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)

Active transport of conjugated and unconjugated electrophiles out of cells is essential for cellular homeostasis. We have previously identified in human tissues a transporter, DNP-SG [S-(2, 4-dinitrophenyl)glutathione] ATPase, capable of carrying out this function [Awasthi et al. (1998) Biochemistry 37, 5231-5238, 5239-5248]. We now report the cloning of DNP-SG ATPase. The sequence of the cDNA clone was identical to that of human RLIP76, a known Ral-binding protein. RLIP76 expressed in E. coli was purified by DNP-SG affinity chromatography. Purified recombinant RLIP76: (1) had ATPase activity stimulated by DNP-SG or doxorubicin (DOX), and the K(m) values of RLIP76 for ATP, DOX, and DNP-SG were similar to those reported for DNP-SG ATPase; (2) upon reconstitution with asolectin as well as with defined lipids, catalyzed ATP-dependent transport of DNP-SG and DOX with kinetic parameters similar to those of DNP-SG ATPase; (3) when transfected into K562 cells, resulted in increased resistance to DOX, and increased ATP-dependent transport of DNP-SG and DOX by inside-out membrane vesicles from transfected cells; (4) direct uptake of purified RLIP76 protein into mammalian cells from donor proteoliposomes confers DOX resistance. These results indicate that RLIP76, in addition to its role in signal transduction, can catalyze transport of glutathione conjugates and xenobiotics, and may contribute to the multidrug resistance phenomenon.
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PMID:Novel function of human RLIP76: ATP-dependent transport of glutathione conjugates and doxorubicin. 1092 26

We have recently shown that RLIP76, a Ral-binding, GTPase-activating protein, is an ATP-dependent transporter of doxorubicin (DOX) as well as glutathione conjugates [Awasthi, S., et al. (2000) Biochemistry 39, 9327-9334]. RLIP76 overexpressed in human cells or transformed E. coli undergoes proteolysis to yield several fragments, including two prominent peptides, N-RLIP76(1-367) and C-RLIP76(410-655), from the N- and C-terminal domains, respectively. To investigate whether the fragmentation of RLIP76 has any relevance to its transport function, we have studied the characteristics of these two peptide fragments. Recombinant N-RLIP76(1-367) and C-RLIP76(410-655) were purified from overexpressing transformed E. coli. While N-RLIP76(1-367) readily underwent proteolysis, showing SDS-gel patterns similar to those of RLIP76, C-RLIP76(410-655) was resistant to such degradation. Both N-RLIP76(1-367) and C-RLIP76(410-655) had ATPase activity (K(m) for ATP, 2.5 and 2.0 mM, respectively) which was stimulated by DNP-SG, DOX, and colchicine (COL). ATP binding to both peptides was confirmed by photoaffinity labeling with 8-azido-ATP that was increased in the presence of compounds that stimulated their ATPase activity. Photoaffinity labeling was also increased in the presence of vanadate, indicating trapping of a reaction intermediate in the ATP binding site. The ATP binding sites in N-RLIP76(1-367) and C-RLIP76(410-655) were identified to be (69)GKKKGK(74) and (418)GGIKDLSK(425), respectively. Mutation of K(74) and K(425) to M residues, in N-RLIP76(1-367) and C-RLIP76(410-655), respectively, abrogated their ATPase activity as well as azido-ATP labeling. Proteoliposomes reconstituted with either N-RLIP76(1-367) or C-RLIP76(410-655) alone did not catalyze ATP-dependent transport of DOX or COL. However, proteoliposomes reconstituted with a mixture of N-RLIP76(1-367) and C-RLIP76(410-655) mediated such transport. Proteoliposomes reconstituted with the mixture of mutant peptides lacking ATPase activity did not exhibit transport activity. Present studies have identified the ATP binding sites in RLIP76, and show that DOX and COL transport can be reconstituted by two fragments of RLIP76.
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PMID:Functional reassembly of ATP-dependent xenobiotic transport by the N- and C-terminal domains of RLIP76 and identification of ATP binding sequences. 1130 Jul 97

