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)

We have investigated the effects of H(+)-ATPase inhibitors, bafilomycin A1 and 7-chloro-4-nitro-benz-2-oxa-1,3 diazole (NBD), and the Golgi inhibitor, brefeldin A, on daunorubicin accumulation and doxorubicin intracellular distribution in the non-P-glycoprotein-mediated multidrug-resistant cell line COR-L23/R. This cell line overexpress a 190 kDa protein which is probably the product of the MRP gene and shows an anthracycline accumulation defect and a drastically altered intracellular anthracycline distribution from the parental cell line COR-L23/P. We found that all three agents could selectively increase the cellular accumulation of daunorubicin in resistant cells. However, these effects were only seen at doses of the modifiers which were equal to or greater than the IC50 of the modifier alone. Effects of the modifiers on the intracellular distribution of doxorubicin fluorescence could, however, be seen at doses lower than those required to produce significant effects on daunorubicin accumulation. However, when used in a continuous MTT chemosensitivity assay none of the agents, used at maximum non-toxic doses, was able to sensitise COR-L23/R cells to doxorubicin or to colchicine. Although these lead compounds are unlikely to be useful as clinical modifiers, development of more selective analogues may prove useful in the modification of non-P-glycoprotein-mediated multidrug resistance.
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PMID:Modification by brefeldin A, bafilomycin A1 and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) of cellular accumulation and intracellular distribution of anthracyclines in the non-P-glycoprotein-mediated multidrug-resistant cell line COR-L23/R. 791 44

Sphinxolides, a newly described family of cytotoxins from the New Caledonian sponge Neosiphonia superstes, bear structural resemblance to scytophycins. We now demonstrate that the cytotoxicity of sphinxolides is associated with cell cycle arrest in G2-M and induction of apoptosis. Like scytophycins and cytochalasins, sphinxolides caused rapid loss of microfilaments in cultured cells, without affecting microtubule organization. Microfilament reassembly was very slow after removal of the sphinxolide, consistent with the slow recovery of cellular proliferation. Sphinxolides potently inhibited actin polymerization in vitro and the microfilament-dependent ATPase activity of purified actomyosin, indicating a direct effect on actin. Importantly, sphinxolides were equally cytotoxic toward MCF-7 human breast carcinoma cells and a subline which overexpresses P-glycoprotein (MCF-7/ADR). Similarly, overexpression of the multidrug resistance-associated protein MRP by HL-60 cells did not confer resistance to the sphinxolides. These studies demonstrate that sphinxolides are potent new antimicrofilament compounds that circumvent multidrug resistance mediated by overexpression of either P-glycoprotein or MRP. Therefore, these agents may be useful in the treatment of drug-resistant tumors.
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PMID:Microfilament depletion and circumvention of multiple drug resistance by sphinxolides. 928 83

The approximately 27 kDa ABC-ATPase, an extraordinarily conserved, unique type of ATPase, acts as a machine to fuel the movement across membranes of almost any type of molecule, from large polypeptides to small ions, via many different membrane-spanning proteins. A particular ABC-ATPase must therefore be tailor-made to function in a complex with its cognate membrane protein, forming a transport pathway appropriate for a specific type of molecule, or in the case of some ABC-transporters, several types of molecule. Molecules to be transported recognise their own transporter, bind and switch on the ATPase, which in turn activates or opens the transport pathway. ABC-dependent transport can be inwards across the membrane, or outwards to the cell exterior, and the ABC-ATPase can fuel transport through pathways which may involve a classical channel (CFTR), a "gateway" mechanism through a proteinacious chamber spanning the bilayer, or conceivably via a pathway at the protein-lipid interface of the outside of the membrane domain. This may be the case for drugs transported by Pgp, a multidrug resistance transporter. In this review, we try to identify the common fundamental principles which unite all ABC-transporters, including the basis of specificity for different transported compounds (allocrites), the interactions between the ATPase and membrane domains, activation of the ATPase and the coupling of consequent conformational changes, to the final movement of an allocrite through a given transport pathway. We discuss the so far limited structural information for the intact ABC-transporter complex and the exciting information from the first crystal structure of an ABC-ATPase. Finally, the action of specific transporters, CFTR (Cl- transport), Pgp, MRP and LmrA, all transporting many different drug molecules and HlyB transporting a large protein toxin are discussed.
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PMID:ABC-ATPases, adaptable energy generators fuelling transmembrane movement of a variety of molecules in organisms from bacteria to humans. 1052 52

