Organic anion-transporting ATPase of rat liver. II. Functional reconstitution of active transport and regulation by phosphorylation.
We have previously purified to homogeneity from rat liver plasma membranes a 90-kDa glycoprotein with S-(2,4-dinitrophenyl)glutathione-stimulated ATPase activity and other properties which identify it as the multispecific organic anion transporter (MOAT) specific for the transport into bile of non-bile acid organic anions (Pikula, S., Hayden, J. B., Awasthi, S., Awasthi, Y. C., and Zimniak, P. (1994) J. Biol. Chem. 269, 27566-27573). In the present communication, we report the functional reconstitution of this protein into artificial proteoliposomes. The reconstituted protein catalyzed time- and concentration-dependent uptake of S-(2,4-dinitrophenyl)glutathione into the vesicles. The transport required the presence of ATP. Phosphorylation of the 90-kDa protein by protein kinase C prior to reconstitution more than tripled the Vmax of transport but did not change the Km for S-(2,4-dinitrophenyl)glutathione. The protein created and, at steady state, maintained a more than 200-fold and 500-fold S-(2,4-dinitrophenyl)glutathione gradient across the membrane for the unphosphorylated and phosphorylated form, respectively. The transport activity of the 90-kDa protein is sufficient to account for the hepatic secretory maximum of non-bile acid organic anions in the rat.
Pikula
S
S
Department of Medicine, University of Arkansas for Medical Sciences, Little Rock 72205.
Hayden
J B
JB
Awasthi
S
S
Awasthi
Y C
YC
Zimniak
P
P
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D027321:Anion Transport Proteins; D000838:Anions / Q000378:metabolism; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008567:Membranes, Artificial; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011510:Proteolipids; D051381:Rats; D014634:Valinomycin / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D027321:Anion Transport Proteins; D000838:Anions; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D008567:Membranes, Artificial; D011510:Proteolipids; C020485:proteoliposomes; D014634:Valinomycin; D014638:Vanadates; D011493:Protein Kinase C; D000251:Adenosine Triphosphatases; D005033:Ethylmaleimide