Organic anion-transporting ATPase of rat liver. I. Purification, photoaffinity labeling, and regulation by phosphorylation.
An ATP-dependent transport system specific for non-bile acid organic anions such as S-(2,4-dinitrophenyl)-glutathione is present in the canalicular plasma membrane of hepatocytes. It has been shown recently that transport of these anions by isolated hepatocytes is modulated by the activity of cellular protein kinase C (Roelofsen, H., Ottenhoff, R., Oude Elferink, R. P., and Jansen, P.L. (1991) Biochem. J. 278, 637-641, 1991). Using a series of affinity chromatography steps, we have purified to apparent homogeneity a 90-kDa glycoprotein which has S-(2,4-dinitrophenyl)glutathione-dependent ATPase activity. As shown by binding to immobilized S-(2,4-dinitrophenyl)glutathione and by photoaffinity labeling with 8-azido-ATP, the binding sites for organic anions and for ATP of the 90-kDa protein are interdependent, a behavior that is consistent with an ATP-dependent transport function. The ATP binding site has been further characterized using a fluorescent ATP analog. The 90-kDa protein was phosphorylated by protein kinase C, and the Vmax (but not the Km) of the S-(2,4-dinitrophenyl)glutathione-dependent ATPase activity increased at least 2-fold upon phosphorylation. On the basis of its enzymatic properties, we propose that the 90-kDa protein is identical with the multispecific organic anion transporter (MOAT) of the hepatic canalicular plasma membrane. The size and oligosaccharide content of the 90-kDa protein indicate that it does not belong to the family of mammalian plasma membrane P-glycoproteins.
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 / Q000302:isolation & purification* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D027321:Anion Transport Proteins; D000838:Anions / Q000378:metabolism*; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D008099:Liver / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000737:chemistry; D008970:Molecular Weight; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats
D027321:Anion Transport Proteins; D000838:Anions; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D000251:Adenosine Triphosphatases