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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Multidrug-resistance-associated protein 1 (MRP1/ABCC1) is a human ATP-binding cassette transporter that confers cell resistance to antitumour drugs. Its NBDs (nucleotide-binding domains) bind/hydrolyse ATP, a key step in the activation of MRP1 function. To relate its intrinsic functional features to the mechanism of action of the full-size transporter, we expressed the N-terminal NBD1 domain (Asn(642) to Ser(871)) in Escherichia coli. NBD1 was highly purified under native conditions and was characterized as a soluble monomer. (15)N-labelling allowed recording of the first two-dimensional NMR spectra of this domain. The NMR study showed that NBD1 was folded, and that Trp(653) was a key residue in the NBD1-ATP interaction. Thus, interaction of NBD1 with ATP/ADP was studied by intrinsic tryptophan fluorescence. The affinity for ATP and ADP were in the same range (K (d(ATP))=118 microM and K (d(ADP))=139 microM). Binding of nucleotides did not influence the monomeric state of NBD1. The ATPase activity of NBD1 was magnesium-dependent and very low [V (max) and K (m) values of 5x10(-5) pmol of ATP x (pmol NBD1)(-1) x s(-1) and 833 microM ATP respectively]. The present study suggests that NBD1 has a low contribution to the ATPase activity of full-length MRP1 and/or that this activity requires NBD1-NBD2 heterodimer formation.
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PMID:Biochemical characterization and NMR studies of the nucleotide-binding domain 1 of multidrug-resistance-associated protein 1: evidence for interaction between ATP and Trp653. 1295 82

In soybean (Glycine max L.), salicylic acid (SA) is converted primarily to SA 2-O-beta-d-glucose (SAG) in the cytoplasm and then accumulates exclusively in the vacuole. However, the mechanism involved in the vacuolar transport of SAG has not been investigated. The vacuolar transport of SAG was characterized by measuring the uptake of [(14)C]SAG into tonoplast vesicles isolated from etiolated soybean hypocotyls. The uptake of SAG was stimulated about six-fold when MgATP was included in the assay media. In contrast, the uptake of SA was only stimulated 1.25-fold by the addition of MgATP and was 2.2-fold less than the uptake of SAG providing an indication that the vacuolar uptake of SA is promoted by glucosylation. The ATP-dependent uptake of SAG was inhibited by increasing concentrations of vanadate (64% inhibition in the presence of 500 microM) but was not very sensitive to inhibition by bafilomycin A(1) (a specific inhibitor of vacuolar H(+)-ATPase; EC 3.6.1.3), and dissipation of the transtonoplast H(+)-electrochemical gradient. The SAG uptake exhibited Michaelis-Menten-type saturation kinetics with a K(m) value of 90 microM for SAG. SAG uptake was inhibited 60% by beta-estradiol 17-(beta-d-glucuronide), but glutathione conjugates and uncharged glucose conjugates were only slightly inhibitory. Based on the characteristics of SAG uptake into soybean tonoplast vesicles it is likely that this uptake occurs through an ATP-binding cassette transporter-type mechanism. However, this vacuolar uptake mechanism is not universal since the uptake of SAG by red beet (Beta vulgaris L) tonoplast vesicles appears to involve an H(+)-antiport mechanism.
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PMID:Uptake of salicylic acid 2-O-beta-D-glucose into soybean tonoplast vesicles by an ATP-binding cassette transporter-type mechanism. 1503 22

The fbpABC operon in Neisseria gonorrhoeae encodes an ATP-binding cassette transporter required for iron uptake from the host ferric binding proteins. The gene for the nucleotide-binding domain (fbpC) expressed in Escherichia coli has intrinsic ATPase activity (0.5 mmol/min/mg) uncoupled from the iron transport process. The FbpC E164D mutant is found to have a 10-fold reduction in specific activity. FbpC is covalently modified by 8-azido-[gamma32P]ATP, indicating that FbpC is a functional ATPase that likely combines with FbpB to form a ferric iron transporter.
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PMID:Characterization of a nucleotide-binding domain associated with neisserial iron transport. 1512 92