Studies have been carried out to examine in vitro drug transport in plasma membrane vesicles isolated from HL60/ADR cells that overexpress MRP. The results demonstrate that glutathione (GSH) enhances transport of daunomycin. A greater increase in transport activity occurs when the reaction is carried out in the presence of both GSH and sodium chloride. Sodium chloride alone has no effect on daunomycin transport. It has also been observed that GSH in the presence of sodium chloride induces a major increase in the transport level of LTC4. Thus far, no metal ion other than sodium chloride has been found to be active in the drug transport system. Kinetic analysis reveals that GSH in the presence of sodium chloride greatly reduces Km and increases Vmax, for daunomycin. Additional studies show that ATPase activity in isolated plasma membrane from HL60/ADR cells is greatly enhanced in the presence of both GSH and sodium chloride. These results suggest the possibility that GSH and sodium chloride stimulate MRP-mediated transport as a result of increased ATPase activity.
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PMID:Multidrug resistance-associated protein (MRP) mediated transport of daunomycin and leukotriene C4 (LTC4) in isolated plasma membrane vesicles. 1065 18

An Ehrlich ascites tumour cell line (EHR2) was selected in vivo for resistance to mitoxantrone (MITOX). The resistant cell line (EHR2/MITOX) was 6123-, 33-, and 30-fold-resistant to mitoxantrone, daunorubicin, and etoposide, respectively, but retained sensitivity to vincristine. The resistant cells showed moderate sensitisation to mitoxantrone on treatment with verapamil or cyclosporin A. Compared with EHR2, the multidrug resistance-associated protein mRNA was increased 13-fold in EHR2/MITOX. Western blot analysis showed an unchanged, weak expression of P-glycoprotein. Topoisomerase IIalpha was reduced to one-third in EHR2/MITOX relative to EHR2 cells, whereas topoisomerase IIbeta was present in EHR2 but could not be detected in EHR2/MITOX. In the resistant subline, net accumulation of MITOX (120 min) and daunorubicin (60 min) was reduced by 43% and 27%, respectively, as compared with EHR2. The efflux of daunorubicin from preloaded EHR2/MITOX cells was significantly increased. EHR2/MITOX microsomes had a significant basal unstimulated ATPase activity. The apparent K(i) value for vanadate inhibition of the ATPase activity in EHR2/MITOX microsomes was not significantly different from the K(i) value for P-glycoprotein-positive cells. However, whereas verapamil (50 microM) inhibited the ATPase activity of EHR2/MITOX microsomes, it stimulated the ATPase activity of microsomes derived from P-glycoprotein-positive cells. In conclusion, the resistance in EHR2/MITOX was multifactorial and appeared to be associated with: 1) a quantitative reduction in topoisomerase IIalpha and beta protein; 2) reduced drug accumulation, probably as a result of increased expression of a novel transport protein with ATPase activity; and 3) increased expression of MRP mRNA.
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PMID:Characterisation of non-P-glycoprotein multidrug-resistant Ehrlich ascites tumour cells selected for resistance to mitoxantrone. 1085 31

cMOAT encodes an ATPase within the family of cMOAT/MRP ATPases that functions as an ATP dependent, multispecific anion transporter within the canalicular surface of hepatocytes that has pharmacologic significance. We describe here the cloning of a murine cMOAT cDNA isolated from mouse liver. The open reading frame of this cDNA incorporates 4627 nucleotides encoding 1309 amino acids with 77.5% and 86.7% identity with the human and rat encoded amino acids, respectively. Northern blotting showed that the expression of cMOAT mRNA occurs primarily in mouse liver in the form of two variants with approximately 5.6 and 7.8 kb of sequence each. cMOAT mRNA was also detected in mouse kidney at a low level but was not detected in other mouse organs or tumors except the Hep 1-6 murine hepatoma where expression was also in the form of the same two mRNA variants.
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PMID:Molecular cloning of the murine cMOAT ATPase. 1100 24

