Inside-out Plasma Membrane Vesicles Research Articles

Exploring the role of the LkABCG36 transporter in lignin accumulation

Lignin is a complex biopolymer formed through the condensation of three monomeric precursors known as monolignols. However, the mechanism underlying lignin precursor transport remains elusive, with uncertainty over whether it occurs through passive diffusion or an active energized process. ATP-binding cassette 36 (ABCG36) plays important roles in abiotic stress resistance. In this study, we investigated the transport functions of LkABCG36 (Larix kaempferi) for lignin precursors and the potential effects of LkABCG36 overexpression in plants. LkABCG36 enhanced the ability of tobacco (Nicotiana tabacum) bright yellow-2 (BY-2) cells to resist monolignol alcohol stress. Furthermore, LkABCG36 overexpression promoted lignin deposition in tobacco plant stem tissue. To understand the underlying mechanism, we measured the BY-2 cell ability to export lignin monomers and the uptake of monolignol precursors in inside-out (inverted) plasma membrane vesicles. We found that the transport of coniferyl and sinapyl alcohols is an ATP-dependent process. Our data suggest that LkABCG36 contributes to lignin accumulation in tobacco stem tissues through a mechanism involving the active transport of lignin precursors to the cell wall. These findings shed light on the lignin biosynthesis process, with important implications for enhancing lignin deposition in plants, potentially leading to improved stress tolerance and biomass production.

ATPase/synthase activity of Paracoccus denitrificans Fo·F1 as related to the respiratory control phenomenon

The time course of ATP synthesis, oxygen consumption, and change in the membrane potential in Paracoccus denitrificans inside-out plasma membrane vesicles was traced. ATP synthesis initiated by the addition of a limited amount of either ADP or inorganic phosphate proceeded up to very low residual concentrations of the limiting substrate. Accumulated ATP did not decrease the rate of its synthesis initiated by the addition of ADP. The amount of residual ADP determined at State 4 respiration was independent of ten-fold variation of Pi or the presence of ATP. The pH-dependence of Km for Pi could not be fitted to a simple phosphoric acid dissociation curve. Partial inhibition of respiration resulted in a decrease in the rate of ATP synthesis without affecting the ATP/ADP reached at State 4. At pH8.0, hydrolysis of ATP accumulated at State 4 was induced by a low concentration of an uncoupler, whereas complete uncoupling results in rapid inactivation of ATPase. At pH7.0, no reversal of the ATP synthase reaction by the uncoupler was seen. The data show that ATP/ADP×Pi ratio maintained at State 4 is not in equilibrium with respiratory-generated driving force. Possible mechanisms of kinetic control and unidirectional operation of the Fo·F1-ATP synthase are discussed.

ModulatoryATPbinding to the E2 state of maize plasma membrane H+‐ATPase indicated by the kinetics of vanadate inhibition

P-type ATPases, as major consumers of cellular ATP in eukaryotic cells, are characterized by the formation of a phosphorylated enzyme intermediate (E2P), a process that is allosterically coupled to translocation of cations against an electrochemical gradient. The catalytic cycle comprises binding of Mg-ATP at the nucleotide-binding domain, phosphorylation of the E1 state (E1), conformational transition to the E2P state, and dephosphorylation through the actuator domain and re-establishment of the E1 state. Recently, it has been suggested that, for several P-type ATPases, Mg-ATP binds to the phosphorylated enzyme, thereby accelerating the transition to the E1 state, before then becoming the enzyme's catalytic substrate. Here, we provide evidence supporting this viewpoint. We employed kinetic models based on steady-state kinetics in the presence and absence of the reversible inhibitor orthovanadate. Vanadate is generally considered to be a conformational probe that specifically binds to the E2 state, arresting the enzyme in a state analogous to the E2P state. Hydrolytic H(+) -ATPase activities were measured in inside-out plasma membrane vesicles isolated from roots and shoots of maize plants. For root enzymes, kinetic models of vanadate inhibition that allow simultaneous binding of Mg-ATP and vanadate to the same enzyme state were most plausible. For shoot enzymes, application of the competitive inhibitor Mg-free ATP attenuated vanadate inhibition, which is consistent with a model in which either Mg-free ATP or Mg-ATP is bound to the enzyme when vanadate binds. Therefore, data from roots and shoots indicate that binding of ATP species before transition to the E1 state plays an important role in the catalytic cycle of plant plasma membrane H(+) -ATPase.