We have recently shown that RLIP76, a ral-binding GTPase activating protein, mediates ATP-dependent transport of glutathione-conjugates (GS-E) and doxorubicin (DOX) (S. Awasthi et al., Biochemistry 39,9327,2000). Transport function of RLIP76 was found to be intact despite considerable proteolytic fragmentation in preparations used for those studies, suggesting either that the residual intact RLIP76 was responsible for transport activity, or that the transport activity could be reconstituted by fragments of RLIP76. If the former were true, intact RLIP76 would have a much higher specific activity for ATP-hydrolysis than the fragmented protein. We have addressed this question by comparing transport properties of recombinant RLIP76 and human erythrocyte membrane RLIP76 purified in buffers treated with either 100 or 500 microM serine protease inhibitor, PMSF. The purity and identity of recombinant and human erythrocyte RLIP76 was established by SDS/PAGE and Western-blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Higher PMSF concentration resulted in lower yield of the 38 kDa band and higher yield of intact RLIP76 from both human and recombinant source. In contrast, the substrate-stimulated ATPase activity in presence of DNP-SG, doxorubicin, daunorubicin, or colchicine were unaffected by increased PMSF; similarly, ATP-dependent transport of doxorubicin in proteoliposomes reconstituted with RLIP76 was unaffected by higher PMSF. These results indicated that limited proteolysis by serine proteases does not abrogate the transport function of RLIP76. Comparison of transport kinetics for daunorubicin between recombinant vs human erythrocyte RLIP76 revealed higher specific activity of transport for tissue purified RLIP76, indicating that additional factors present in tissue purified RLIP76 can modulate its transport activity.
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PMID:Purification and functional reconstitution of intact ral-binding Gtpase activating protein, RLIP76, in artificial liposomes. 1173 24

Transport of xenobiotics and their metabolites by ATP-binding cassette (ABC) transporters particularly P-glycoprotein (Pgp) and the multidrug resistance associated protein (MRP1) has been extensively studied during last decade. Our recent studies demonstrate that RLIP76, a previously known GTPase-activating protein catalyzes ATP-dependent, uphill transport of anionic glutathione conjugates as well as of weakly cationic anthracyclines including doxorubicin (Adriamycin), a widely used drug in cancer chemotherapy. RLIP76 has inherent ATPase activity, which is stimulated by doxorubicin and glutathione conjugates. RLIP76 does not meet the criteria for classical ABC proteins such as MRP1 or Pgp, but similar to ABC proteins, it has two ATP-binding sequences, (69)GKKKGK(74) and (418)GGIKDLSK(425). Mutations in these sequences abrogate its ATP-binding, ATPase activity, and transport function. Purified RLIP76 when reconstituted in proteoliposomes mediates ATP-dependent saturable transport of doxorubicin and glutathione conjugates. Transfection of K562 cells with RLIP76 confers these cells resistance to doxorubicin and 4-hydroxynonenal. Cells enriched with RLIP76 also acquire resistance to radiation toxicity. RLIP76 also catalyzes the transport of physiologic ligands such as leukotrienes (LTC4) and the conjugate of 4-hydroxynonenal and glutathione. In some cells (e.g., erythrocytes and lung cancer cells), the majority of transport activity for Adriamycin and glutathione conjugates including LTC4 is accounted for by RLIP76. These studies strongly suggest that RLIP76-mediated transport of organic ions has physiological and toxicological relevance and that it may play an important role in the mechanism of drug resistance.
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PMID:RLIP76, a novel transporter catalyzing ATP-dependent efflux of xenobiotics. 1243 96

RLIP76 functions as an ATP-dependent transporter of amphiphilic chemotherapeutic drugs such as doxorubicin (DOX, adriamycin), as well as of glutathione-conjugates of endogenous electrophilic toxins such as 4-hydroxynonenal (4HNE). RLIP76 couples transport and ATP-hydrolysis with a 1:1 stoichiometry, making the ATPase activity of RLIP76 an excellent surrogate for its transport activity. Present studies were performed to determine the relationship of the RLIP76 ATPase activity with DOX and 4HNE resistance in a panel of 13 native human lung cancer cell lines. RLIP76 was purified from each cell line and homogeneity demonstrated by SDS-PAGE and amino acid composition analysis. Anti-RLIP76 antibodies were shown by Ouchterlony double immunodiffusion tests to be non-cross-reactive with any other proteins including P-glycoprotein (Pgp) or multidrug resistance associated protein (MRP). These antibodies completely immunoprecipitated ATPase activity of purified RLIP76 fractions, further confirming homogeneity of purified RLIP76. RLIP76 ATPase purified from NSCLC cell lines was about 2-fold more active than that from SCLC in the absence of the stimulator dinitrophenyl S-glutathione (206+/-47, n=7 vs. 94+/-22, n=6, nmol/min/mg protein, respectively), or in its presence (340+/-60, n=7 vs. 186+/-32, n=6, nmol/min/mg; p<0.01). Partial tryptic digest revealed a 44 kDa internal fragment of RLIP76 beginning at Thr-294 in NSCLC cell lines. This fragment was absent from all SCLC, suggesting the possibility that the activity of RLIP76 in SCLC and NSCLC is differentially regulated through post-translational modifications. Taken together, these findings suggest that RLIP76 activity is a general determinant of 4HNE and DOX resistance, and that its activity contributes to the drug-resistant phenotype of NSCLC.
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PMID:Role of RLIP76 in lung cancer doxorubicin resistance: I. The ATPase activity of RLIP76 correlates with doxorubicin and 4-hydroxynonenal resistance in lung cancer cells. 1252 36

