ABCB4 p.Ser430Thr
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PMID: 10831598
[PubMed]
Urbatsch IL et al: "Conserved walker A Ser residues in the catalytic sites of P-glycoprotein are critical for catalysis and involved primarily at the transition state step."
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49
EXPERIMENTAL PROCEDURES Introduction of S430A, S430T, S1073A, and S1073T Mutations into Mouse mdr3 cDNA-Site-directed mutagenesis used the commercially available Altered Sites II method from Promega.
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ABCB4 p.Ser430Thr 10831598:49:47
status: NEW57 The S430T mutation was obtained as follows.
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ABCB4 p.Ser430Thr 10831598:57:4
status: NEW64 Mutations were transferred into plasmid pHIL-mdr3.5-His6 (24) using the following restriction fragments: S430A and S430T, BglII- SmaI; S1073A and S1073T, SpeI-SnaBI.
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ABCB4 p.Ser430Thr 10831598:64:115
status: NEW140 We generated mutations S430A, S430T, S1073A, and S1073T in mouse MDR3 Pgp, in order to study the catalytic role of these Ser residues.
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ABCB4 p.Ser430Thr 10831598:140:30
status: NEW159 Fig. 2 shows results obtained for wild-type, S430T, and S1073T mutants.
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ABCB4 p.Ser430Thr 10831598:159:45
status: NEW160 It is seen that maximal MgATPase activity of S430T and S1073T was around 80% of wild-type, and was obtained at equimolar concentration of Mg2ϩ and ATP as in wild-type.
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ABCB4 p.Ser430Thr 10831598:160:45
status: NEW180 Inhibition of MgATPase Activity of S430T and S1073T Mutant Pgp by Vanadate, Fluoroaluminate, and Beryllium Fluoride-Vi and AlFx are transition state analogs that trap MgADP tenaciously in the catalytic sites of Pgp, while BeFx is a ground-state analog that traps MgADP and mimics bound MgATP.
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ABCB4 p.Ser430Thr 10831598:180:35
status: NEW190 Fig. 4 shows results for wild-type, S430A, and S430T with 2.5 M nucleotide.
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ABCB4 p.Ser430Thr 10831598:190:47
status: NEW194 From Fig. 4 it is clear that, in mutants and wild-type, degree of labeling was the same in the S430A, S430T, and wild-type Pgp.
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ABCB4 p.Ser430Thr 10831598:194:102
status: NEW200 Metal dependence of ATPase activity of wild-type, S430T, and S1073T mutant Pgp.
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ABCB4 p.Ser430Thr 10831598:200:50
status: NEW202 E, wild-type; ƒ, S430T; Ⅺ, S1073T.
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ABCB4 p.Ser430Thr 10831598:202:23
status: NEW204 TABLE I MgATPase activity of wild-type and mutant Pgp preparations Mutant Pgp Specific ATPase activity KM(MgATP)a Kapp(verapamil)b No verapamil Plus verapamila mol/min/mg mM M Wild-type 0.3 4.0 (13-fold) 0.44 6.0 S430A NSc NSc S430T 0.26 3.3 (13-fold) 0.53 3.0 S1073A NSc NSc S1073T 0.18 3.2 (18-fold) 0.47 5.0 a Verapamil was included at 150 M, shown to be sufficient for maximal stimulation of activity.
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ABCB4 p.Ser430Thr 10831598:204:243
status: NEW212 Fig. 5A shows photolabeling of lipid-activated wild-type Pgp by 8-azido-[32 P]ATP. Fig. 5B shows that other nucleotide substrates, including natural ATP, competed well with 8-azido- ATP. Fig. 5 (C-F) shows results obtained with S430A, S430T, S1073A, and S1073T mutants.
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ABCB4 p.Ser430Thr 10831598:212:235
status: NEW220 Vi Trapping of Mg-[␣-32 P]ADP-Development of the centrifuge column procedure for use with pure Pgp allowed Vi trapping procedures to be carried out with the natural substrate MgATP, and for stoichiometry of trapped nucleotide to be calculated for the first time with pure protein. Fig. 6 shows that wild-type, S430T, and S1073T mutants each trapped Mg[␣- 32 P]ADP to the extent of 0.90-0.96 mol/mol of Pgp.
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ABCB4 p.Ser430Thr 10831598:220:317
status: NEW227 This showed that direct photolabeling is strongly disfavored in the transition state conformation.4 Vi Trapping of 8-Azido-[␣-32 P]ADP with Different Metals-It was shown above that S430T and S1073T mutants showed a different spectrum of ATPase activities from wild-type with varied metal cofactors and, interestingly, that S430A and S1073A mutants, which were inactive with MgATP as substrate, nevertheless showed very low hydrolysis activity with MnATP, CoATP and CaATP (Fig. 3).
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ABCB4 p.Ser430Thr 10831598:227:188
status: NEW229 With wild-type, S430T, and S1073T, Vi trapping occurred with all four divalent metals, but not in presence of EDTA.
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ABCB4 p.Ser430Thr 10831598:229:16
status: NEW243 TABLE II Inhibition of S430T and S1073T mutant Pgp by vanadate, fluoroaluminate, and beryllium fluoride, and reactivation rate after inhibition ATPase activity was assayed at 37 °C in presence of 1 mM NaATP, 3 mM MgCl2, 150 M verapamil, for 20 min, pH 7.5, at 37 °C in presence of Vi, AlFx, or BeFx added at varying concentration.
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ABCB4 p.Ser430Thr 10831598:243:23
status: NEW247 Mutant Pgp Vanadate Fluoroaluminate Beryllium fluoride IC50 a t1/2 b IC50 a t1/2 b IC50 a t1/2 b M min M min M min Wild-type 1.0 81 5.0 5.4 9.0 38 S430T 2.0 89 10 5.7 10 39 S1073T 2.0 73 10 5.5 15 40 a IC50, concentration at which 50% inhibition was observed.
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ABCB4 p.Ser430Thr 10831598:247:171
status: NEW248 b t1/2, half-time for reactivation of ATPase at 37 °C. DISCUSSION General-In this work we examined the mutants S430A, S430T, S1073A, and S1073T, affecting the Ser residue at the end of the Walker A sequence of the Nand C-terminal ATP-binding sites of mouse MDR3 P-glycoprotein.
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ABCB4 p.Ser430Thr 10831598:248:124
status: NEW269 After irradiation, protein was run on SDS gels and subjected to autoradiography. A, wild-type Pgp; B, S430A mutant; C, S430T mutant.
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ABCB4 p.Ser430Thr 10831598:269:119
status: NEW272 Panels A and C-F, Pgp (2 g) was incubated with 2.5 M 8-azido-[␣-32 P]ATP and 150 M verapamil in presence of increasing concentration of MgCl2 (the zero Mg2ϩ lane had 1 mM EDTA), irradiated by UV light, and the protein run on SDS gels then subjected to autoradiography. A, wild-type; C, S430A; D, S430T; E, S1073A; F, S1073T.
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ABCB4 p.Ser430Thr 10831598:272:333
status: NEW