Rhodamine 123 binds to multiple sites in the multidrug resistance protein (MRP1)

The mechanisms of MRP1-drug binding and transport are not clear. In this study, we have characterized the interaction between MRP1 and rhodamine 123 (Rh123) using the photoreactive-iodinated analogue, [(125)I]iodoaryl azido-rhodamine 123 (or IAARh123). Photoaffinity labeling of plasma membranes from HeLa cells transfected with MRP1 cDNA (HeLa-MRP1) with IAARh123 shows the photolabeling of a 190 kDa polypeptide not labeled in HeLa cells transfected with the vector alone. Immunoprecipitation of a 190 kDa photolabeled protein with MRP1-sepcific monoclonal antibodies (QCRL-1, MRPr1, and MRPm6) confirmed the identity of this protein as MRP1. Analysis of MRP1-IAARh123 interactions showed that photolabeling of membranes from HeLa-MRP1 with increasing concentrations of IAARh123 was saturable, and was inhibited with excess of IAARh123. Furthermore, the photoaffinity labeling of MRP1 with IAARh123 was greatly reduced in the presence of excess Leukotreine C(4) or MK571, but to a lesser extent with excess doxorubicin, colchicine or chloroquine. Cell growth assays showed 5-fold and 14-fold increase in the IC(50) of HeLa-MRP1 to Rh123 and the Etoposide VP16 relative to HeLa cells, respectively. Analysis of Rh123 fluorescence in HeLa and HeLa-MRP1 cells with or without ATP suggests that cross-resistance to Rh123 is in part due to reduced drug accumulation in the cytosol of HeLa-MRP1 cells. Mild digestion of purified IAARh123-photolabeled MRP1 with trypsin showed two large polypeptides (approximately 111 and approximately 85 kDa) resulting from cleavage in the linker domain (L1) connecting the multiple-spanning domains MSD0 and MSD1 to MSD2. Exhaustive proteolysis of purified IAARh123-labeled 85 and 111 kDa polypeptides revealed one (6 kDa) and two (approximately 6 plus 4 kDa) photolabeled peptides, respectively. Resolution of total tryptic digest of IAARh123-labeled MRP1 by HPLC showed three radiolabeled peaks consistent with the three Staphylococcus aureus V8 cleaved peptides from the Cleveland maps. Together, the results of this study show direct binding of IAARh123 to three sites that localize to the N- and C-domains of MRP1. Moreover, IAARh123 provides a sensitive and specific probe to study MRP1-drug interactions.     

Major photoaffinity drug binding sites in multidrug resistance protein 1 (MRP1) are within transmembrane domains 10-11 and 16-17

MRP1 is an ABC (or ATP binding cassette) membrane transport protein shown to confer resistance to structurally dissimilar drugs. Studies of MRP1 topology suggested the presence of a hydrophobic N-domain with five potential membrane-spanning domains linked to an MDR1-like core (MSD1-NBD1-L1-MSD2-NBD2) by an intracellular linker domain (L0). MRP1-mediated multidrug resistance is thought to be due to enhanced drug efflux. However, little is known about MRP1-drug interaction and its drug binding site(s). We previously developed several photoreactive probes to study MRP1-drug interactions. In this report, we have used eight MRP1-HA variants that were modified to have hemagglutinin A (HA) epitopes inserted at different sites in MRP1 sequence. Exhaustive in-gel digestion of all IAARh123 photoaffinity-labeled MRP1-HA variants revealed the same profile of photolabeled peptides as seen for wild type MRP1. Photolabeling of the different MRP1-HA variants followed by digestion with increasing concentrations of trypsin or Staphylococcus aureus V8 protease (1:800 to 1:5 w/w) and immunoprecipitation with anti-HA mAb identified two small photolabeled peptides ( approximately 6-7 kDa) from MRP1-HA(574) and MRP1-HA(1222). Based on the location of the HA epitopes in the latter variants together with molecular masses of the two peptides, the photolabeled amino acid residues were localized to MRP1 sequences encoding transmembranes 10 and 11 of MSD1 (Ser(542)-Arg(593)) and transmembranes 16 and 17 of MSD2 (Cys(1205)-Glu(1253)). Interestingly, the same sequences in MRP1 were also photolabeled with a structurally different photoreactive drug, IACI, confirming the significance of transmembranes 10, 11, 16 and 17 in MRP1 drug binding. Taken together, the results in this study provide the first delineation of the drug binding site(s) of MRP1. Furthermore, our findings suggest the presence of common drug binding site(s) for structurally dissimilar drugs.     

