[38] ATPase activity of Chinese hamster P-glycoprotein

Senior, Alan E.; Al‐Shawi, Marwan K.; Urbatsch, Ina L.

doi:10.1016/s0076-6879(98)92040-7

Cited by 72 publications

(96 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Substrate-stimulated ATPase activity has been analyzed in detail for P-glycoprotein (31,33). However, many puzzling questions remain unanswered: First, different values of the ATPase activity have been reported.…”

Section: Discussionmentioning

confidence: 99%

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

Gorbulev

Abele

Tampé

2001

Proc. Natl. Acad. Sci. U.S.A.

115

114

View full text Add to dashboard Cite

The transporter associated with antigen processing (TAP) is essential for intracellular transport of protein fragments into the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. On the cell surface, these peptide-MHC complexes are monitored by cytotoxic T lymphocytes. To study the ATP hydrolysis of TAP, we developed an enrichment and reconstitution procedure, by which we fully restored TAP function in proteoliposomes. A TAPspecific ATPase activity was identified that could be stimulated by peptides and blocked by the herpes simplex virus protein ICP47. Strikingly, the peptide-binding motif of TAP directly correlates with the stimulation of the ATPase activity, demonstrating that the initial peptide-binding step is responsible for TAP selectivity. ATP hydrolysis follows Michaelis-Menten kinetics with a maximal velocity V max of 2 mol͞min per mg TAP, corresponding to a turnover number of approximately 5 ATP per second. This turnover rate is sufficient to account for the role of TAP in peptide loading of MHC molecules and the overall process of antigen presentation. Interestingly, sterically restricted peptides that bind but are not transported by TAP do not stimulate ATPase activity. These results point to coordinated dialogue between the peptide-binding site, the nucleotide-binding domain, and the translocation site via conformational changes within the TAP complex.membrane proteins ͉ transport mechanism ͉ ATPase ͉ antigen processing A n essential step in the major histocompatibility complex (MHC) class I-mediated cellular immune defense is the transport of antigenic peptides from the cytoplasm into the endoplasmic reticulum (ER) (reviewed by ref. 1). The transporter associated with antigen processing (TAP) translocates peptides that are mainly derived from the proteasomal degradation into the ER (reviewed by ref. 2). Subsequently, peptides bind to MHC class I molecules and are presented at the cell surface, where they are recognized by cytotoxic T lymphocytes.TAP belongs to the family of ATP-binding cassette (ABC) transporters that, under consumption of ATP, translocate a vast spectrum of substrates across membranes (3). On the basis of sequence comparison, TAP fits in the B subfamily of human ABC transporters (www.gene.ucl.ac.uk͞users͞hester͞abc.html), including the well-characterized P-glycoprotein (ABCB1). The TAP complex forms a heterodimer of TAP1 (ABCB2) and TAP2 (ABCB3), where each subunit comprises a hydrophobic transmembrane domain and a cytoplasmic nucleotide-binding domain (NBD) containing the Walker A͞B and C-loop consensus sequences. The transmembrane domains are thought to form a channel by which peptides are transported across the ER membrane driven by ATP hydrolysis. The coupling between NBDs, the substrate-binding site, and the translocation pathway is not understood and is a central topic to be studied for all ABC transporters.TAP is part of a macromolecular peptide translocation and loading complex that comprises TAP1, TAP2, tapasin, MHC class I hea...

show abstract

Section: Discussionmentioning

confidence: 99%

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

Gorbulev

Abele

Tampé

2001

Proc. Natl. Acad. Sci. U.S.A.

115

114

View full text Add to dashboard Cite

show abstract

“…Both ATP sites are essential for the drug transport function of Pgp (7-10) but are believed to hydrolyze ATP in an alternate sequence (11)(12)(13). Pgp possesses a basal rate of ATP hydrolysis that is stimulated upon interaction with many transport substrates as well as with several Pgp modulators (14).…”

Section: Hydrolysismentioning

confidence: 99%

“…Vanadate (Vi), a phosphate analog, replaces phosphate at the catalytic site (30) when present during ATP hydrolysis. Due to its low rate of dissociation, vanadate stabilizes Pgp in a catalytic intermediate (mimicking a conformational state) that immediately follows the first ATP hydrolytic event (11,18,19,22,23). Trapping of ADP and vanadate at the catalytic site, following ATP hydrolysis, leads to a dramatic decrease in the affinity of Pgp for its transport substrates, such as […”

Section: Hydrolysismentioning

confidence: 99%

“…Vanadate (Vi), a phosphate analog, replaces phosphate at the catalytic site (30) when present during ATP hydrolysis. Due to its low rate of dissociation, vanadate stabilizes Pgp in a catalytic intermediate (mimicking a conformational state) that immediately follows the first ATP hydrolytic event (11,18,19,22,23 (18,19). This is accompanied by an experimentally detectable conformational change (15,17).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Allosteric Modulation Bypasses the Requirement for ATP Hydrolysis in Regenerating Low Affinity Transition State Conformation of Human P-glycoprotein

