ABCMdb

PMID: 8910331

Loo TW, Clarke DM
Inhibition of oxidative cross-linking between engineered cysteine residues at positions 332 in predicted transmembrane segments (TM) 6 and 975 in predicted TM12 of human P-glycoprotein by drug substrates.
J Biol Chem. 1996 Nov 1;271(44):27482-7., 1996-11-01 [PubMed]

Sentences

No. Mutations Sentence Comment

66 ABCB1 p.Phe335Cys ABCB1 p.Phe978Cys Accordingly, site-directed mutagenesis was used to change the codon for each residue surrounding Phe-335 and Phe-978 to cysteine (Fig. 1B). Login to comment 116 ABCB1 p.Leu975Cys ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys ABCB1 p.Leu332Cys B, the above experiment was repeated with Cys-less P-glycoprotein(His)10, mutants containing only one cysteine (L332C or L975C), or a mutant containing both cysteines (L332C ϩ L975C). Login to comment 117 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys C, mutant L332C ϩ L975C was treated with (lane 2) or without (lane 1) copper phenanthroline for 15 min at 37 °C and then solubilized with SDS sample buffer. Login to comment 138 ABCB1 p.Met949Cys ABCB1 p.Leu332Val Each residue at equivalent positions in predicted TM6 (residues Leu-332-Val-338) and TM11 (residues Met-949-Cys-956) was systematically changed to cysteine followed by treatment of the protein with oxidant. Login to comment 151 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys To test for this possibility, the cDNAs coding for Cys-less P-glycoprotein and mutant L332C ϩ L975C were transfected into NIH 3T3 cells, and drug-resistant colonies were selected in the presence of 45 nM colchicine or 5 nM vinblastine. Login to comment 156 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys ABCB1 p.Phe335Cys ABCB1 p.Phe978Cys A, membranes prepared from cells transfected with vector alone (control) or cotransfected with cDNA for mutant L332C in the Cys-less NH2-terminal half-molecule A52 and the cDNA for mutant L975C in the Cys-less COOH-terminal half-molecule A52 or with cDNA for mutant F335C in the NH2-terminal half-molecule A52 and the cDNA for mutant F978C in the COOH-terminal half-molecule A52 were treated with oxidant for various intervals and at different temperatures. Login to comment 158 ABCB1 p.Leu975Cys ABCB1 p.Leu975Cys ABCB1 p.Leu975Cys ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys ABCB1 p.Leu332Cys B, HEK 293 cells were co-transfected with cDNAs encoding Cys-less NH2-and COOH-terminal half-molecules A52; the cDNAs of mutant L332C in the NH2-terminal half-molecule A52 and the Cys-less COOH-terminal half-molecule A52; the cDNAs of Cys-less NH2-terminal half-molecule-A52 and mutant L975C in the Cys-less COOH-terminal half-molecule A52 or with the cDNAs of the mutant L335C in the NH2-terminal half-molecule A52 and mutant L975C in the COOH-terminal half-molecule A52. Login to comment 167 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys As shown in Fig. 5, the ATPase activities of both mutants (Cys-less and L332C ϩ L975C) were similar in the presence of 5 mM colchicine, 1 mM verapamil, and 100 ␮M vinblastine. Login to comment 168 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys Compared with the wild-type enzyme, both Cys-less and L332C ϩ L975C mutants had lower drug-stimulated ATPase activities. Login to comment