P-glycoprotein Is Stably Inhibited by Vanadate-induced Trapping of Nucleotide at a Single Catalytic Site

Using plasma membranes from a multidrug-resistant Chinese hamster ovary cell line, which are highly enriched in Pgp, we show that vanadate-induced trapping of nucleotide at a single catalytic site produces stably inhibited Pgp, with t for reactivation of ATPase activity of 84 min at 37°C and >30 h at 4°C

Ina L. Urbatsch; Banumathi Sankaran; Joachim Weber; Alan E. Senior

2002

Scholarcy highlights

  • P-glycoprotein shows drug-stimulated ATPase activity
  • Using plasma membranes from a multidrug-resistant Chinese hamster ovary cell line, which are highly enriched in Pgp, we show that vanadate-induced trapping of nucleotide at a single catalytic site produces stably inhibited Pgp, with t for reactivation of ATPase activity of 84 min at 37°C and >30 h at 4°C
  • The finding that vanadate trapping of nucleotide at just one site/Pgp is sufficient to give full inhibition of ATPase activity shows that the two predicted nucleotide sites can not function independently as catalytic sites
  • Plasma membranes were preincubated with varied concentrations of vanadate in the presence of ATP and MgSO4 and the Pgp-ATPase activity determined after removal of unbound ligands
  • Experiments were carried out usingATP orUTP with either magnesium or cobalt ions in the preincubation phase in order to trap radioactive nucleotide in the catalytic sites, the samples eluting from centrifuge columns were irradiated at 254 nm in order to test whether direct photolabeling of the Pgp by nucleotide occurred
  • Inhibition of ATPase activity was fully reversible with a half-life of 84 min at 37°C, but reactivation was very slow at 4°C with a half-life of >30 h
  • We note that vanadate-induced inhibition was demonstrated to occur in purified, reconstituted Pgp, with characteristics the same as those seen in plasma membrane Pgp. the procedure of vanadate trapping of nucleotide may be applied with advantage to studies of pure Pgp. We thank Sumedha Bhagat for excellent technical assistance and Dr E

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