We have recently demonstrated that a previously known Ral-binding GTPase activating protein, RLIP76, can also catalyze ATP-dependent transport of various structurally unrelated xeno- and endobiotics irrespective of their net charge (Awasthi et al., 2000, Biochemistry, 39: 9327). RLIP76 is a non-ATP binding cassette (ABC) protein but it has two ATP-binding sites and shows basal ATPase activity which is stimulated in the presence of its transport substrates (allocrites) such as doxorubicin (DOX) and S-(2,4-dinitrophenyl) glutathione (DNP-SG). Proteoliposomes reconstituted with purified RLIP76 catalyze ATP-dependent, saturable transport of DOX, as well as of glutathione-conjugates including leukotrienes (LTC4) and the GSH-conjugate of 4-hydroxynonenal (GS-HNE). In erythrocytes the majority of transport activity for DOX, GS-HNE, and LTC4 is accounted for by RLIP76. Cells exposed to mild oxidative stress show a rapid and transient induction of RLIP76 resulting in an increased efflux of GS-HNE and acquire resistance to oxidative stress mediated toxicity and apoptosis. Cells transfected with RLIP76 acquire resistance to DOX through increased efflux of the drug suggesting its possible role in the mechanisms of drug-resistance. In this article, we discuss the significance of transport functions of RLIP76 highlighting its role in the defense mechanisms against oxidative injury, and modulation of signaling mechanisms.
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PMID:Transport functions and physiological significance of 76 kDa Ral-binding GTPase activating protein (RLIP76). 1254 92

ATP-dependent transport of doxorubicin (DOX) by recombinant human RLIP76 has been demonstrated previously in reconstituted proteoliposomes. In the preceding communication, we demonstrated that the ATPase activity of RLIP76 was 2-fold higher in NSCLC as compared with SCLC, and it correlated with their inherent DOX resistance. Present studies were performed to determine whether greater ATPase activity of RLIP76 in NSCLC translated into greater RLIP76 mediated DOX transport, and to determine the overall contribution of RLIP76 toward total DOX transport. Consistent with the greater RLIP76 ATPase activity in NSCLC, DOX transport in artificial proteoliposomes reconstituted with purified RLIP76 from NSCLC was 1.8-fold greater than in SCLC. Anti-RLIP76 antibodies completely abrogated DOX transport in these RLIP76 proteoliposomes, whereas anti-MRP or anti-Pgp antibodies had no effect on transport. DOX transport studies in crude membrane vesicles from SCLC and NSCLC also showed a 2.1-fold higher rate of transport in NSCLC as compared with SCLC. Anti-RLIP76 IgG, which recognized only RLIP76 in crude extracts of both SCLC and NSCLC, inhibited 67+/-4% (n=12 cell lines) of total DOX transport in crude membrane vesicles from both SCLC and NSCLC. Inhibition of DOX transport by anti-MRP and anti-Pgp antibody was 35+/-7% (n=12) and 2+/-0.3% (n=12), respectively. The mixture of the three antibodies inhibited DOX transport by 95+/-3% (n=12). These studies demonstrate that DOX transport activity of RLIP76 is significantly greater in NSCLC as compared with SCLC, and that RLIP76 is the major DOX transporter in lung cancer cell lines.
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PMID:Role of RLIP76 in lung cancer doxorubicin resistance: II. Doxorubicin transport in lung cancer by RLIP76. 1263 60