Multilocus sequence typing (MLST) was used to obtain insights into the genetic relationships between 14 vancomycin-resistant Enterococcus faecium (VREF) isolates from humans (hospitalized patients, 5 strains) and nonhuman sources (meat and poultry, 9 strains) in northern Italy over the period 1993-2001. The typing scheme (Homan et al., 2002, J. Clin. Microb., 40:1963-1971) based on seven housekeeping genes--adk (adenylate kinase), atpA (ATP synthase, alpha subunit), ddl (D-alanine-D-alanine ligase), gyd (glyceraldehyde-3-phosphate dehydrogenase), gdh (glucose-6-phosphate dehydrogenase), purK (phosphoribosylaminoimidazole carboxylase ATPase subunit), and pstS (phosphate ATP-binding cassette transporter)--was used. In the 14 VREF analyzed, the number of unique alleles ranged from 1 (gyd) to 8 (atpA). Isolates from hospitalized patients were defined by the unique allele purK 1. Nine sequence types (STs) were identified. All of the epidemic strains isolated over the period 2000-2001 showed identical or closely related pulsed-field gel electrophoresis (PFGE) patterns and clustered in the same ST78. These strains shared six of the seven alleles with the strain CA20 representative of the 1993-1999 outbreaks, which PFGE indicated as being unrelated to those of the recent outbreaks. MLST confirmed the unrelatedness of human and nonhuman strains already detected by PFGE. All isolates clustered in three main genetic lineages: group A comprised two of the three isolates from meat; group C the human strains of all outbreaks and one poultry strain; and group B four of the five poultry strains and one meat strain. All human strains carried the esp gene and clustered in the C1 sublineage that has been described as having emerged recently worldwide.
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PMID:Vancomycin-resistant Enterococcus faecium isolates causing hospital outbreaks in northern Italy belong to the multilocus sequence typing C1 lineage. 1525 26

Fungal ATP-binding cassette transporter regulation was investigated using Candida glabrata Cdr1p and Pdh1p expressed in Saccharomyces cerevisiae. Rephosphorylation of Pdh1p and Cdr1p was protein kinase A inhibitor-sensitive but responded differentially to Tpk isoforms, stressors, and glucose concentration. Cdr1p Ser(307), which borders the nucleotide binding domain 1 ABC signature motif, and Ser(484), near the membrane, were dephosphorylated on glucose depletion and independently rephosphorylated during glucose exposure or under stress. The S484A enzyme retained half the wild type ATPase activity without affecting azole resistance, but the S307A enzyme was unstable to plasma membrane isolation. Studies of pump function suggested conformational interaction between Ser(484) and Ser(307). An S307A/S484A double mutant, which failed to efflux the Cdr1p substrate rhodamine 6G, had a fluconazole susceptibility 4-fold greater than the Cdr1p expressing strain, twice that of the S307A mutant, but 64-fold less than the control null strain. Stable intragenic suppressors indicative of homodimer nucleotide binding domain 1-nucleotide binding domain 1 interactions partially restored rhodamine 6G pumping and increased fluconazole and rhodamine 6G resistance in the S307A/S484A mutant. Nucleotide binding domain 1 of Cdr1p is a sensor of important physiological stimuli.
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PMID:Phosphorylation of candida glabrata ATP-binding cassette transporter Cdr1p regulates drug efflux activity and ATPase stability. 1549 68

