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)

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug-MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on K(m) values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). V(max) values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.
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PMID:Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol. 1702 89

Lipid rafts are cholesterol- and shingolipid-enriched membrane microdomains implicated in membrane signaling and trafficking. To assess renal epithelial raft functions through the characterization of their associated membrane proteins, we have isolated lipid rafts from rat kidney by sucrose gradient fractionation after detergent treatment. The low-density fraction was enriched in cholesterol, sphingolipid, and flotillin-1 known as lipid raft markers. Based on proteomic analysis of the low-density fraction, the protein with the highest significance score was the alpha-subunit of Na(+)-K(+)-ATPase (NKA), whose raft association was validated by simultaneous immunoblotting. The beta-subunit of NKA was copurified from the low-density fraction. To test the role of lipid rafts in sorting and membrane delivery of renal-transporting epithelia, we have chosen to study thick ascending limb (TAL) epithelium for its high NKA activity and the property to be stimulated by antidiuretic hormone (ADH). Cultured rabbit TAL cells were studied. Cholesterol depletion and detergent extraction at warmth caused a shift of NKA to the higher-density fractions. Comparative preparations from blood monocytes revealed the absence of NKA from rafts in these nonpolarized cells. Short-term exposure of rabbit TAL cells to ADH (1 h) caused translocation and enhanced raft association of NKA via cAMP activation. Preceding cholesterol depletion prevented this effect. TAL-specific, glycosylphosphatidylinositol-anchored Tamm Horsfall protein was copurified with NKA in the same raft fraction, suggesting functional interference between these products. These results may have functional implications regarding the turnover, trafficking, and regulated surface expression of NKA as the major basolateral ion transporter of TAL.
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PMID:Role of lipid rafts in membrane delivery of renal epithelial Na+-K+-ATPase, thick ascending limb. 1708 58

The human ABCG2 multidrug transporter provides protection against numerous toxic compounds and causes multidrug resistance in cancer. Here we examined the effects of changes in membrane cholesterol on the function of this protein. Human ABCG2 was expressed in mammalian and in Sf9 insect cells, and membrane cholesterol depletion or enrichment was achieved by preincubation with beta cyclodextrin or its cholesterol-loaded form. We found that mild cholesterol depletion of intact mammalian cells inhibited ABCG2-dependent dye and drug extrusion in a reversible fashion, while the membrane localization of the transporter protein was unchanged. Cholesterol enrichment of cholesterol-poor Sf9 cell membrane vesicles greatly increased ABCG2-driven substrate uptake, substrate-stimulated ATPase activity, as well as the formation of a catalytic cycle intermediate (nucleotide trapping). Interestingly, modulation of membrane cholesterol did not significantly affect the function of the R482G or R482T substrate mutant ABCG2 variants, or that of the MDR1 transporter. The selective, major effect of membrane cholesterol on the wild-type ABCG2 suggests a regulation of the activity of this multidrug transporter during processing or in membrane micro-domain interactions. The experimental recognition of physiological and pharmacological substrates of ABCG2, as well as the fight against cancer multidrug resistance may be facilitated by demonstrating the key role of membrane cholesterol in this transport activity.
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PMID:Membrane cholesterol selectively modulates the activity of the human ABCG2 multidrug transporter. 1766 39

Na+,K+-ATPase (porcine alpha1/His10*beta1 or human alpha1/porcine His10*beta1) has been expressed in Pichia pastoris and purified by Co2+-chelate affinity resin chromatography, yielding about 80% pure, functional, and stable protein in a single step. The protein was eluted in nonionic detergents together with a phosphatidylserine. Size exclusion chromatography showed that the protein eluted in n-dodecyl beta-d-maltoside is an alpha1/beta1 protomer, whereas that in octaethylene glycol dodecyl monoether contains a mixture of alpha1/beta1 protomer and higher order oligomers. The Na+,K+-ATPase activity (8-16 (mumol/min)/mg of protein) is similar in both detergents. Thus, the minimal functional unit is the alpha1/beta1 protomer, and activity is unaffected by the presence of oligomeric forms. Screening of phospholipids for stabilization of the Na+,K+-ATPase activity shows that (a) acid phospholipids are required and phosphatidylserine is somewhat better than phosphatidylinositol and (b) optimal stabilization is achieved with asymmetric phosphatidylserines having saturated (18:0 >or= 16:0) and unsaturated (18:1 > 18:2) side chains at sn-1 an sn-2 positions, respectively. In the presence of phosphatidylserine, cholesterol stabilizes the protein at 37 degrees C, but not at 0 degrees C. Cholesterol also increases the "apparent affinity" of the phosphatidylserine and stabilizes optimally in the presence of phosphatidylserines with a saturated fatty acyl chain at the sn-1 position. Ergosterol is a poor stabilizer. We propose that phosphatidylserine and cholesterol interact specifically with each other near the alpha1/beta1 subunit interface, thus stabilizing the protein. These interactions do not seem to affect Na+,K+-ATPase activity.
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PMID:Stabilization of Na(+),K(+)-ATPase purified from Pichia pastoris membranes by specific interactions with lipids. 1793 86

