ABCMdb

ABCB1 p.Glu393Cys

None has been submitted yet.

Publications

PMID: 12820887 [PubMed] Gabriel MP et al: "Communication between the nucleotide binding domains of P-glycoprotein occurs via conformational changes that involve residue 508."

No. Sentence Comment

70 In each case, the primer introduced the desired cysteine residue (underlined) and also a silent, Table 1: Oligonucleotide Sequences Employed to Introduce Single Cysteine Mutations into Pgpa mutation restriction site sequence (5'-3') E393C -EcoRI GA AAT TTG tgt TTC AGA AAT R395C -EcoRI TTG GAg TTC tGt AAT GTT CAC M450C +HinCII GAG GGG tgt GTC AGT GTT GAc GGA CAG S452C +HinCII GGG ATG GTC tGT GTT GAc GGA CAG a Lowercase is used to denote mutated bases, while underlined triplets denote introduced codons for cysteine. Login to comment
149 Therefore, our investigations concentrated on three mutations located on the R-helical subdomain (I500C, N508C, K578C) and two on the surface of the -sheet ABC-specific subdomain (E393C, S452C). Login to comment
156 Unfortunately, mutants E393C (5 ( 0.4 µg) and S452C (13 ( 3 µg) produced significantly lower yields despite several attempts to increase expression by producing higher titer recombinant baculovirus. Login to comment
182 To Table 2: The Effects of Cysteine Replacement on Characteristics of ATP Hydrolysisa basal ATPase activity stimulated ATPase activity Pgp isoform n Km (mM) Vmax (µmol/min/mg) Km (mM) Vmax (µmol/min/mg) fold stimulation Cys-less 10 0.52 ( 0.05 0.29 ( 0.06 0.37 ( 0.04 0.83 ( 0.12 2.9 ( 0.2 E393C 3 0.39 ( 0.03 0.47 ( 0.25 0.18 ( 0.01 1.41 ( 0.48 3.4 ( 0.8 S452C 4 0.39 ( 0.03 0.49 ( 0.13 0.31 ( 0.04 1.19 ( 0.37 2.4 ( 0.7 I500C 4 0.20 ( 0.03 0.24 ( 0.07 0.24 ( 0.04 0.51 ( 0.21 2.6 ( 0.2 N508C 4 0.54 ( 0.12 0.21 ( 0.09 0.38 ( 0.03 0.52 ( 0.10 3.2 ( 0.5 K578C 4 0.64 ( 0.17 0.31 ( 0.13 0.35 ( 0.05 0.54 ( 0.21 2.0 ( 0.2 a ATPase activity was measured for each mutant isoform of Pgp as described in Materials and Methods. Login to comment
188 Table 3: The Effects of Cysteine Replacement on Drug Stimulation or Inhibition of ATP Hydrolysisa nicardipine vinblastine XR9576 vanadate Pgp isoform EC50 (µM) EC50 (µM) IC50 (µM) IC50 (µM) Cys-less 6.3 ( 0.1 4.6 ( 1.3 0.79 ( 0.27 3.4 ( 1.3 E393C 4.4 ( 1.4 10.8 ( 4.2 0.79 ( 0.16 4.6 ( 1.1 S452C 2.9 ( 0.8 8.2 ( 3.4 0.61 ( 0.24 4.9 ( 0.4 I500C 3.0 ( 0.6 5.4 ( 0.7 0.80 ( 0.19 5.1 ( 0.9 N508C 5.8 ( 0.9 6.2 ( 2.9 0.55 ( 0.15 7.4 ( 1.8 K578C 7.7 ( 0.5 12.4 ( 2.1 0.57 ( 0.11 4.3 ( 0.8 a ATPase activity was measured for each Pgp isoform in the presence of 2 mM Mg‚ATP with varying concentrations (3 nM to 100 µM) of either stimulatory drugs (nicardipine, vinblastine) or inhibitors (XR9576, vanadate). Login to comment
197 None of the mutants E393C (2.3 ( 2.1%), S452C (2.2 ( 1.9%), I500C (1.9 ( 1.2%), nor K578C (3.1 ( 1.1%) displayed significant inhibition of the drug stimulated ATPase activity in the presence of up to 100 µM NEM (data not shown). Login to comment
209 The E393C and S452C mutant proteins were labeled with [3H]-NEM; however, the presence of contaminating bands at around 140 kDa precluded accurate quantitation. Login to comment
220 Table 4: The Ability of [3H]-NEM to Label Pgp Isoformsa Pgp isoform fraction labeledc wild type 1.00 ( 0.06 Cys-less 0.08 ( 0.05 E393C n/db S452C n/d I500C 0.29 ( 0.08 N508C 0.33 ( 0.02 K578C 0.03 ( 0.05 a The various purified Pgp (250 ng) isoforms were incubated with 1 µM [3 H]NEM for 1 h at 20 °C and then subjected to SDS-PAGE (8%) and autoradiography to determine the relative accessibilities of the introduced cysteine residues to covalent modification. Login to comment
293 In contrast, the E393C, S452C, and I500C isoforms were labeled by NEM, yet this did not perturb the basal or drug stimulated ATPase activity. Login to comment