Ca2+/H+ exchange in the plasma membrane of Arabidopsis thaliana leaves

A large number of plant Ca2+/H+ exchangers have been identified in endomembranes, but far fewer have been studied for Ca2+/H+ exchange in plasma membrane so far. To investigate the Ca2+/H+ exchange in plasma membrane here, inside-out plasma membrane vesicles were isolated from Arabidopsis thaliana leaves using aqueous two-phase partitioning method. Ca2+/H+ exchange in plasma membrane vesicles was measured by Ca2+-dependent dissipation of a pre-established pH gradient. The results showed that transport mediated by the Ca2+/H+ exchange was optimal at pH 7.0, and displayed transport specificity for Ca2+ with saturation kinetics at Km = 47 μM. Sulfate and vanadate inhibited pH gradient across vesicles and decreased the Ca2+-dependent transport of H+ out of vesicles significantly. When the electrical potential across plasma membrane was dissipated with valinomycin and potassium, the rate of Ca2+/H+ exchange increased comparing to control without valinomycin effect, suggesting that the Ca2+/H+ exchange generated a membrane potential (interior negative), i.e. that the stoichiometric ratio for the exchange is greater than 2H+:Ca2+. Eosin Y, a Ca2+-ATPase inhibitor, drastically inhibited Ca2+/H+ exchange in plasma membrane as it does for the purified Ca2+-ATPase in proteoliposomes, indicating that measured Ca2+/H+ exchange activity is mainly due to a plasma membrane Ca2+ pump. These suggest that calcium (Ca2+) is transported out of Arabidopsis cells mainly through a Ca2+-ATPase-mediated Ca2+/H+ exchange system that is driven by the proton-motive force from the plasma membrane H+-ATPase.

Dysfunction of the Fusion of Pre-Synaptic Plasma Membranes and Synaptic Vesicles Caused by Oxidative Stress, and its Prevention by Vitamin E

To define whether hyperoxia induces the dysfunction of membrane fusion between synaptic vesicles with pre-synaptic plasma membranes in the nerve terminals, and whether vitamin E prevents this abnormal event, we investigated the influence of hyperoxia on the fusion ability of isolated synaptic vesicles and the inside-out type pre-synaptic plasma membrane vesicles from rat brain using the fluorescence tracing method. The membrane fusion ability of both membranes from rats subjected to hyperoxia was markedly decreased compared with the membranes from a normal rat. Rats subjected to hyperoxia in the form of oxidative stress showed significant increases in the levels of thiobarbituric acid reactive substances (TBARS), conjugated dienes, and protein carbonyl moieties in both synaptic vesicles and pre-synaptic plasma membranes. When rats were supplemented with vitamin E, these abnormalities were inhibited even when rats were subjected to hyperoxia.

Kinetics of oxidative phosphorylation catalyzed by inside-out plasma membrane vesicles of Paracoccus denitrificans

Coupled inside-out plasma membrane vesicles of Paracoccus denitrificans are capable of proton-motive force (pmf)-required, pmf-generating ATP hydrolysis [1]. The steady-state ATP hydrolysis, catalyzed by P. denitrificans FO·F1 (PFO·F1) proceeds via compulsory sequential mechanism where ADP leaves the enzyme–products (ADP and Pi) complex first followed by irreversible release of Pi [2]. Inasmuch operation of PFO·F1 ATPase (synthase) is macroscopically reversible it seems imperative to reconcile the steady-state kinetics of pmf-generating ATP hydrolysis with that of pmf-utilising ATP synthesis. To reach this goal the dependencies of the initial steadystate rates of oxidative phosphorylation on ADP and Pi concentrations and on pmf were measured. ATP synthesis rate showed simple hyperbolic dependence on either substrate (within the concentration ranges of 1.5–50 μM for ADP and 10–500 μM for Pi) with no mutual dependence of apparent Km values thus suggesting random formation of the enzyme–ADP·Pi complex. When the ‘third substrate’, pmf was varied by limitation of succinate or NADH oxidation rates an apparent ‘ping-pong’ mechanism was evident: a decrease of the maximal rates caused by a decrease of pmf resulted in proportional decrease of apparent Km values for either Pi or ADP. This pattern suggests that an irreversible step, presumably ATP formation at the enzyme active site, precedes the involvement of pmf in the overall reaction (likely at the product release step). Comparison of the steady-state kinetics of ATP hydrolysis [2] and synthesis shows that macroscopic reversibility of the PFO·F1 ATPase (synthase) reaction cannot be ascribed to operation of single microscopically reversible enzyme species. We propose that apparent equilibrium between any given pmf and intracellular (intramitochondrial) phosphoryl potential ([ATP]/[ADP]·[Pi]) is maintained by ‘futile cycle’ of ATP synthesis and hydrolysis catalyzed by kinetically (and structurally) distinct FO·F1 species. Supported by the Russian Foundation for Fundamental Research grant 08-04-00594.

