Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasma membrane P-glycoprotein is known as an ATP-dependent drug efflux pump that confers multidrug resistance to tumor cells. None of the reported purification procedures worked properly for our P-glycoprotein-overproducing cell lines, i.e. murine lymphoid leukemia P388/ADR25, rat hepatoma AS30-D/COL10, and human lymphoblastic leukemia CEM/VLB5 cells. We have thus developed a general procedure for efficient purification of P-glycoprotein by combining solubilization with sodium dodecyl sulfate and chromatography on ceramic hydroxyapatite. This procedure was successful for the three cell lines and yielded 70% of the P-glycoprotein present in the starting plasma membranes with more than 99% purity. After exchanging sodium dodecyl sulfate into dodecyl maltoside and reconstitution into liposomes, purified P-glycoprotein exhibited a specific ATPase activity of about 200 nmol/min/mg, which was very similar to that obtained for P-glycoprotein solubilized and purified with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. This ATPase activity was sensitive to orthovanadate inhibition and stimulated by verapamil and other drugs. More importantly, drug transport properties of the reconstituted P-glycoprotein were comparable with those of P-glycoprotein embedded in plasma membranes. Since it is virtually devoid of lipids, this preparation is suitable for both functional and structural investigations.
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PMID:Efficient purification and reconstitution of P-glycoprotein for functional and structural studies. 891 May 34

The MDR1 protein (P-glycoprotein) is a membrane ATPase whose expression results in resistance to several anti-tumor drugs. It has been proposed that the MDR1 protein, in addition to its pumplike properties, can function as (Gill et al. Cell 71: 23-32, 1992; Altenberg et al. Cancer Res. 54:618-622, 1994) or mediate the activity of (Hardy et al. EMBO J. 14: 68-75, 1995) a hypotonic stress-induced Cl- current. In addition, one study found that drug transport and Cl- channel-associated functions of MRD1 were separable and mutually exclusive and that, when cells were swelled, the MDR1 protein could not transport substrate. This hypothesis was tested in four pairs of isogenic cell lines with MDR1 transfectants expression 8,000-55,000 MDR1 antibody binding sites per cell. Cytoplasmic exclusion of rhodamine 123 was used as an indicator of MDR1 function to measure the effect of hypotonic stress, MDR1 inhibitors, and Cl- channel blockers on MRD1 transport function. It was found that MDR1 activity and its inhibition by cyclosporine A or flufenamic acid were unaffected by hypotonicity alone or in combination with Cl- channel blockers.
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PMID:MDR1/P-glycoprotein function. I. Effect of hypotonicity and inhibitors on rhodamine 123 exclusion. 896 46

P-Glycoprotein functions as an ATP-driven active efflux pump for many natural products and chemotherapeutic drugs. Hydrophobic peptides have been shown to block drug uptake by P-glycoprotein, indicating that they might be transport substrates. The present study examines the interaction of the synthetic peptide series NAc-LnY-amide with the multidrug transporter. Several peptides in this series caused up to 3.5-fold enhancement of colchicine accumulation in membrane vesicles from multidrug resistant (MDR) cells, which suggests the existence of novel interactions between the binding sites for peptides and drug. Peptides did not stimulate vinblastine transport, which was inhibited as expected for competing substrates. These peptides displayed modest stimulatory effects on the ATPase activity of P-glycoprotein. None blocked azidopine photoaffinity labelling, showing that they probably occupy a binding site separate from that for the drug. Studies with 125I-labelled NAc-LLY-amide showed that it was transported by P-glycoprotein in both membrane vesicles and reconstituted proteoliposomes. Uptake of the peptide was rapid, saturable, osmotically sensitive and occurred against a concentration gradient. The enhancing effect of NAc-LLY-amide on colchicine transport was reciprocated, i.e. colchicine greatly increased the transport of labelled peptide by P-glycoprotein. Peptide transport was also modulated, both positively and negatively, by other MDR spectrum drugs. It is concluded that linear hydrophobic peptides are indeed transported by P-glycoprotein, and some have interactions with drug substrates that result in mutual stimulation of transport.
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PMID:Synthetic hydrophobic peptides are substrates for P-glycoprotein and stimulate drug transport. 897 48

