Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase.

Sarkadi, Balázs; Price, Elmer M.; Boucher, Richard C.; Germann, Ursula A.; Scarborough, Gene A.

doi:10.1016/s0021-9258(18)42909-2

Cited by 613 publications

(127 citation statements)

References 33 publications

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“…Sf9 cells were cultured and infected with a recombinant baculovirus stock as described earlier. 7 In case of cholesterol-loaded membranes, cells were collected and treated with 10 × volume Hanks' balanced salt solution (HBSS; Gibco, Invitrogen, Carlsbad, CA) containing 1 mM cholesterol@RAMEB, for 30 min at 37 °C. After 30-min treatment, cells were washed and resuspended in HBSS.…”

Section: Methodsmentioning

confidence: 99%

“…After 30-min treatment, cells were washed and resuspended in HBSS. Purified membrane vesicles from cholesterol-treated and untreated baculovirus-infected Sf9 cells were prepared according to the method described by Sarkadi et al 7 Membrane protein content was determined using the BCA method (Pierce Biotechnology, Rockford, IL).…”

Section: Methodsmentioning

confidence: 99%

“…6 BSEP transporter function is often studied in vitro in membrane-based vesicular transport assays applying canalicular membrane vesicles 1 or using sandwich cultured hepatocytes as a cellular model. 6 For high-throughput applications, efflux transporters are commonly expressed in insect cell lines (e.g., Sf9) taking advantage of the robust baculovirus insect cell system, 7 and purified membranes are utilized in vesicular transport and ATPase assays. 8 After preparation, these membrane products can be stored for at least 1 year at -80 °C (without thawing) and used as biochemical "reagents" in the various assays.…”

Section: Introductionmentioning

confidence: 99%

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Mouse Bsep ATPase Assay: A Nonradioactive Tool for Assessment of the Cholestatic Potential of Drugs

et al. 2008

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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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Mouse Bsep ATPase Assay: A Nonradioactive Tool for Assessment of the Cholestatic Potential of Drugs

et al. 2008

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“…Whether a compound is an inhibitor or a transport substrate for ABCG/PDR transporters is solely determined by the affinity that sets the on versus off rates, and by the kinetics underlying the interactions in the binding zones [ 33 ]. Indeed, biochemistry data also suggest that certain inhibitors inhibit ATPase activity [ 349 , 350 , 351 ]. Binding of the second ATP molecule functions as a molecular glue that triggers complete NBD dimerization, thus inducing the conformational switch of the TMDs into a substrate-occluded state (3).…”

Section: Model For a Conserved Catalytic Transport Cycle Of Abcg/pdr Transportersmentioning

confidence: 99%

Multidrug Resistance in Mammals and Fungi—From MDR to PDR: A Rocky Road from Atomic Structures to Transport Mechanisms

Khunweeraphong

Kuchler

2021

IJMS

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Multidrug resistance (MDR) can be a serious complication for the treatment of cancer as well as for microbial and parasitic infections. Dysregulated overexpression of several members of the ATP-binding cassette transporter families have been intimately linked to MDR phenomena. Three paradigm ABC transporter members, ABCB1 (P-gp), ABCC1 (MRP1) and ABCG2 (BCRP) appear to act as brothers in arms in promoting or causing MDR in a variety of therapeutic cancer settings. However, their molecular mechanisms of action, the basis for their broad and overlapping substrate selectivity, remains ill-posed. The rapidly increasing numbers of high-resolution atomic structures from X-ray crystallography or cryo-EM of mammalian ABC multidrug transporters initiated a new era towards a better understanding of structure–function relationships, and for the dynamics and mechanisms driving their transport cycles. In addition, the atomic structures offered new evolutionary perspectives in cases where transport systems have been structurally conserved from bacteria to humans, including the pleiotropic drug resistance (PDR) family in fungal pathogens for which high resolution structures are as yet unavailable. In this review, we will focus the discussion on comparative mechanisms of mammalian ABCG and fungal PDR transporters, owing to their close evolutionary relationships. In fact, the atomic structures of ABCG2 offer excellent models for a better understanding of fungal PDR transporters. Based on comparative structural models of ABCG transporters and fungal PDRs, we propose closely related or even conserved catalytic cycles, thus offering new therapeutic perspectives for preventing MDR in infectious disease settings.

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“…Purified membrane vesicles from baculovirusinfected Sf9 cells were prepared essentially as described previously. 30 Membrane protein content was determined using the BCA method (Pierce Biotechnology, Rockford, IL). The presence of the MRP2 transporter was confirmed with Western blot, using the MRP2-specific monoclonal antibody M 2 III-5 (Alexis Corp, Lausen, Switzerland).…”

Section: Membrane Preparationmentioning

confidence: 99%

Characterization of 5(6)-Carboxy-2,′7′-Dichlorofluorescein Transport by MRP2 and Utilization of this Substrate as a Fluorescent Surrogate for LTC4

et al. 2008

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KRISZTINA HEREDI-SZABO, EMESE KIS, EVA MOLNAR, ANDRAS GYORFI, and PETER KRAJCSIMRP2 (ABCC2) is an efflux transporter expressed on the apical membrane of polarized cells. This protein has a major role in the biliary elimination of toxic compounds from the liver. As MRP2 transports many endogenous compounds, including LTC4 as well as xenobiotics and toxic phase II metabolites, blockade of this transporter may cause the accumulation of these compounds in the hepatocyte, resulting in hepatotoxicity. The vesicular transport assay is a great tool to study drug-drug and drug-endogenous compound interactions of ABC transporters. In this assay, inside-out membrane vesicles are used, so the test compound can readily access the transporter. As MRP2 transports many ionic compounds that are difficult to investigate in a whole-cell system because of permeability reasons, the vesicular transport assay is a good choice for screening MRP2-mediated interactions. LTC4 is not an optimal substrate for high-throughput screening for MRP2 interactors, even though it is an important MRP2 substrate. Therefore, the transport of a drug surrogate, 5(6)-carboxy-2,′7′-dichlorofluorescein (CDCF), by MRP2 was characterized using the vesicular transport assay. The data indicate that CDCF proves to be an ideal substrate for MRP2 vesicular transport assay with its optimal detection and transport properties. (Journal of Biomolecular Screening 2008:295-301)

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Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase.

Cited by 613 publications

References 33 publications

Mouse Bsep ATPase Assay: A Nonradioactive Tool for Assessment of the Cholestatic Potential of Drugs

Mouse Bsep ATPase Assay: A Nonradioactive Tool for Assessment of the Cholestatic Potential of Drugs

Multidrug Resistance in Mammals and Fungi—From MDR to PDR: A Rocky Road from Atomic Structures to Transport Mechanisms

Characterization of 5(6)-Carboxy-2,′7′-Dichlorofluorescein Transport by MRP2 and Utilization of this Substrate as a Fluorescent Surrogate for LTC4

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