Detergents as intrinsic P-glycoprotein substrates and inhibitors

We assessed the interaction of three electrically neutral detergents (Triton X-100, C 12EO 8, and Tween 80) with P-glycoprotein (ABCB1, MDR1) and identified the molecular elements responsible for this interaction. To this purpose we titrated P-glycoprotein in inside-out plasma membrane vesicles of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) with the detergents below their critical micelle concentration, CMC. The P-glycoprotein ATPase measured as a function of the detergent concentration yielded bell-shaped activity curves which were evaluated with a two-site binding model. The lipid–water partition coefficient and the transporter-water binding constant of the detergents were measured independently. Knowledge of these two parameters allowed assessment of the free energy of detergent binding to P-glycoprotein in the lipid membrane, Δ G tl 0, that reflects the direct detergent–transporter affinity. It increased as the number of ethoxyl groups increased, suggesting that these hydrogen bond acceptor groups are the key elements for the detergent–transporter interaction in the lipid membrane. The free energy of binding to P-glycoprotein per ethoxyl group (EO) was determined as approximately Δ G EO 0 = − 1.6 kJ/mol. The present findings moreover document that, depending on the concentration applied, detergents are intrinsic substrates for, or inhibitors of P-glycoprotein.

Ultrastructural analysis of lysosome reactions to inside-out cell membrane vesicles in a cell-free system

Lysosome reactions were ultrastructurally analyzed using a cell-free system with inside-out cell membrane vesicles (IOVs) prepared from rat erythrocyte ghosts in an alkaline buffer and with wheat germ agglutinin-coated colloidal gold particles (WGA-CGs). The submembranous surface coat in the ghosts was depleted from the IOVs' outer surfaces. When lysosomes from rat liver were incubated with these IOVs, some of the trilaminar membranes of the lysosomes and IOVs came into close contact and formed a five-laminar structure without an intermembranous gap. In other reactions, the membranes of both structures formed one continuous trilaminar membrane along the margin of contact and ruffling five-laminar structures in other regions. Several lysosomes exhibited invaginating hollows or projections that entrapped or encircled the IOVs. Similar five-laminar structures were seen at a few points of contact between the IOVs and the hollowing or projecting membranes. In contrast, such reactions were much rarer when IOVs with reconstituted spectrins and actins on their outer surface were used. The formation of tubuliform pits with membrane-bound WGA-CGs was also observed after the incubation of lysosomes with WGA-CGs. These observations suggest that lysosomes fuse with cytoskeleton-depleted IOVs, wrap arm-like projections around them, enclose them by invagination or incorporate their membrane-bound macromolecules through the process of tubuliform invagination. Furthermore, the fusion and wrapping processes are not necessarily independent.

Regulation of Function by Dimerization through the Amino-terminal Membrane-spanning Domain of Human ABCC1/MRP1

Overexpression of some ATP-binding cassette (ABC) membrane transporters such as ABCB1/P-glycoprotein/MDR1 and ABCC1/MRP1 causes multidrug resistance in cancer chemotherapy. It has been thought that half-ABC transporters with one nucleotide-binding domain and one membrane-spanning domain (MSD) likely work as dimers, whereas full-length transporters with two nucleotide-binding domains and two or three MSDs function as monomers. In this study, we examined the oligomeric status of the human full-length ABC transporter ABCC1/MRP1 using several biochemical approaches. We found 1) that it is a homodimer, 2) that the dimerization domain is located in the amino-terminal MSD0L0 (where L0 is loop 0) region, and 3) that MSD0L0 has a dominant-negative function when coexpressed with wild-type ABCC1/MRP1. These findings suggest that ABCC1/MRP1 may exist and function as a dimer and that MSD0L0 likely plays some structural and regulatory functions. It is also tempting to propose that the MSD0L0-mediated dimerization may be targeted for therapeutic development to sensitize ABCC1/MRP1-mediated drug resistance in cancer chemotherapy.