Maki¹,

Moitra²,

Ghosh

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

The human P-glycoprotein (Pgp) 3 functions as an ATP-driven drug transporter conferring multidrug resistance in cancer cells and restricting bioavailability of many antimicrobial and anticancer agents (1, 2). It is a 1280-amino acid integral membrane protein of the ATP-binding cassette (ABC) transporter family (3, 4), with two highly homologous halves, each containing a hydrophobic transmembrane domain and a relatively hydrophilic cytosolic domain. Each hydrophobic domain contains six putative transmembrane helices that, in conjunction with the transmembrane regions of the other half, form the drug-translocating pathway (substrate site) across the lipid bilayer (1, 5). The cytosolic domains each contain three consensus sequences (3) that together contribute to the formation of two ATP binding/hydrolysis sites (ATP sites) (6).Both ATP sites are essential for the drug transport function of Pgp (7-10) but are believed to hydrolyze ATP in an alternate sequence (11-13). Pgp possesses a basal rate of ATP hydrolysis that is stimulated upon interaction with many transport substrates as well as with several Pgp modulators (14). Binding and hydrolysis of ATP induce a conformational change in that is coupled to substrate translocation across the lipid bilayer and its subsequent dissociation (18 -24). The two ATP sites of Pgp were believed to be functionally equivalent with drug translocation and regeneration of the transporter directly coupled to a single round of ATP binding or/and hydrolysis (12,25,26). However, evidence suggests that although the two ATP sites assume similar structural conformation, they have distinct functional roles within a single catalytic turnover of (18,19), whereas hydrolysis by the other resets the transporter for the subsequent round of transport activity (27,28). Vanadate (Vi), a phosphate analog, replaces phosphate at the catalytic site (30) when present during ATP hydrolysis. Due to its low rate of dissociation, vanadate stabilizes Pgp in a catalytic intermediate (mimicking a conformational state) that immediately follows the first ATP hydrolytic event (11,18,19,22,23 (18,19). This is accompanied by an experimentally detectable conformational change (15,17). Chemical cross-linking with thiol reagents and cysteine-scanning mutagenesis revealed increased accessibility of the drug-translocating pathway from the extracellular side of the lipid bilayer due to vanadate trapping (22).Interestingly, the [ 125 I]IAAP-binding site once transformed to its low affinity state remains unaltered even after dissociation of vanadate and

show abstract

“…In both assays, the amount of P i liberated was determined by a colorimetric assay (at A 630 ) essentially as described in [19,20], using K 2 HPO 4 as a standard. Alternatively, ATPase activity was measured employing a continuous ATP-regenerating assay, where ATP hydrolysis is coupled to the consumption of NADH [21]. Assays were performed at 22 • C in a temperature-controlled 96-well plate in a spectrophotometer (Polar Start Galaxy, BMG).…”

Section: Atpase Activity Assaymentioning

confidence: 99%

Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli

Benabdelhak

Schmitt

Horn

et al. 2005

Biochemical Journal

View full text Add to dashboard Cite

The ATPase activity of the ABC (ATP-binding cassette) ATPase domain of the HlyB (haemolysin B) transporter is required for secretion of Escherichia coli haemolysin via the type I pathway. Although ABC transporters are generally presumed to function as dimers, the precise role of dimerization remains unclear. In the present study, we have analysed the HlyB ABC domain, purified separately from the membrane domain, with respect to its activity and capacity to form physically detectable dimers. The ATPase activity of the isolated ABC domain clearly demonstrated positive co-operativity, with a Hill coefficient of 1.7. Furthermore, the activity is (reversibly) inhibited by salt concentrations in the physiological range accompanied by proportionately decreased binding of 8-azido-ATP. Inhibition of activity with increasing salt concentration resulted in a change in flexibility as detected by intrinsic tryptophan fluorescence. Finally, ATPase activity was sensitive towards orthovanadate, with an IC 50 of 16 µM, consistent with the presence of transient dimers during ATP hydrolysis. Nevertheless, over a wide range of protein or of NaCl or KCl concentrations, the ABC ATPase was only detected as a monomer, as measured by ultracentrifugation or gel filtration. In contrast, in the absence of salt, the sedimentation velocity determined by analytical ultracentrifugation suggested a rapid equilibrium between monomers and dimers. Small amounts of dimers, but apparently only when stabilized by 8-azido-ATP, were also detected by gel filtration, even in the presence of salt. These data are consistent with the fact that monomers can interact at least transiently and are the important species during ATP hydrolysis.

show abstract

[38] ATPase activity of Chinese hamster P-glycoprotein

Cited by 72 publications

References 22 publications

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

Allosteric Modulation Bypasses the Requirement for ATP Hydrolysis in Regenerating Low Affinity Transition State Conformation of Human P-glycoprotein

Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli

Contact Info

Product

Resources

About