Photoaffinity labeling under non-energized conditions of a specific drug-binding site of the ABC multidrug transporter LmrA from Lactococcus lactis

The Lactococcus lactis multidrug resistance ABC transporter protein LmrA has been shown to confer resistance to structurally and functionally diverse antibiotics and anti-cancer drugs. Using a previously characterized photoreactive drug analogue of Rhodamine 123 (iodo-aryl azido-Rhodamine 123 or IAARh123), direct and specific photoaffinity labeling of LmrA in enriched membrane vesicles could be achieved under non-energized conditions. This photoaffinity labeling of LmrA occurs at a physiologically relevant site as it was inhibited by molar excess of ethidium bromide>Rhodamine 6G>vinblastine>doxorubicin>MK571 (a quinoline-based drug) while colchicine had no effect. The MDR-reversing agents PSC 833 and cyclosporin A were similarly effective in inhibiting IAARh123 photolabeling of LmrA and P-glycoprotein. In-gel digestion with Staphyloccocus aureus V8 protease of IAARh123-photolabeled LmrA revealed several IAARh123 labeled polypeptides, in addition to a 6.8kDa polypeptide that comprises the last two transmembrane domains of LmrA.     

Effects of plant sterols on human multidrug transporters ABCB1 and ABCC1

The effects of dietary plant sterols on human drug efflux transporters P-glycoprotein (P-gp, ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) were investigated using P-gp-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural phytosterols found in foods, herbs, and dietary supplements such as β-sitosterol, campesterol, stigmasterol, fucosterol, and z-guggulsterone were investigated. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-gp, increased in the presence of guggulsterone in KB-C2 cells. The efflux of rhodamine 123 from KB-C2 cells was inhibited by guggulsterone. Guggulsterone also increased the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. The ATPase activities of P-gp and MRP1 were stimulated by guggulsterone. These results suggest that guggulsterone, a natural dietary hypolipidemic agent have dual inhibitory effects on P-gp and MRP1 and the potencies to cause food-drug interactions.     

Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a “facial” detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K = 0.06 mM^− 1), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.     

Interaction of nonionic detergents with phospholipids in hepatic microsomes at subsolubilizing concentrations as studied by 31P-NMR

The effect of low concentrations of nonionic detergents with different critical micelle concentrations such as Triton X-100, Brij 35 and octylglucoside on rabbit liver microsomes is studied by means of ³¹P-NMR, ¹H-NMR, dynamic light scattering and functional investigations. Hexane phosphonic acid diethyl ester was used as a phosphorus membrane probe molecule to monitor the interaction of detergent molecules with microsomal phospholipids by ³¹P-NMR. This method is more sensitive than ³¹P-NMR of phospholipids alone and permitted the estimation of the maximum number of detergent molecules which can be incorporated in microsomes without the formation of mixed micelles outside the membrane. These membrane saturation concentrations were determined to be 0.07 (Brij 35), 0.1 (Triton X-100) and 0.4 (octylglucoside) (molar ratio of detergent/total phospholipids). Above these detergent concentrations, mixed micelles consisting of detergent and membrane constituents are formed, coexisting with the microsomes up to the membrane solubilization concentration. The results indicate a dependence of the membrane saturation concentration on the critical micelle concentration of the detergent and a preferential removal of phosphatidylcholine over phosphatidylethanolamine from the microsomes by all detergents studied.     

A positively charged amino acid proximal to the C-terminus of TM17 of MRP1 is indispensable for GSH-dependent binding of substrates and for transport of LTC4