Biological rafts were identified and isolated at 37 degrees C and neutral pH. The strategy for isolating rafts utilized membrane tension to generate large domains. For lipid compositions that led only to microscropically unresolvable rafts in lipid bilayers, membrane tension led to the appearance of large, observable rafts. The large rafts converted back to small ones when tension was relieved. Thus, tension reversibly controls raft enlargement. For cells, application of membrane tension resulted in several types of large domains; one class of the domains was identified as rafts. Tension was generated in several ways, and all yielded raft fractions that had essentially the same composition, validating the principle of tension as a means to merge small rafts into large rafts. It was demonstrated that sphingomyelin-rich vesicles do not rise during centrifugation in sucrose gradients because they resist lysis, necessitating that, contrary to current experimental practice, membrane material be placed toward the top of a gradient for raft fractionation. Isolated raft fractions were enriched in a GPI-linked protein, alkaline phosphatase, and were poor in Na(+)-K(+) ATPase. Sphingomyelin and gangliosides were concentrated in rafts, the expected lipid raft composition. Cholesterol, however, was distributed equally between raft and nonraft fractions, contrary to the conventional view.
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PMID:Raft composition at physiological temperature and pH in the absence of detergents. 1799 86

Employing detergent-free sucrose-density gradient fractionation method we isolated cholesterol-rich lighter membrane fractions containing approximately 10% of protein, approximately 30% of cholesterol in membranes of ventricular myocardium. Cholesterol-rich lighter membrane fractions contain >70% of Na, K-ATPase and caveolins 1 and 3 and <10% of beta-actin. Treatment of hypothyroid rats with T(3) increased the relative abundance of both alpha1 and beta1 Na, K-ATPase subunits in total membranes by 4- to 5-fold (with no change in caveolin-3), and resulted in 1.9-fold increase in enzyme activity. T(3)-induced Na, K-ATPase subunits were preferentially distributed to the lighter fractions (#s 4, 5 and 6); and increased abundance of alpha1 and beta1 were 34-70% and 43-68%, respectively. We conclude that the activity of Na, K-ATPase is not uniform in cardiac membranes, and while a significant amount of Na, K-ATPase is present in cardiac cholesterol-rich membrane fractions, the intrinsic activity is significantly less than the enzyme present in relatively cholesterol-poor membranes.
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PMID:Effect of thyroid hormone on the distribution and activity of Na, K-ATPase in ventricular myocardium. 1845 53

Studies on Cd hepatotoxicity have focused mainly on induction of cytochrome P(450) system and related enzymes. In the present study young adult male rats given a single intra-peritoneal injection of Cd (0.84mg Cd/kg body weight) and effects on kinetic parameters rat liver microsomal Na(+), K(+)-ATPase and G6Pase were evaluated at the end of 1 month and 1 week. The substrate and temperature kinetics parameters were examined and attempts were made to seek correlation with changes in lipid/phospholipid profiles. The Na(+), K(+) ATPase activity decreased only in 1 week Cd-treated group but recovered at the end of 1 month. The activity resolved in two distinct kinetic components in control as well as the experimental groups. In 1 week Cd-treated group the K(m) value of both the components was unchanged, whereas V(max) value decreased. In 1-month Cd-treated group V(max) value only of component I increased. The catalytic efficiency of both the components was not affected in the experimental groups. In 1-week Cd-treated group the energy of activations at high-temperature range (E(H)) and low-temperature range (E(L)) decreased, whereas for 1-month Cd-treated group the energies of activations did not change. The G6Pase activity measured at 37 degrees C was high only in 1-month Cd-treated group. The activity resolved in two kinetically distinguishable components in control as well as in the experimental groups. K(m) value of component I decreased in both the Cd-treated groups. In 1-month Cd-treated group the V(max) value of component II increased. The catalytic efficiency of G6Pase was unchanged despite changes in K(m) and V(max). In 1-week Cd-treated group the E(H) and E(L) decreased, whereas only E(L) showed decrease in 1-month Cd-treated group. Cholesterol (CHL) content increased in both the Cd-treated groups. Content of lysophospholipid (Lyso), spinghomyelin (SPM) and phosphatidic acid (PA) increased, whereas phosphatidylcholine (PC) and phosphatidylserine (PS) decreased in 1-week Cd-treated group. In 1-month Cd group the Lyso, SPM, and PC increased while PC, phosphatidylethanolamine (PE) and PA decreased. In conclusion, Cd has short-term effects on microsomal Na(+), K(+)-ATPase which are reversed by the end of 1 month and that G6Pase does not seem to be a target of Cd insult.
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PMID:Effect of treatment with cadmium on kinetic properties of Na(+), K(+)-ATPase and glucose-6-phosphatase activity in rat liver microsomes a correlative study on influence of lipid/phospholipid make-up. 1883 18