Miltefosine (hexadecylphosphocholine) is used for topical treatment of breast cancers. It has been shown previously that a high percentage of breast carcinomas express MDR1 or MRP. We investigated the sensitivity of MDR1 -expressing cells to treatment with miltefosine. We show that cells overexpressing MDR1 (NCI/ADR-RES, KB-8-5, KB-C1, CCRF/VCR1000, CCRF/ADR5000) were less sensitive to miltefosine treatment when compared to the sensitive parental cell lines. HeLa cells transfected with MDR1 exhibited resistance to the compound, indicating that expression of this gene is sufficient to reduce the sensitivity to miltefosine. The resistance of MDR1-expressing cells to miltefosine was less pronounced than that to adriamycin or vinblastine. Expression of MDR2 did not correlate with the resistance to miltefosine. As shown by a fluorescence quenching assay using MIANS-labelled P-glycoprotein (PGP), miltefosine bound to PGP with a K(d)of approximately 7 microM and inhibited PGP-ATPase activity with an IC(50)of approximately 35 microM. Verapamil was not able to reverse the resistance to miltefosine. Concentrations of miltefosine up to approximately 60 microM stimulated, whereas higher concentrations inhibited the transport of [3H]-colchicine with an IC(50)of approximately 297 microM. Binding studies indicated that miltefosine seems to interact with the transmembrane domain and not the cytosolic nucleotide-binding domain of PGP. These data indicate that expression of MDR1 may reduce the response to miltefosine in patients and that this compound interacts with PGP in a manner different from a number of other substrates.
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PMID:MDR1 causes resistance to the antitumour drug miltefosine. 1135 55

Overexpression of the multidrug resistance-associated protein (MRP1) causes multidrug resistance in cultured cells. MRP1 transports a large number of glutathione, glucuronide, and sulfate-conjugated organic anions by an ATP-dependent efflux mechanism. Six other MRP proteins exist (MRP2-7), and mutations in some of these genes cause major pathological conditions in humans. A detailed characterization of the structure and mechanism of action of these proteins requires an efficient expression system from which large amounts of active protein can be obtained. We report the expression of a recombinant MRP1 in the methylotrophic yeast Pichia pastoris. The protein is expressed in the membrane fraction of these cells, as a stable and underglycosylated 165 kDa peptide. Expression levels are very high, and 30 times superior to those seen in multidrug-resistant HeLa/MRP1 transfectants. MRP1 expressed in P. pastoris binds 8-azido[alpha-(32)P]ATP in a Mg(2+)-dependent and EDTA-sensitive fashion, which can be competed by a molar excess of ADP and ATP. Under hydrolysis conditions (at 37 degrees C), orthovanadate induces trapping of the 8-azido[alpha-(32)P]nucleotide in MRP1, which can be further modulated by known MRP1 ligands. MRP1 is also labeled by a photoactive analogue of rhodamine 123 (IAARh123) in P. pastoris/MRP1 membranes, and this can be competed by known MRP1 ligands. Finally, MRP1-positive membrane vesicles show ATP-dependent uptake of LTC(4). Thus, MRP1 expressed in P. pastoris is active and shows characteristics of MRP1 expressed in mammalian cells, including drug binding, ligand-modulated formation of the MRP1-MgADP-P(i) intermediate (ATPase activity), and ATP-dependent substrate transport. The successful expression of catalytically active and transport-competent MRP1 in P. pastoris should greatly facilitate the efficient production and isolation of the wild type or inactive mutants of MRP1, or of other MRP proteins for structural and functional characterization.
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PMID:Functional expression of multidrug resistance protein 1 in Pichia pastoris. 1144 77