MRP1 is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. Our recent study demonstrated that GSH is required for the labeling of MRP1(932)(-)(1531) with a photoanalogue of agosterol A (AG-A) and suggested that GSH interacts with the L(0) region of MRP1. In this study, we further characterized the GSH-dependent binding site of azido AG-A on MRP1. Coexpression of the N- and C-terminal halves of MRP1 (residues 1-1222, TM1-16, and 1223-1531, TM17, respectively) in Sf21 insect cells reconstituted a functional drug transporter with a K(m) for LTC(4) (97 nM) similar to that of intact MRP1. In membrane vesicles from those cells, GSH-dependent photolabeling of the MRP1 fragment (1-1222) required the coexpression of the C-terminal MRP1 fragment (1223-1531). An MRP1 fragment extending from residue 1 to 1295 however could be photolabeled by azido AG-A in a GSH-dependent manner. These data indicate that amino acids 1223-1295 of MRP1 are required for AG-A binding to MRP1 in a GSH-dependent manner. However, cross-linking of the photolabel to MRP1 occurs at a more upstream site. An arginine residue at position 1249 of MRP1 was shown to be important for the GSH-dependent binding of AG-A to MRP1. Mutation of this arginine to alanine (R1249A) resulted in a decreased level of GSH-dependent azido AG-A photolabeling of MRP1. Furthermore, this mutant attenuated MRP1 function by decreasing the level of LTC(4) substrate transport and impairing resistance to the drug vincristine (VCR). In summary, this study demonstrates that a region of MRP1 (amino acids 1223-1295), which includes TM helix 17, is required for azido AG-A binding to MRP1 in a GSH-dependent manner. A GSH-dependent drug binding site may exist in this region. Furthermore, our findings suggest that the charged amino acid Arg(1249) proximal to the C-terminus of TM helix 17 is indispensable for MRP1-substrate interaction and the function of MRP1.     

Detergent-insoluble glycosphingolipid/cholesterol microdomains of the myelin membrane

Glycosphingolipids and cholesterol form lateral assemblies, or lipid 'rafts', within biological membranes. Lipid rafts are routinely studied biochemically as low-density, detergent-insoluble complexes (in non-ionic detergents at 4 degrees C; DIGs, detergent-insoluble glycosphingolipid/cholesterol microdomains). Recent discrepancies recommended a re-evaluation of the conditions used for the biochemical analysis of lipid rafts. We have investigated the detergent insolubility of several known proteins present in the glycosphingolipid/cholesterol-rich myelin membrane, using four detergents representing different chemical classes (TX-100, CHAPS, Brij 96 and TX-102), under four conditions: detergent extraction of myelin either at (i) 4 degrees C or (ii) 37 degrees C, or at 4 degrees C after pre-extraction with (iii) saponin or (iv) methyl-beta-cyclodextrin (MbetaCD). Each detergent was different in its ability to solubilize myelin proteins and in the density of the DIGs produced. Brij 96 DIGs floated to a lower density than other detergents tested, possibly representing a subpopulation of DIGs in myelin. DIGs pre-extracted with saponin were denser than DIGs pre-extracted with MbetaCD. Furthermore, pre-extraction with MbetaCD solubilized proteolipid protein (known to associate with cholesterol), whereas pre-extraction with saponin did not, suggesting that saponin is less effective as a cholesterol-perturbing agent than is MbetaCD. These results demonstrate that DIGs isolated by different detergents are not necessarily comparable, and that these detergent-specific DIGs may represent distinct biochemical, and possibly physiological, entities based on the solubilities of specific lipids/proteins in each type of detergent.     

Biophysical approaches in the study of biomembrane solubilization: quantitative assessment and the role of lateral inhomogeneity

Detergents are amphiphilic molecules widely used to solubilize biological membranes and/or extract their components. Nevertheless, because of the complex composition of biomembranes, their solubilization by detergents has not been systematically studied. In this review, we address the solubilization of erythrocytes, which provide a relatively simple, robust and easy to handle biomembrane, and of biomimetic models, to stress the role of the lipid composition on the solubilization process. First, results of a systematic study on the solubilization of human erythrocyte membranes by different series of non-ionic (Triton, CxEy, Brij, Renex, Tween), anionic (bile salts) and zwitterionic (ASB, CHAPS) detergents are shown. Such quantitative approach allowed us to propose R_e ^sat—the effective detergent/lipid molar ratio in the membrane for the onset of hemolysis as a new parameter to classify the solubilization efficiency of detergents. Second, detergent-resistant membranes (DRMs) obtained as a result of the partial solubilization of erythrocytes by TX-100, C₁₂E₈ and Brij detergents are examined. DRMs were characterized by their cholesterol, sphingolipid and specific proteins content, as well as lipid packing. Finally, lipid bilayers of tuned lipid composition forming liposomes were used to investigate the solubilization process of membranes of different compositions/phases induced by Triton X-100. Optical microscopy of giant unilamellar vesicles revealed that pure phospholipid membranes are fully solubilized, whereas the presence of cholesterol renders the mixture partially or even fully insoluble, depending on the composition. Additionally, Triton X-100 induced phase separation in raft-like mixtures, and selective solubilization of the fluid phase only.     