P-glycoprotein functions as an ATP-driven efflux pump for antitumor agents. C219 is a monoclonal antibody which recognizes regions near both ATP binding domains in each half of P-glycoprotein. In this study, we have demonstrated that C219 inhibits the ATPase activity of P-glycoprotein based on the following findings: 1) the inhibition of total ATPase activity by C219 was selective to P-glycoprotein-positive membranes; 2) the C219-sensitive fraction of ATPase correlated the expression of P-glycoprotein; and 3) modulators of P-glycoprotein ATPase, verapamil and cyclosporin A, affected the C219-sensitive fraction of ATPase. The photolabeling of P-glycoprotein with 8-azido-[alpha-32P]ATP was inhibited by C219, suggesting that the inhibition of ATP binding by C219 reduced the activity. Since C219 interacts with P-glycoprotein ATPase, C219 might become a useful tool for studying the role of P-glycoprotein ATPase.
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PMID:Functional modulation of ATPase of P-glycoprotein by C219, a monoclonal antibody against P-glycoprotein. 901 91

One of the major causes of multidrug resistance in human cancers is expression of the P-glycoprotein multidrug transporter, which acts as an efflux pump for a diverse range of natural products, chemotherapeutic drugs, and hydrophobic peptides. In the present study, fluorescence techniques were used to probe the nucleotide binding domains (NBD) of P-glycoprotein. The transporter was labeled at two conserved cysteine residues, one within each NBD, using the thiol-reactive fluor 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS), and collisional quenching was used to assess solvent accessibility of the bound probe. Acrylamide was a poor quencher, which suggests that MIANS is buried in a relatively inaccessible region of the protein. Iodide ion was a highly effective quencher, whereas Cs+ was not, demonstrating the presence of a positive charge in the region close to the ATP binding site. The fluorescent nucleotide derivative 2'(3')-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) was hydrolysed slowly by P-glycoprotein, with a V(max) approximately 20-fold lower than that for unmodified ATP, and a K(M) of 81 microM. TNP-ATP and TNP-ADP inhibited P-glycoprotein ATPase activity, indicating that they interact with the NBD, whereas TNP-AMP was a very poor inhibitor. When TNP-nucleotides bound to P-glycoprotein, their fluorescence intensity was enhanced in a concentration-dependent manner. Both TNP-ATP and TNP-ADP bound to P-glycoprotein with substantially higher affinity than ATP, with K(d) values of 43 and 36 microM, respectively. Addition of ATP led to only partial displacement of TNP-ATP. Resonance energy transfer was observed between cysteine-bound MIANS and TNP-ATP/ADP, which indicated that the two fluorescent groups are located close to each other within the catalytic site of P-glycoprotein.
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PMID:Fluorescence studies on the nucleotide binding domains of the P-glycoprotein multidrug transporter. 906 12

P-glycoprotein (P-gp) mediates a multidrug resistance (MDR) phenotype in tumor cell lines selected with lipophilic cytotoxic drugs. Transport studies using purified P-glycoprotein reconstituted into defined liposomes have shown energy-dependent drug efflux of structurally dissimilar drugs. In this report, we have examined the effects of N-ethylmaleimide, a potent inhibitor of the P-gp ATPase, on P-gp drug binding in intact MDR cells and in plasma membranes. Our results show that short term treatment of MDR cells with 1-50 microM N-ethylmaleimide led to a concentration dependent increase in P-gp photoaffinity labeling with iodoaryl-azidoparazosin (IAAP). In addition, N-ethylmaleimide increases [3H] vinblastine accumu-lation in drug-resistant but not in sensitive cells. Comparison of IAAP photolabeled P-gp from intact cells with or without N-ethylmaleimide treatment did not show differences in the pattern of IAAP photolabeled peptides. Thus, the observed increase in P-gp photolabeling with IAAP in N-ethylmaleimide treated cells is not due to photolabeling at different sites. Incubation of MDR cells with [14C] N-ethylmaleimide showed that P-gp is directly modified at several Cysteine residues, as found from a complete proteolytic digestion of [14C] Nethylmaleimide labeled P-gp. The comparison of V8 staphylococcus aureas peptides from [14C] Nethylmaleimide or IAAP modified P-gp showed some peptides to co-migrate on SDS PAGE. However, modification of plasma membranes from drug resistant cells treated with N-ethylmaleimide did not show a dose-dependent increase in P-gp photolabeling with IAAP as seen with intact MDR cells. Interestingly, N-ethylmaleimide increases P-gp phosphorylation by inhibiting the turnover of Pgp phosphates. However, inhibition of P-gp phosphorylation with calyculin A did not show an increase in P-gp photolabeling in MDR cells. Taken together, the results of this study suggest that N-ethylmaleimide potentiates P-gp photolabeling with IAAP by inhibiting P-gp ATPase thereby increasing the local concentration of IAAP in intact MDR cells. Furthermore, inhibition of P-gp ATPase by N-ethylmaleimide does not lead to conformational changes that affects P-gp drug binding.
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PMID:N-ethylmaleimide increases P-glycoprotein photoaffinity labeling with iodoaryl-azidoprazosin in multidrug resistant cells. 906 77

