ABCMdb

PMID: 16505485

Maki N, Moitra K, Ghosh P, Dey S
Allosteric modulation bypasses the requirement for ATP hydrolysis in regenerating low affinity transition state conformation of human P-glycoprotein.
J Biol Chem. 2006 Apr 21;281(16):10769-77. Epub 2006 Feb 27., 2006-04-21 [PubMed]

Sentences

No. Mutations Sentence Comment

50 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala Baculovirus Expression of Human Pgp-Recombinant baculoviruses BV-MDR1-(His6) (19) and BV-MDR1-(His6)-F983A (32) harboring the wild type and the Pgp F983A mutant, respectively, were used to infect High Five insect cells grown in Excell 400 medium as described (34). Login to comment 86 ABCB1 p.Phe983Ala No modulator-induced regeneration of the [125 I]IAAP binding was observed in the Pgp F983A mutant that is impaired in interaction with cis-(Z)-flupentixol (Fig. 1, A (bottom) and B). Login to comment 93 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala A, isolated membranes (0.1 mg/ml protein) from High Five insect cells, infected eitherwithrecombinantbaculovirusBV-MDR1(His6) or with BV-MDR1(His6)-F983A, were vanadate-trapped with 1.25 mM 8-azido-ATP (8-N3-ATP) and 0.25mM sodiumorthovanadate.Vanadate-trapped Pgp, either the wild type (top three panels) or the Pgp F983A mutant (bottom panel), in isolated membranes was pelleted, resuspended, and incubated in a vanadate- and nucleotide-free medium for the indicated time periods in the absence (second panel from top) and presence of either 1.25 mM 8-azido-ATP (top) or 25 ␮M cis-(Z)-flupentixol (third panel from top and bottom panel), prior to photocross-linking with 5 nM [125 I]IAAP. Login to comment 97 ABCB1 p.Phe983Ala Solid bar, none; open bar, 1.25 mM 8-azido-ATP; hatched bar, 25 ␮M cis-(Z)-flupentixol plus 0.25 mM EDTA; gray bar, F983A/25 ␮M cis-(Z)-flupentixol plus 0.25 mM EDTA. Login to comment