Nucleotide binding and nucleotide hydrolysis properties of the ABC transporter MRP6 (ABCC6)

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.     

Ultrastructure of human erythrocyte GLUT1

This study was undertaken to examine GLUT1 quaternary structure. Independent but complementary methodologies were used to investigate the influence of membrane-solubilizing detergents on GLUT1/lipid/detergent micelle hydrodynamic radii. Hydrodynamic size analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze-fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. GLUT1 forms a multimeric complex in octyl glucoside that dissociates upon addition of reductant. Some detergents (e.g., CHAPS and dodecyl maltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). These complexes do not reassemble as larger oligomers when dissociating detergents are subsequently replaced with nondissociating detergents such as octyl glucoside or cholic acid. When dissociating detergents are replaced with lipids, the resulting proteoliposomes catalyze protein-mediated sugar transport, and the subsequent addition of solubilizing, nondissociating detergents generates higher (tetrameric) GLUT1 aggregation states. These findings suggest that some detergents stabilize while others destabilize GLUT1 quaternary structure. GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer.     

Glutathione-dependent binding of a photoaffinity analog of agosterol A to the C-terminal half of human multidrug resistance protein

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD(0)), which is connected to a P-glycoprotein-like core region (DeltaMRP) by a cytoplasmic linker domain zero (L(0)). It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD(0) and L(0) are not known. We synthesized [(125)I]11-azidophenyl agosterol A ([(125)I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C(932-1531)) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C(4). Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L(0) is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L(0) of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.     

Dissociation, aggregation of sesame alpha-globulin in nonionic detergent solution.

Nonionic detergents Triton X-100 and Brij 36T induce dissociation and aggregation of the protein sesame alpha-globulin above the critical micelle concentrations (cmc) of the detergents. Spectrophotometric titration in Triton shows no change in the pKInt value of the tyrosyl groups at 1x10-3 M detergent where both dissociation and aggregation of the protein are observed. Fluorescence measurement does not indicate any change in the environment of the tryptophan groups of the protein in Brij. Viscosity measurements show no major conformational change of the protein in the detergent solution. Binding measurements suggest that perhaps micelles of the detergent predominantly bind to the protein. The detergent micelles preferentially bind to the exposed hydrophobic surfaces of the protein subunits. The association of the protein detergent complex through electrostatic interaction is probably responsible for the formation of the aggregates.     

The minimal structural requirement of concanavalin A that retains its functional aspects

A systematic investigation of the effects of several commonly used detergents on the conformation and function of concanavalin A at pH 7 in solution form was made by using circular dichroism (CD), intrinsic fluorescence, 1-anilino 8-sulphonic acid (ANS) binding, dynamic light scattering (DLS) and sugar inhibition assay. In the presence of 6.0 mM sodium dodecyl sulphate (SDS), an anionic detergent, and 0.8 mM cetyl tri methyl ammonium bromide (CTAB), a cationic detergent, intermediate states of concanavalin A were obtained having a negative CD peaks at 222 and 208 nm respectively, a characteristic of alpha-helix. These states also retained tertiary contacts with altered tryptophan environment and high ANS binding (exposed hydrophobic area) which can be characterized as molten globule states. Concanavalin A in the presence of 5.0 mM 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulphonate (CHAPS), a zwitterionic detergent, and 0.07 mM brij-35, a non-ionic detergent, also exists in intermediate states. These intermediates (molten globules) had high ANS binding with native-like secondary (inherent beta-sheet) and tertiary structure. The intermediate states were characterized further by means of dynamic light-scattering measurements and kinetic data. To study the possible functional requirement of the minimum structure, the intermediate states characterized in the presence of detergents were shown to retain the activity with polysaccharide (dextran). The pattern of activity observed was brij-35 > CHAPS > CTAB > SDS. The specific binding and activity of concanavalin A with ovalbumin was investigated as a function of time by turbidity measurements. Cationic and anionic detergents showed significant effects on the structure of concanavalin A as compared with zwitterionic and non-ionic detergents.     