Multidrug resistance protein 1 (MRP1) is a member of the "C" branch of the ATP-binding cassette transporter superfamily. The NH(2)-proximal nucleotide-binding domain (NBD1) of MRP1 differs functionally from its COOH-proximal domain (NBD2). NBD1 displays intrinsic high-affinity ATP binding and little ATPase activity. In contrast, ATP binding to NBD2 is strongly dependent on nucleotide binding by NBD1, and NBD2 is more hydrolytically active. We have demonstrated that occupancy of NBD2 by ATP or ADP markedly decreased substrate binding by MRP1. We have further explored the relationship between nucleotide and substrate binding by examining the effects of various ATP analogs and ADP trapping, as well as mutations in conserved functional elements in the NBDs, on the ability of MRP1 to bind the photoactivatable, high-affinity substrate cysteinyl leukotriene C(4) (LTC(4))(.) Overall, the results support a model in which occupancy of both NBD1 and NBD2 by ATP results in the formation of a low-affinity conformation of the protein. However, nonhydrolyzable ATP analogs (beta,gamma-imidoadenosine 5'-triphosphate and adenylylmethylene diphosphonate) failed to substitute for ATP or adenosine 5'-O-(thiotriphosphate) (ATPgammaS) in decreasing LTC(4) photolabeling. Furthermore, mutations of the signature sequence in either NBD that had no apparent effect on azido-ATP binding abrogated the formation of a low-affinity substrate binding state in the presence of ATP or ATPgammaS. We suggest that the effect of these mutations, and possibly the failure of some ATP analogs to decrease LTC(4) binding, may be attributable to an inability to elicit a conformational change in the NBDs that involves interactions between the signature sequence and the gamma-phosphate of the bound nucleotide.
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PMID:Functional interactions between nucleotide binding domains and leukotriene C4 binding sites of multidrug resistance protein 1 (ABCC1). 1575 10

Gefitinib (Iressa) is a selective epidermal growth factor receptor tyrosine kinase inhibitor and is used for the treatment of lung cancer. Recently, we discovered that it inhibits the breast cancer resistance protein, which is an ATP-binding cassette transporter. P-glycoprotein (Pgp) also pumps multiple types of drugs out of the cell using energy generated from ATP, and confers multidrug resistance on cancer cells. This study was designed to examine whether gefitinib inhibits the function of Pgp. We used multidrug resistant PC-6/PTX lung cancer and MCF-7/Adr breast cancer cells which overexpress Pgp and measured their drug sensitivity and drug-efflux function by tetrazolium assay and flowcytometry, respectively. In addition, the drug-stimulated ATPase activity of Pgp was measured using insect membranes that express human Pgp. Epidermal growth factor receptor was expressed in MCF-7/Adr, but not in PC-6/PTX cells, and the overexpression of Pgp did not confer resistance to gefitinib to both cell types. However, clinically achievable levels of gefitinib moderately reversed the Pgp-mediated resistance to paclitaxel and docetaxel in Pgp overexpressing cells. In addition, gefitinib increased the intracellular accumulation of the Pgp substrate rhodamine-123 in resistant cells, and activated ATPase in a preparation of pure Pgp-expressing membrane. These findings suggest that gefitinib directly interacts with Pgp and inhibits its function. Gefitinib may clinically inhibit the excretion of Pgp substrate drugs including anticancer agents, and its drug-interaction should therefore be considered.
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PMID:Gefitinib, an EGFR tyrosine kinase inhibitor, directly inhibits the function of P-glycoprotein in multidrug resistant cancer cells. 1595 94

The ATP-binding cassette is the most abundant family of transporters including many medically relevant members and gathers both importers and exporters involved in the transport of a wide variety of substrates. Although three high resolution three-dimensional structures have been obtained for a prototypic exporter, MsbA, two have been subjected to much criticism. Here, conformational changes of BmrA, a multidrug bacterial transporter structurally related to MsbA, have been studied. A three-dimensional model of BmrA, based on the "open" conformation of Escherichia coli MsbA, was probed by simultaneously introducing two cysteine residues, one in the first intracellular loop of the transmembrane domain and the other in the Q-loop of the nucleotide-binding domain (NBD). Intramolecular disulfide bonds could be created in the absence of any effectors, which prevented both drug transport and ATPase activity. Interestingly, addition of ATP/Mg plus vanadate strongly prevented this bond formation in a cysteine double mutant, whereas ATP/Mg alone was sufficient when the ATPase-inactive E504Q mutation was also introduced, in agreement with additional BmrA models where the ATP-binding sites are positioned at the NBD/NBD interface. Furthermore, cross-linking between the two cysteine residues could still be achieved in the presence of ATP/Mg plus vanadate when homobifunctional cross-linkers separated by more than 13 Angstrom were added. Altogether, these results give support to the existence, in the resting state, of a monomeric conformation of BmrA similar to that found within the open MsbA dimer and show that a large motion is required between intracellular loop 1 and the nucleotide-binding domain for the proper functioning of a multidrug ATP-binding cassette transporter.
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PMID:The Q-loop disengages from the first intracellular loop during the catalytic cycle of the multidrug ABC transporter BmrA. 1610 40