P-Glycoprotein Kinetics Measured in Plasma Membrane Vesicles and Living Cells

P-glycoprotein (MDR1, ABCB1) is an ATP-dependent efflux transporter of a large variety of compounds. To understand P-glycoprotein in more detail, it is important to elucidate its activity in the cellular ensemble as well as in plasma membrane vesicles (under conditions where other ATP dependent proteins are blocked). We measured P-glycoprotein activity in inside-out vesicles formed from plasma membranes of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) for comparison with previous measurements of P-glycoprotein activity in living NIH-MDR1-G185 cells. In plasma membrane vesicles activity was measured by monitoring phosphate release upon ATP hydrolysis and in living cells by monitoring the extracellular acidification rate upon ATP synthesis via glycolysis. P-glycoprotein was stimulated as a function of the concentration with 19 structurally different drugs, including local anesthetics, cyclic peptides, and cytotoxic drugs. The concentrations of half-maximum P-glycoprotein activation, K1, were identical in inside-out plasma membrane vesicles and in living cells and covered a broad range of concentrations (K1 approximately (10(-8)-10(-3)) M). The influence of the pH, drug association, and vesicle aggregation on the concentration of half-maximum P-glycoprotein activation was investigated. The turnover numbers in plasma membrane vesicles and in living cells were also approximately identical if the latter were measured in the presence of pyruvate. However, in the absence of pyruvate they were higher in living cells. The rate of ATP hydrolysis/ATP synthesis decreased exponentially with decreasing free energy of drug binding from water to the transporter, DeltaG0(tw)(1) (or increasing binding affinity). This suggests that drug release from the transmembrane domains has to occur before ATP is hydrolyzed for resetting the transporter.

Effects of methotrexate on calcium flux in rat liver mitochondria, microsomes and plasma membrane vesicles

The metabolic effects of methotrexate in perfused livers are similar to those exerted by hormones acting through Ca 2+-dependent mechanisms. The aim of the present study was to determine whether the effects of methotrexate are mediated by a direct action on cellular Ca 2+ fluxes. Methotrexate did not affect the ATP-dependent 45Ca 2+ uptake by mitochondria, microsomes and inside-out plasma membrane vesicles and Ca 2+ efflux from plasma membrane vesicles. However, methotrexate was able to stimulate 45Ca 2+ release from preloaded microsomes. The amount of Ca 2+ released by methotrexate was similar to that induced by IP 3. Methotrexate could be acting through the capacitative calcium entry mechanism.

Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system

Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 μM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.

The antimalarial drug resistance protein Plasmodium falciparum chloroquine resistance transporter binds chloroquine.

Recently, mutations in the novel polytopic integral membrane protein PfCRT were shown to cause chloroquine resistance (CQR) in the malarial parasite Plasmodium falciparum. PfCRT is not a member of the well-known family of ABC proteins that have previously been associated with other drug resistance phenomena. Thus, the mechanism(s) whereby mutant PfCRT molecules confer antimalarial drug resistance is (are) unknown. Previously, we succeeded in overexpressing PfCRT to high levels in Pichia pastoris yeast by synthesizing a codon-optimized version of the pfcrt gene. Using purified membranes and inside-out plasma membrane vesicles (ISOV) isolated from strains harboring either wild-type or CQR-associated mutant PfCRT, we now show that under deenergized conditions the PfCRT protein specifically binds the antimalarial drug chloroquine (CQ) with a K(D) near 400 nM but does not measurably bind the related drug quinine (QN) at physiologically relevant concentrations. Transport studies using ISOV show that QN is passively accumulated as expected on the basis of previous measurement of the ISOV DeltapH for the different strains. However, passive accumulation of CQ is lower than expected for ISOV harboring mutant PfCRT, despite higher DeltapH for these ISOV.