How Amphipols Embed Membrane Proteins: Global Solvent Accessibility and Interaction with a Flexible Protein Terminus

Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded β-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins.     

A structural characterization of gap junctions isolated from mouse liver

Mouse liver gap junctions have been isolated by using an anionic detergent, n-dodecanoyl sarcosine, in combination with non-ionic polyoxyethylene ethers (Brij 35 and Brij 58) and (W-1) detergents. Purified gap junctions are obtained in a sucrose step gradient containing 1-o-n-octyl-beta-D-glucopyranoside. This procedure is aimed at minimizing proteolysis. The junctions thus isolated have a hexagonal lattice of connexons with a lattice constant of 7.6-8.4 nm. Presence of a major Mr 26,000 gap junctional protein has been confirmed by SDS-PAGE.     

Solubilization of UDP-glucose collagen glucosyltransferase activities from chick embryo liver: Golgi apparatus, smooth and rough endoplasmic reticulum.

1. The choice of a suitable detergent for solubilization of UDP-glucose collagen glucosyltransferase (GGT) activities from chick embryo liver has been investigated. Several detergents were used (zwitterionic detergent as Chaps, and non-ionic detergents as Triton X-100, Nonidet P 40, Brij 35). 2. All the detergents with GGT activities were tested in Golgi apparatus, smooth and rough endoplasmic reticulum (SER, RER). 3. 80-100% GGT Golgi apparatus activity was easily solubilized at low concentrations in surfactant (0.5 mg/ml). 25-78% of SER and RER GGT activities were extracted at this concentration. 4. A higher level of detergent (5 mg/ml) was necessary to release all GGT activities of SER and RER. Protein extraction was identical to GGT activities.     

Amphilphilic nature of spiralin, the major protein of the Spiroplasma citri cell membrane.

Spiralin could not be solubilized in the absence of detergents, and it was shown by charge-shift crossed immunoelectrophoresis that this protein was capable of binding detergents under nondenaturing conditions. These properties indicate the amphiphilic nature of spiralin, which therefore should be regarded as an intrinsic membrane protein. The efficiency of mild (ionic and neutral) detergents to solubilize spiralin was as follows: deoxycholate greater than lauroyl sarcosinate, cholate, taurocholate, taurodeoxycholate greater than Triton X-100 greater than Brij 58 greater than Tween 20, indicating that mild ionic detergents were more effective than neutral ones. Solubilization of spiralin was quantitative with sodium deoxycholate. It was also shown that although a membrane protein is not extractable by a given detergent from the membrane, this does not necessarily mean that the protein is not soluble in this detergent.     

Photolabeling of human and murine multidrug resistance protein 1 with the high affinity inhibitor [125I]LY475776 and azidophenacyl-[35S]glutathione

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent transporter of structurally diverse organic anion conjugates. The protein also actively transports a number of non-conjugated chemotherapeutic drugs and certain anionic conjugates by a presently poorly understood GSH-dependent mechanism. LY475776is a newly developed (125)I-labeled azido tricyclic isoxazole that binds toMRP1 with high affinity and specificity in a GSH-dependent manner. The compound has also been shown to photolabel a site in the COOH-proximal region of MRP1's third membrane spanning domain (MSD). It is presently not known where GSH interacts with the protein. Here, we demonstrate that the photactivateable GSH derivative azidophenacyl-GSH can substitute functionally for GSH in supporting the photolabeling of MRP1 by LY475776 and the transport of another GSH-dependent substrate, estrone 3-sulfate. In contrast to LY475776, azidophenacyl-[(35)S] photolabels both halves of the protein. Photolabeling of the COOH-proximal site can be markedly stimulated by low concentrations of estrone 3-sulfate, suggestive of cooperativity between the binding of these two compounds. We show that photolabeling of the COOH-proximal site by LY475776 and the labeling of both NH(2)- and COOH- proximal sites by azidophenacyl-GSH requires the cytoplasmic linker (CL3) region connecting the first and second MSDs of the protein, but not the first MSD itself. Although required for binding, CL3 is not photolabeled by azidophenacyl-GSH. Finally, we identify non-conserved amino acids in the third MSD that contribute to the high affinity with which LY475776 binds to MRP1.