ABCMdb

PMID: 11287418

Sauna ZE, Smith MM, Muller M, Ambudkar SV
Functionally similar vanadate-induced 8-azidoadenosine 5'-[alpha-(32)P]Diphosphate-trapped transition state intermediates of human P-glycoprotin are generated in the absence and presence of ATP hydrolysis.
J Biol Chem. 2001 Jun 15;276(24):21199-208. Epub 2001 Apr 3., 2001-06-15 [PubMed]

Sentences

No. Mutations Sentence Comment

55 ABCB1 p.Asp555Asn ABCB1 p.Asp1200Asn Preparation of Crude Membranes from HeLa Cells Expressing Mutant and Wild-type Pgp-A 70-80% confluent monolayer of HeLa cells was infected with vTF7-3 and transfected with pTM1-MDR1 (wild type) or pTM1-MDR1 bearing the homologous mutations at positions 555 (D555N) and 1200 (D1200N) as described previously (9). Login to comment 56 ABCB1 p.Asp555Asn ABCB1 p.Asp1200Asn The vaccinia virus expression vectors pTM1-MDR1 (wild type) and pTM1-MDR1(D555N) and pTM1-MDR1(D1200N) were provided by C. A. Hrycyna and M. M. Gottesman, Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda. Login to comment 224 ABCB1 p.Asp555Asn ABCB1 p.Asp1200Asn The Pgp mutants D555N and D1200N in Walker B domain of ATP sites do not show Vi-induced trapping of [␣-32 P]8-azido-ADP using either [␣-32 P]8-azido-ADP or [␣-32 P]8-azido-ATP. Login to comment 225 ABCB1 p.Asp555Asn ABCB1 p.Asp1200Asn Crude membranes were prepared from HeLa cells transiently expressing the pTM1-MDR1 (designated wild type) and pTM1-MDR1 bearing the homologous mutations in Walker B region of ATP sites at positions 555 and 1200 (designated D555N and D1200N), respectively. Login to comment 230 ABCB1 p.Asp555Asn ABCB1 p.Asp555Asn ABCB1 p.Asp1200Asn ABCB1 p.Asp1200Asn Lane 1, wild-type Pgp membranes labeled with [␣-32 P]8-azido-ATP; lane 2, D555N membranes labeled with [␣-32 P]8-azido-ATP; lane 3, D1200N membranes labeled with [␣-32 P]8-azido-ATP; lane 4, wild-type membranes labeled with [␣-32 P]8-azido-ADP; lane 5, D555N membranes labeled with [␣-32 P]8-azido-ADP; and lane 6, D1200N membranes labeled with [␣-32 P]8-azido-ADP. Login to comment 290 ABCB1 p.Asp555Asn ABCB1 p.Asp1200Asn The mutations in either the N-terminal ATP site (D555N) or the C-terminal site (D1200N) abolish Vi-induced trapping of [␣-32 P]8-azido-ADP by both the hydrolysis and non-hydrolysis routes (Fig. 6). Login to comment