P-glycoprotein. ATP hydrolysis by the N-terminal nucleotide-binding domain.
Two ATP-binding domains are found in members of the family of ATP-dependent transport proteins, which includes P-glycoprotein and cystic fibrosis transmembrane conductance regulator. To investigate the involvement of the two ATP-binding domains in the ATPase activity of P-glycoprotein, full-length and the 5'-half of human MDR1 cDNA, which encodes P-glycoprotein, were fused with the Escherichia coli lacZ gene and expressed in NIH3T3 cells. Immunoprecipitated full-length P-glycoprotein beta-galactosidase showed ATPase activity with apparent specific activity of 180 nmol/mg/min, a value higher than previously reported, in the presence of phospholipids, suggesting that stabilization of the transmembrane domains is necessary for ATP hydrolysis. N-terminal half P-glycoprotein-beta-galactosidase also showed ability to hydrolyze ATP but with slightly lower specific activity. Both ATPase activities showed similar characteristics when the effect of several inhibitors was analyzed, indicating that the N-terminal ATP-binding domain contains all residues necessary to hydrolyze ATP without interacting with the C-terminal ATP-binding domain.
Department of Agricultural Chemistry, Kyoto University, Japan.
A M
AM
Shimabuku
T
T
Nishimoto
K
K
Ueda
T
T
Komano
D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008562:Membrane Glycoproteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D001616:beta-Galactosidase; D000251:Adenosine Triphosphatases