ABCMdb

ABCB1 p.Cys1074Ala

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Publications

PMID: 10581363 [PubMed] Ueda K et al: "Comparative aspects of the function and mechanism of SUR1 and MDR1 proteins."

No. Sentence Comment

467 8-Azido-ATP binding with the wild-type MDR1 was inhibited by 100 WM NEM, while 8-azido-ATP binding with the C431A/C1074A mutant form was not, suggesting that the cysteines of Walker A motifs in both NBFs are responsible for the e¡ects of NEM on ATP binding. Login to comment
469 However, ATP-binding to the C1074A mutant form was signi'cantly reduced by the same treatment, similar to the wild-type MDR1. Login to comment
465 8-Azido-ATP binding with the wild-type MDR1 was inhibited by 100 WM NEM, while 8-azido-ATP binding with the C431A/C1074A mutant form was not, suggesting that the cysteines of Walker A motifs in both NBFs are responsible for the e&#a1;ects of NEM on ATP binding. Login to comment

PMID: 9733949 [PubMed] Takada Y et al: "Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein."

No. Sentence Comment

51 The C431A/C1074A double-mutant form of P-glycoprotein, in which the cysteine residues of the WA motif in both NBFs were replaced by alanine, trapped nucleotides even after treatment with 100 WM NEM (Fig. 1B). Login to comment
52 Also, these cysteine-to-alanine mutations did not a¡ect the function of P-glycoprotein, because the pattern and degree of multidrug resistance conferred by the C431A/ C1074A double-mutant form were similar to those conferred by the wild-type protein (data not shown). Login to comment
53 By contrast, vanadate-induced nucleotide trapping of the C431A and C1074A mutant forms, in which the cysteine residue of the WA motif in only one of the NBFs was replaced by alanine, was a¡ected by NEM (Fig. 1C,D). Login to comment
54 Nucleotide trapping with the C431A mutant form was inhibited by 10 WM NEM (Fig. 1C), and nucleotide trapping with the C1074A mutant form was inhibited by 50 WM NEM (Fig. 1D). Login to comment
56 E¡ects of NEM on vanadate-induced nucleotide trapping in P-glycoprotein. Plasma membrane proteins (about 20 Wg) from stable KB-3-1 transfectants expressing equivalent amounts of the wild-type human P-glycoprotein (A), the C431A/C1074A double-mutant form, in which the cysteine residues of Walker A in both NBFs were replaced by alanine (B), or the C431A (C) or C1074A (D) single-mutant form were treated with NEM at 1 WM (lane 2), 10 WM (lane 3), 50 WM (lane 4), or 100 WM (lane 5), or with NEM (lane 1). Login to comment
61 A, P-glycoprotein-S; B, C431A/C1074A; C, C431A; D, C1074A. Experiments were done in duplicate. Login to comment
64 P-Glycoprotein-S was labeled by 5 WM biotin maleimide and speci'cally inhibited by the presence of 100 WM NEM (Fig. 2A), but the C431A/C1074A mutant form was labeled little if at all (Fig. 2B). Login to comment
65 The C431A and C1074A mutant forms were also both labeled in an NEM-dependent manner by biotin maleimide (Fig. 2C,D), suggesting that biotin maleimide at a concentration of 5 WM specifically and uniformly labels the WA cysteines in both NBFs, as previously reported [16]. Login to comment
70 8-Azido-ATP binding with the C431A/C1074A mutant form was not inhibited by 100 WM NEM, but possibly increased (Fig. 3B), whereas 8-azido-ATP binding of the C431A mutant form appeared not to be a¡ected (Fig. 3C). Login to comment
71 However, ATP binding to the C1074A mutant form was signi'cantly reduced by treatment with 100 WM NEM (Fig. 3D), similar to the wild-type P-glycoprotein. Login to comment
77 A, P-glycoprotein-S; B, C431A/C1074A; C, C431A; D, C1074A. Experiments were done in triplicate. Fig. 4. Login to comment
94 The C431A/C1074A mutant form of P-glycoprotein was indistinguishable from the wild-type in its function. Login to comment
95 Also the C431A/C1074A mutant P-glycoprotein trapped nucleotides even after treatment with 100 WM NEM, indicating that the other 've cysteines were probably not accessible to NEM. Login to comment
97 However, the C431A/C1074A mutant P-glycoprotein showed an increase in ATP binding after NEM treatment (Fig. 3B), indicating that NEM modi'cation of other cysteine residues outside NBFs may allosterically a¡ect ATP binding in a positive manner. Login to comment
111 However, NEM treatment signi'cantly reduced 8-azido-ATP binding in the C1074A mutant P-glycoprotein. Login to comment