Both P-glycoprotein nucleotide-binding sites are catalytically active.
The technique of vanadate trapping of nucleotide was used to study catalytic sites of P-glycoprotein (Pgp) in plasma membranes from multidrug-resistant Chinese hamster ovary cells. Vanadate trapping of Mg- or Co-8-azido-nucleotide (1 mol/mol of Pgp) caused complete inhibition of Pgp ATPase activity, with reactivation rates at 37 degrees C of 1.4 x 10(-3) s-1 (t1/2 = 8 min) or 3.3 x 10(-4) s-1 (t1/2 = 35 min), respectively. UV irradiation of the inhibited Pgp yielded permanent inactivation of ATPase activity and specific photolabeling of Pgp. Mild trypsin digestion showed that the two nucleotide sites were labeled in equal proportion. The results show that both nucleotide sites in Pgp are capable of nucleotide hydrolysis, that vanadate trapping of nucleotide at either site completely prevents hydrolysis at both sites, and that vanadate trapping of nucleotide in the N- or C-terminal nucleotide sites occurs non-selectively. A minimal scheme is presented to explain inhibition by vanadate trapping of nucleotide and to describe the normal catalytic pathway. The inhibited Pgp-Mg-nucleotide.vanadate complex is probably an analog of the catalytic transition state, implying that when one nucleotide site assumes the catalytic transition state conformation the other site cannot do so and suggesting that the two sites may alternate in catalysis.
Urbatsch
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IL
Department of Biochemistry, University of Rochester Medical Center, New York 14642, USA.
Sankaran
B
B
Bhagat
S
S
Senior
A E
AE
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D016466:CHO Cells; D002384:Catalysis; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D006224:Cricetinae; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism / Q000502:physiology; D014638:Vanadates / Q000378:metabolism / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; D009711:Nucleotides; D010710:Phosphates; D003035:Cobalt; D014638:Vanadates; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008274:Magnesium