Cholesterol promotes basal and verapamil-induced ATPase activity of P-glycoprotein (P-gp). We investigated whether these effects are related to each other and to the impact of the sterol on bilayer fluidity and verapamil membrane affinity. P-gp was reconstituted in egg-phosphatidylcholine (PhC) liposomes with or without cholesterol, 1,2-dipalmitoyl-phosphatidylcholine (DPPC), alpha-tocopherol (alpha-Toc) or 2,2,5,7,8-pentamethyl-6-chromanol (PMC). Basal and verapamil-induced ATPase activities were studied with an enzymatic assay. Membrane fluidity was characterized with diphenyl-hexatriene anisotropy measurements and membrane affinity by equilibrium dialysis. DPPC (70% mol/mol) decreased the fluidity of PhC bilayers to the same level as 20% cholesterol. PMC (20%) and alpha-Toc (20%) decreased the fluidity to lesser extents. alpha-Toc and PMC, but not DPPC increased the verapamil membrane affinity. While 20% cholesterol strikingly enhanced the basal ATPase activity, none of the other constituents had a similar effect. In contrast, verapamil stimulation of P-gp ATPase activity was not only enabled by cholesterol but also by alpha-Toc and DPPC. PMC had no effect. In conclusion, cholesterol exerts distinct effects on basal and verapamil-induced ATPase activity. The influence on basal ATPase activity is sterol-specific while its effect on verapamil-induced ATPase activity is unspecific and not related to its influence on membrane fluidity and on verapamil membrane affinity.
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PMID:Cholesterol-mediated activation of P-glycoprotein: distinct effects on basal and drug-induced ATPase activities. 1893 60

The mouse ortholog of the human bile salt export pump (BSEP) transporter was expressed in a baculovirus-infected insect cell (Sf9) system to study the effect of membrane cholesterol content on the transporter function. The transport activity of cholesterol-loaded mouse Bsep-HAM-Sf9 vesicles was determined in a vesicular transport assay with taurochenodeoxycholate (TCDC), a known BSEP substrate. Mouse Bsep transports TCDC at a high rate that can be sensitively detected in the ATPase assay. Cholesterol upload of the Sf9 membrane potentiates both TCDC transport and TCDC-stimulated ATPase activities. Inhibitory effect of BSEP interactors on probe substrate transport was tested in both vesicular transport and ATPase assays using cholesterol-loaded membrane vesicles. A good rank order correlation was found between IC(50) values measured in TCDC-stimulated mBsep ATPase assay and in the human BSEP vesicular transport assay utilizing taurocholate (TC) as probe substrate. This upgraded form of the mouse Bsep-HAM ATPase assay is a user friendly, sensitive, nonradioactive method for early high-throughput screening of drugs with BSEP-related cholestatic potential. It may complement the human BSEP-mediated taurocholate vesicular transport inhibition assay.
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PMID:Mouse Bsep ATPase assay: a nonradioactive tool for assessment of the cholestatic potential of drugs. 1902 16

Resistance to a broad spectrum of structurally diverse chemotherapeutic drugs (multidrug resistance; MDR) is a major impediment to the treatment of cancer. One cause of MDR is the expression at the tumor cell surface of P-glycoprotein (Pgp), which functions as an ATP-powered multidrug efflux pump. Since Pgp interacts with its substrates after they partition into the lipid bilayer, changes in membrane physicochemical properties may have substantial effects on its functional activity. Various interactions between cholesterol and Pgp have been suggested, including a role for the protein in transbilayer movement of cholesterol. We have characterized several aspects of Pgp-cholesterol interactions, and found that some of the previously reported effects of cholesterol result from inhibition of Pgp ATPase activity by the cholesterol-extracting reagent, methyl-beta-cyclodextrin. The presence of cholesterol in the bilayer modulated the basal and drug-stimulated ATPase activity of reconstituted Pgp in a modest fashion. Both the ability of drugs to bind to the protein and the drug transport and phospholipid flippase functions of Pgp were also affected by cholesterol. The effects of cholesterol on drug binding affinity were unrelated to the size of the compound. Increasing cholesterol content greatly altered the partitioning of hydrophobic drug substrates into the membrane, which may account for some of the observed effects of cholesterol on Pgp-mediated drug transport. Pgp does not appear to mediate the flip-flop of a fluorescent cholesterol analogue across the bilayer. Cholesterol likely modulates Pgp function via effects on drug-membrane partitioning and changes in the local lipid environment of the protein.
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PMID:Interaction of the P-glycoprotein multidrug efflux pump with cholesterol: effects on ATPase activity, drug binding and transport. 1904 91


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