VX-710 or (S)-N[2-Oxo-2-(3,4,5-trimethoxyphenyl)acetyl]-piperidine-2-carboxylic acid 1,7-bis(3-pyridyl)-4-heptyl ester, a novel non-macrocyclic ligand of the FK506-binding protein FKBP12, was evaluated for its ability to reverse P-glycoprotein-mediated multidrug resistance in vitro. VX-710 at 0.5-5 microM restored sensitivity of a variety of multidrug resistant cells to the cytotoxic action of doxorubicin, vincristine, etoposide or paclitaxel, including drug-selected human myeloma and epithelial carcinoma cells, and human MDR1 cDNA-transfected mouse leukemia and fibroblast cells. Uptake experiments showed that VX-710 at 0.5-2.5 microM fully restored intracellular accumulation of [14C]doxorubicin in multidrug resistant cells, suggesting that VX-710 inhibits the drug efflux activity of P-glycoprotein. VX-710 effectively inhibited photoaffinity labeling of P-glycoprotein by [3H]azidopine or [125I]iodoaryl azidoprazosin with EC50 values of 0.75 and 0.55 microM. Moreover, P-glycoprotein was specifically labeled by a tritiated photoaffinity analog of VX-710 and unlabeled VX-710 inhibited analog binding with an EC50 of 0.75 microM. VX-710 also stimulated the vanadate-inhibitable P-glycoprotein ATPase activity 2- to 3-fold in a concentration-dependent manner with an apparent k(a) of 0.1 microM. These data indicate that a direct, high-affinity interaction of VX-710 with P-glycoprotein prevents efflux of cytotoxic drugs by the MDR1 gene product in multidrug resistant tumor cells.
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PMID:Cellular and biochemical characterization of VX-710 as a chemosensitizer: reversal of P-glycoprotein-mediated multidrug resistance in vitro. 907 9

Multidrug resistance due to P-glycoprotein is a serious impediment to successful chemotherapy of cancer. Numerous compounds are known that inhibit the drug-exporting function of P-glycoprotein. Understanding the mechanisms of action of these chemosensitizers is made difficult by the complexity of the in vivo cell systems usually employed. To examine the direct effects of chemosensitizers, we have developed a system in which purified and reconstituted P-glycoprotein transports. Hoechst 33342 from the lipid membrane to the aqueous interior of proteoliposomes, requiring ATP hydrolysis (Shapiro AB and Ling V, J Biol Chem 270: 16167-16175, 1995). Here, we use this system to understand the effect on P-glycoprotein of quercetin, one of three flavonoids that have been reported to have the unique property of stimulating drug transport by P-glycoprotein in vivo (Phang et al., Cancer Res 53: 5977-5981, 1993). Since flavonoids are abundant in food, it is important to understand their effects on the function of P-glycoprotein because of the implications for cancer chemotherapy. In our hands, quercetin inhibited P-glycoprotein-mediated Hoechst 33342 efflux and enhanced accumulation, as measured by flow cytometry, by multidrug-resistant CHRC5 cells. In the purified system, quercetin strongly inhibited Hoechst 33342 transport by P-glycoprotein, at least in part by inhibiting the ATPase activity of P-glycoprotein required for transport. We conclude that the previously reported stimulatory effect of quercetin on drug efflux from multidrug-resistant cells is not a direct effect on P-glycoprotein. The ATPase domain of P-glycoprotein may be an attractive target for new chemosensitizing agents.
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PMID:Effect of quercetin on Hoechst 33342 transport by purified and reconstituted P-glycoprotein. 910 11