Isothiocyanates (ITCs) are non-nutrient constituents abundant in cruciferous vegetables and are effective in blocking carcinogenesis in a variety of tissues. ITCs permeate into cells rapidly and accumulate in cells primarily as glutathione (GSH) conjugates. We have demonstrated recently that certain ITCs are inhibitors of ABCG2 (breast cancer resistance protein, BCRP), an ATP-binding cassette transporter that plays an important role in drug absorption and disposition as well as in the development of multidrug resistance in cancer cells. The purpose of this study was to investigate the mechanisms of interactions between ITCs and BCRP and elucidate the transport of phenethyl isothiocyanate (PEITC) by BCRP. Inside-out membrane vesicles were prepared from human breast cancer BCRP-overexpressing MCF-7/MX100 and the parental MCF-7/sensitive cells. The ATPase study using 100 muM ITCs showed that ITCs are potential inhibitors of BCRP ATPase activity. The transport of (14)C-PEITC into BCRP-overexpressing MCF-7/MX100 cell vesicles was ATP-dependent and inhibited by fumitremorgin C (FTC), a specific inhibitor of BCRP, indicating that PEITC is a substrate for BCRP. In the control MCF-7/sensitive cell vesicles, no ATP-dependent and FTC-inhibited transport of (14)C-PEITC was observed. Taken together, the results of this investigation provided evidence that ITCs are potential inhibitors of BCRP ATPase and PEITC, in its unchanged form, is transported by BCRP. These data may be important in elucidating the interaction of ITCs and cellular transporters and in understanding the potential food-drug interaction.
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PMID:Membrane transport of dietary phenethyl isothiocyanate by ABCG2 (breast cancer resistance protein). 1619 94

ATP-binding cassette (ABC) transporters couple ATP binding and hydrolysis to the movement of substances across the membrane; conformational changes clearly play an important role in the transporter mechanism. Previously, we have shown that a dimer of MalK, the ATPase subunit of the maltose transporter from Escherichia coli, undergoes a tweezers-like motion in a transport cycle. The MalK monomer consists of an N-terminal nucleotide binding domain and a C-terminal regulatory domain. The two nucleotide-binding domains in a dimer are either open or closed, depending on whether ATP is present, while the regulatory domains maintain contacts to hold the dimer together. In this work, the structure of MalK in a posthydrolysis state is presented, obtained by cocrystallizing MalK with ATP-Mg(2+). ATP was hydrolyzed in the crystallization drop, and ADP-Mg(2+) was found in the resulting crystal structure. In contrast to the ATP-bound form where two ATP molecules are buried in a closed interface between the nucleotide-binding domains, the two nucleotide-binding domains of the ADP-bound form are open, indicating that ADP, unlike ATP, cannot stabilize the closed form. This conclusion is further supported by oligomerization studies of MalK in solution. At low protein concentrations, ATP promotes dimerization of MalK, whereas ADP does not. The structures of dimeric MalK in the nucleotide-free, ATP-bound, and ADP-bound forms provide a framework for understanding the nature of the conformational changes that occur in an ATP-binding cassette transporter hydrolysis cycle, as well as how conformational changes in MalK are coupled to solute transport.
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PMID:ATP hydrolysis is required to reset the ATP-binding cassette dimer into the resting-state conformation. 1632 9


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