Naproxen affects Ca 2+ fluxes in mitochondria, microsomes and plasma membrane vesicles

There is substantial evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) affect cellular processes regulated by Ca 2+ ions, including the metabolic responses of the liver to Ca 2+-dependent hormones. The aim of the present study was to determine whether the effects of naproxen are mediated by a direct action on cellular Ca 2+ fluxes. The effects of naproxen on 45 Ca 2+ fluxes in mitochondria, microsomes and inside-out plasma membrane vesicles were examined. Naproxen strongly impaired the mitochondrial capacity to retain 45 Ca 2+ and inhibited also ATP-dependent 45 Ca 2+ uptake by microsomes. Naproxen did not modify 45 Ca 2+ uptake by inside-out plasma membrane vesicles, but it inhibited the hexokinase/glucose-induced Ca 2+ efflux from preloaded vesicles. Additional assays performed in isolated mitochondria revealed that naproxen causes mitochondrial uncoupling and swelling in the presence of Ca 2+ ions. These effects were prevented by EGTA, ruthenium red and cyclosporin A, indicating that naproxen acts synergistically with Ca 2+ ions by promoting the mitochondrial permeability transition. The experimental results suggest that naproxen may impair the metabolic responses to Ca 2+-dependent hormones acting by at least two mechanisms: (1) by interfering with the supply of external Ca 2+ through a direct action on the plasma membrane Ca 2+ influx, and (2) by affecting the refilling of the agonist-sensitive internal stores, including endoplasmic reticulum and mitochondria.

Role of multidrug resistance protein 2 (MRP2, ABCC2) in alkylating agent detoxification: MRP2 potentiates glutathione S-transferase A1-1-mediated resistance to chlorambucil cytotoxicity.

Our previous studies have shown that the glutathione S-transferases (GSTs) can operate in synergy with the efflux transporter multidrug resistance protein 1 (MRP1, ABCC1) to confer resistance to the cyto- and genotoxicities of some anticancer drugs and carcinogens. The current study was designed to determine whether the alternative efflux transporter, MRP2 (ABCC2), can also potentiate GST-mediated detoxifications in HepG2 cells. HepG2 cells, which express high-level MRP2 but not MRP1, were stably transduced with GST expression vectors under tetracycline-repressible transcriptional control. MRP2 was able to support GSTA1-1-mediated resistance to chlorambucil (CHB) cytotoxicity in HepG2 cells. Resistance was GST isozyme-specific in that GSTP1a-1a and GSTM1a-1a failed to confer protection from CHB toxicity. Moreover, inhibition of MRP2 with sulfinpyrazone completely reversed GSTA1-1-associated resistance, indicating that MRP2-efflux function is required to potentiate GSTA1-1-mediated resistance. Relative transport by MRP1 versus MRP2 of monoglutathionyl-CHB (CHB-SG) was examined using inside-out plasma membrane vesicles derived from MCF7 cells transduced with MRP1 or MRP2 expression vectors. Both MRP1 and MRP2 transported CHB-SG efficiently, at the levels of protein expressed, with similar Vmax and with Km of 0.39 and 10 microM, respectively. We conclude that detoxification of CHB by GSTA1-1 requires the removal of the glutathione conjugate formed and that either MRP1 or MRP2 can serve this efflux function. These findings have implications for the role of MRP2 in detoxification of alkylating agents in the apical epithelium of liver and kidney where it is highly expressed as well as the role of MRP2 in the emergence of alkylating drug resistance in cancer cells.

Separate Analysis of Twin-arginine Translocation (Tat)-specific Membrane Binding and Translocation in Escherichia coli

The twin-arginine translocation (Tat) pathway exports those precursor proteins to the periplasmic space of bacteria that harbor a twin-arginine (RR) consensus motif in their signal sequences. We have reproduced translocation of several Tat substrates into inside-out plasma membrane vesicles from Escherichia coli. Translocation proceeding at an efficiency of up to 20% occurs specifically via the Tat pathway as indicated by (i) its requirement for elevated levels of the TatABC proteins in the membrane vesicles, (ii) competition by an intact twin-arginine signal peptide, and (iii) susceptibility toward dissipation of the transmembrane H(+) gradient. The latter treatment, while blocking translocation, still allows for functional membrane association of Tat precursors. This is shown by the finding that translocation of isolated membrane-bound Tat precursor is restored upon re-energization of the vesicles.

Ceramide and sphingosine have an antagonistic effect on the plasma-membrane Ca2+-ATPase from human erythrocytes.