Our studies have shown that RLIP76 (RALBP1), a 76 kDa Ral-binding, Rho/Rac-GAP and Ral effector protein, is a novel multispecific transporter of xenobiotics as well as GS-Es. Like previously characterized ABC transporters, it mediates ATP-dependent transport of structurally unrelated amphiphilic xenobiotics and displays inherent ATPase activity, which is stimulated by its substrate allocrites. It does not have significant sequence homology with ABC transporters and differs from the ABC transporters in several other important aspects, including (i) lack of any close homologs in humans, (ii) lack of a classical Walker domain, (iii) integral membrane association without clearly defined transmembrane domains and (iv) its role as a direct link to Ras/Ral/Rho and EGF-R signaling through its multifunctional nature, including GAP activity, regulation of exocytosis as well as clathrin-coated pit-mediated receptor endocytosis. Its multifunctional nature derives from the presence of multiple motifs, including a Rho/Rac GAP domain, a Ral effector domain binding motif, 2 distinct ATP-binding domains, a H(+)-ATPase domain, PKC and tyrosine kinase phosphorylation sites and the ability to undergo fragmentation into multiple smaller peptides which participate as components of macromolecular functional complexes. One of the physiologic functions of RLIP76 is regulation of intracellular concentration of the electrophilic intermediates of oxidative lipid metabolism by mediating efflux of GS-E formed from oxidative degradation of arachidonic acid, including leukotrienes and the 4HNE-GSH conjugate. RLIP76-mediated transport of amphiphilic chemotherapeutic agents such as anthracyclines and vinca alkaloids as well as GS-E produced during oxidative metabolism places this multifunctional protein in a central role as a resistance mechanism for preventing apoptosis caused by chemotherapeutic agents and a variety of external/internal stressors, including oxidative stress, heat shock and radiation.
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PMID:Transport of glutathione conjugates and chemotherapeutic drugs by RLIP76 (RALBP1): a novel link between G-protein and tyrosine kinase signaling and drug resistance. 1286 21

Increased active transport of LTC(4) observed frequently in multidrug-resistant cancer cells have been attributed to ABC-transporter proteins particularly, MRP1. We have demonstrated recently that a novel non-ABC transporter, RLIP76 (RALBP1) can also mediate ATP-dependent transport of GSH-conjugates (GS-E) as well as doxorubicin (DOX). We demonstrate RLIP76 reconstituted in artificial liposomes can catalyze ATP-dependent transport of LTC(4), which can be modulated by PKC-alpha. The ATPase activity of E. coli expressed homogenous RLIP76 was stimulated in a saturable fashion by LTC(4) with half maximal stimulation at 130 nM. Proteoliposomes reconstituted with RLIP76 catalyzed temperature and osmolar sensitive ATP-dependent transport of LTC(4) with K(m) values of 5.1 mM and 210 nM for ATP and LTC(4), respectively. V(max) for transport was found to be 3.2 nmol/min/mg. Colchicine inhibited LTC(4) transport to 50% at 5.8 microM. PKC-alpha catalyzed phosphorylation of RLIP76 and increased its transport activity by 2-3-fold. Membrane vesicles prepared from the small (SCLC) and non-small (NSCLC) lung cancer cell lines as well as HL-60 (leukemia) and U937 (lymphoma) cell lines exhibited ATP-dependent transport of LTC(4), which was inhibited by anti-RLIP76 antibodies. The rate of transport of LTC(4) in SCLC (H69, H378) was half of that observed in NSCLC cell lines but after transfection with RLIP76, the transport rate of LTC(4) in H69 became comparable to that in NSCLC cell lines. Anti-RLIP76 antibodies inhibited LTC(4) transport by 67-81% in all 8 cell lines examined, whereas N-19 anti-MRP1 antibodies inhibited transport of LTC(4) by only 11-26%. These results suggest that RLIP76 is the major LTC(4) transporter in cancer cells and that its transport activity is regulated by PKC-alpha-mediated phosphorylation.
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PMID:RLIP76 (RALBP1)-mediated transport of leukotriene C4 (LTC4) in cancer cells: implications in drug resistance. 1538 49

RLIP76, a stress-responsive, multi-functional protein with multi-specific transport activity towards glutathione-conjugates (GS-E) and chemotherapeutic agents is frequently overexpressed in malignant cells. Our recent studies suggest that it plays a prominent anti-apoptotic role selectively in cancer cells. The present studies were performed to compare RLIP76 activity towards glutathione-conjugates in recombinant and K562 human erythroleukemia cells. The purity and identity of recombinant and K562 RLIP76 was established by SDS-PAGE and Western blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Comparison of ATPase activity and transport kinetics for DNP-SG and GS-HNE between recombinant vs. K562 RLIP76 revealed higher specific activity of ATPase and transport for recombinant purified RLIP76, indicating that additional factors present in recombinant purified RLIP76 can modulate its transport activity.
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PMID:Functional reconstitution of RLIP76 catalyzing ATP-dependent transport of glutathione-conjugates. 1908 90


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