Sequences and available structures were compared for all the widely distributed representatives of the P-loop GTPases and GTPase-related proteins with the aim of constructing an evolutionary classification for this superclass of proteins and reconstructing the principal events in their evolution. The GTPase superclass can be divided into two large classes, each of which has a unique set of sequence and structural signatures (synapomorphies). The first class, designated TRAFAC (after translation factors) includes enzymes involved in translation (initiation, elongation, and release factors), signal transduction (in particular, the extended Ras-like family), cell motility, and intracellular transport. The second class, designated SIMIBI (after signal recognition particle, MinD, and BioD), consists of signal recognition particle (SRP) GTPases, the assemblage of MinD-like ATPases, which are involved in protein localization, chromosome partitioning, and membrane transport, and a group of metabolic enzymes with kinase or related phosphate transferase activity. These two classes together contain over 20 distinct families that are further subdivided into 57 subfamilies (ancient lineages) on the basis of conserved sequence motifs, shared structural features, and domain architectures. Ten subfamilies show a universal phyletic distribution compatible with presence in the last universal common ancestor of the extant life forms (LUCA). These include four translation factors, two OBG-like GTPases, the YawG/YlqF-like GTPases (these two subfamilies also consist of predicted translation factors), the two signal-recognition-associated GTPases, and the MRP subfamily of MinD-like ATPases. The distribution of nucleotide specificity among the proteins of the GTPase superclass indicates that the common ancestor of the entire superclass was a GTPase and that a secondary switch to ATPase activity has occurred on several independent occasions during evolution. The functions of most GTPases that are traceable to LUCA are associated with translation. However, in contrast to other superclasses of P-loop NTPases (RecA-F1/F0, AAA+, helicases, ABC), GTPases do not participate in NTP-dependent nucleic acid unwinding and reorganizing activities. Hence, we hypothesize that the ancestral GTPase was an enzyme with a generic regulatory role in translation, with subsequent diversification resulting in acquisition of diverse functions in transport, protein trafficking, and signaling. In addition to the classification of previously known families of GTPases and related ATPases, we introduce several previously undetected families and describe new functional predictions.
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PMID:Classification and evolution of P-loop GTPases and related ATPases. 1191 78

Mutations in the MRP gene family member MRP6 cause pseudoxanthoma elasticum (PXE) in humans, a disease affecting elasticity of connective tissues. The normal function of MRP6, including its physiological substrate(s), remains unknown. To address these issues, recombinant rat Mrp6 (rMrp6) was expressed in the methylotrophic yeast Pichia pastoris. The protein was expressed in the membrane fraction as a stable 170 kDa protein. Its nucleotide binding and hydrolysis properties were investigated using the photoactive ATP analogue 8-azido-[alpha-(32)P]ATP and compared to those of the drug efflux pump MRP1. rMrp6 can bind 8-azido-[alpha-(32)P]ATP in a Mg(2+)-dependent and EDTA-sensitive fashion. Co(2+), Mn(2+), and Ni(2+) can also support 8-azido-[alpha-(32)P]ATP binding by rMrp6 while Ca(2+), Cd(2+), and Zn(2+) cannot. Under hydrolysis conditions (at 37 degrees C), the phosphate analogue beryllium fluoride (BeF(x)()) can stimulate trapping of the 8-azido-[alpha-(32)P]adenosine nucleotide in rMrp6 (and in MRP1) in a divalent cation-dependent and temperature-sensitive fashion. This suggests active ATPase activity, followed by trapping and photo-cross-linking of the 8-azido-[alpha-(32)P]ADP to the protein. By contrast to MRP1, orthovanadate-stimulated nucleotide trapping in rMrp6 does not occur in the presence of Mg(2+) but can be detected with Ni(2+) ions, suggesting structural and/or functional differences between the two proteins. The rMrp6 protein can be specifically photolabeled by a fluorescent photoactive drug analogue, [(125)I]-IAARh123, with characteristics similar to those previously reported for MRP1 (1), and this photolabeling of rMrp6 can be modulated by several structurally unrelated compounds. The P. pastoris expression system has allowed demonstration of ATP binding and ATP hydrolysis by rMrp6. In addition to providing large amounts of active protein for detailed biochemical studies, this system should also prove useful to identify potential rMrp6 substrates in [(125)I]-IAARh123 photolabeling competition studies, as well as to study the molecular basis of PXE mutations, which are most often found in the NBD2 of MRP6.
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PMID:Nucleotide binding and nucleotide hydrolysis properties of the ABC transporter MRP6 (ABCC6). 1206 97


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