Most cancer deaths result from the cancer's either being intrinsically resistant to chemotherapeutic drugs or becoming resistant after being initially sensitive. Often, in cells grown in cell culture, drug resistance correlates with the presence of one or more of the so-called P-glycoproteins or multidrug resistance proteins, products of the mdr family of genes. This review is largely concerned with the transport kinetics of the P-glycoproteins. We first present a brief overview of the P-glycoproteins, their properties, and their clinical significance. Later sections of the review expand on this material with special emphasis on the substrates of P-glycoprotein and how they cross the cell membrane, on the transport kinetics of the P-glycoprotein, on reversers of its action, and on its activity as an ATPase. In a final section, we consider the mechanism of action of P-glycoprotein as an actively transporting membrane pump. The characteristic of P-glycoprotein considered the most difficult to explain is its very broad specificity (or lack of specificity), but there are precedents for this property in well-known proteins such as serum albumin, which binds a range of molecular types, including substrates and reversers of P-glycoprotein, seemingly as broad as does P-glycoprotein. Pointing out this analogy does not provide a molecular explanation for the substrate-binding properties of P-glycoprotein but does make those properties more assimilable.
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PMID:Kinetics of the multidrug transporter (P-glycoprotein) and its reversal. 911 23

P-glycoprotein, a plasma membrane protein overexpressed in multidrug-resistant (MDR) cells, exhibits in vitro an ATPase activity and is responsible for the energy-dependent efflux of structurally unrelated cytotoxic drugs (like vinblastine) and various MDR-reversing agents (like verapamil and progesterone) from these MDR cells. To investigate the mechanism of P-glycoprotein interaction with various compounds, we measured the P-glycoprotein ATPase activity on membrane vesicles prepared from the MDR cell line DC-3F/ADX, and we studied the effects of vinblastine, verapamil and progesterone on this ATPase activity. The basal P-glycoprotein ATPase activity is increased by verapamil and progesterone, with respective half-maximal activating concentrations of approximately 1.5 microM and approximately 25 microM, and activation factors of approximately 1.7 and approximately 2.2. Vinblastine inhibits the activation of P-glycoprotein ATPase induced by verapamil or progesterone with an inhibition constant approximately 0.5 microM in both cases. This demonstrates that vinblastine has a specific modulating site on P-glycoprotein. The combined modulation of P-glycoprotein ATPase by vinblastine and verapamil reveals that these two drugs are mutually exclusive. Since these two molecules have different effects both on the basal P-glycoprotein ATPase activity and on the MgATP concentration dependence of P-glycoprotein ATPase activity, they could bind P-glycoprotein either on different and overlapping sites, or on distant but interacting sites. In contrast, the combined modulation of P-glycoprotein ATPase by vinblastine and progesterone reveals a non-competitive relationship between these two drugs, and hence shows that they can independently and simultaneously bind P-glycoprotein on distinct sites. Since verapamil and progesterone are mutual inhibitors of P-glycoprotein ATPase stimulation in a non-competitive manner, these two molecules can also bind independently P-glycoprotein on separated sites. This is confirmed here by the observation of a synergistic effect when mixtures of verapamil and progesterone are tested for the modulation of P-glycoprotein ATPase. Three MDR-related molecules, taken as models for interaction with P-glycoprotein, appear thus to bind on at least two different separated specific sites. These results favor a multisite model rather than a universal site model to describe the broad substrate specificity characterizing P-glycoprotein function.
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PMID:Competitive and non-competitive inhibition of the multidrug-resistance-associated P-glycoprotein ATPase--further experimental evidence for a multisite model. 911 38


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