. Nucleotide requirement and osmotic sensitivity of [3H]taurocholate transport indicates true transport into the vesicle lumen. Optimized conditions for reconstitution included the addition of a high concentration of an osmolyte (glycerol) and the presence of exogenous phospholipids (0.3%) during solubilization. Highest transport rates were obtained by reconstitution into acetone/ether-precipitated Escherichia coli phospholipid supplemented with 20% cholesterol and by use of octylglucoside concentrations between 30 mM and 50 mM. Taurocholate transport was non-competitively inhibited by vanadate (Ki = 39 microM). The kinetic parameters of cyclosporin A inhibition (Ki = 2.6 microM for taurocholate and 4.3 microM for leukotriene C4 transport) as well as the affinities of taurocholate (Km = 12 microM) and leukotriene C4 (Km = 0.5 microM) in the proteoliposome system indicate that the reconstitution resulted in functionally active transport systems, which are representative of ATP-dependent transport in the intact plasma membrane.
Büchler
M
M
Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Böhme
M
M
Ortlepp
H
H
Keppler
D
D
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002784:Cholesterol / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D017997:Leukotriene C4 / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013656:Taurocholic Acid / Q000378:metabolism
D001647:Bile Acids and Salts; D008081:Liposomes; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids; C020485:proteoliposomes; D017997:Leukotriene C4; D013656:Taurocholic Acid; C024990:asolectin; D000255:Adenosine Triphosphate; D002784:Cholesterol; C002630:alpha,beta-methyleneadenosine 5'-triphosphate