The plasma-membrane Ca(2+)-ATPase is a key enzyme in the regulation of the intracellular Ca(2+) concentration. On the other hand, sphingolipids have been recognized recently as important second messengers, acting in many systems in combination with Ca(2+). In view of the fact that the Ca(2+)-ATPase is stimulated by ethanol, and since sphingolipids possess free hydroxy groups, we decided to study the possible effect of ceramide and sphingosine on this calcium pump. Here we show that ceramide stimulates the Ca(2+)-ATPase in a dose-dependent manner and additively to the activation observed in the presence of calmodulin or ethanol, when compared with any of these effectors added alone. Ceramide affects both the affinity for Ca(2+) and the V(max) of the enzyme. Furthermore, this second messenger also stimulates Ca(2+) transport in inside-out plasma-membrane vesicles from erythro cytes. Conversely, sphingosine, which is reported to act in many systems antagonistically with ceramide, showed an inhibitory effect on Ca(2+)-ATPase activity. This inhibition was also observed on the calmodulin-stimulated enzyme. These results, taken together, suggest that ceramide and sphingosine act antagonistically on the plasma-membrane Ca(2+)-ATPase. This is in accordance with the frequently reported opposite effect of these sphingolipids on intracellular Ca(2+) concentration.

Enhanced ATP-dependent copper efflux across the root cell plasma membrane in copper-tolerant Silene vulgaris.

We studied copper uptake in inside-out plasma membrane vesicles derived from roots of copper-sensitive, moderately copper-tolerant and highly copper-tolerant populations of Silene vulgaris (Amsterdam, Marsberg and Imsbach, respectively). Plasma membrane vesicles were isolated using the two-phase partitioning method and copper efflux was measured using direct filtration experiments. Vesicles derived from Imsbach plants accumulated two and three times more copper than those derived from Marsberg and Amsterdam plants, respectively. This accumulation was ATP-dependent. Also, 9-amino-6-chloro-2-methoxyacridine fluorescence quenching rates upon copper addition decreased in the order Imsbach>Marsberg>Amsterdam. Our results support the hypothesis that efflux of copper across the root plasma membrane plays a role in the copper tolerance mechanism in S. vulgaris.

Transmembrane topography of plasma membrane constituents in mung bean ( Vigna radiata L.) hypocotyl cells : I. Transmembrane distribution of phospholipids

The transmembrane distribution of phospholipids (PLs) in the plasma membrane (PM) of mung bean ( Vigna radiata L.) hypocotyl cells was investigated using annexin V-fluorescein isothiocyanate, porcine pancreas phospholipase A 2, and 31P-nuclear magnetic resonance (NMR) spectroscopy. Phosphatidylserine was not located on the cell surface of mung bean protoplasts. However, phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid were found to be almost symmetrically distributed across right-side-out PM vesicles obtained by aqueous two-phase partitioning by porcine pancreas phospholipase A 2 assay. 31P-NMR assay showed that the amount of PLs is about equal in the outer and the inner leaflets of the right-side-out PM vesicles. These results suggest that the topography of PM PLs might not contribute to well-known asymmetrical properties of the outer and inner surfaces of higher plant PMs. It is also indicated that inside-out PM vesicles created by Brij 58-treatment do not retain the native PL topography on dithionate reduction of 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled PLs incorporated in the PM vesicles.

Influences of glutathione on anionic substrate efflux in tumour cells expressing the multidrug resistance-associated protein, MRP1

The ATP-dependent transport of natural product drugs, e.g. vincristine, by multidrug resistance-associated protein (MRP1) requires reduced glutathione (GSH), whilst that of anionic substrates does not. The present results suggest, however, that GSH can modulate transport of anionic species. Efflux of fluorescent anionic substrates was measured from adherent MRP1-expressing human multidrug-resistant lung tumour cells, COR-L23/R, and drug-sensitive parental cells. As expected, much greater efflux of calcein, methylfluorescein-glutathione (GS-MF), and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) was observed from the resistant cells. Unexpectedly, lowering GSH levels in COR-L23/R cells by inhibiting GSH synthesis with buthionine sulfoximine decreased efflux of calcein and of GS-MF (3-fold and 1.6-fold) but not efflux of BCECF. Transport of the anionic conjugate dinitrophenyl-glutathione ([ 3H]DNP-SG) was investigated by following its uptake into inside-out plasma membrane vesicles prepared from the MRP1-expressing cells. At least 90% of the ATP-dependent uptake was blockable by the anti-MRP1 antibody QCRL-3 and 100 μM vincristine inhibited uptake but only in the presence of 1–3 mM GSH, suggesting MRP1 to be the protein primarily responsible for this transport. Agents shown to reduce efflux of calcein from resistant cells, i.e. indomethacin, MK-571, and probenecid, also inhibited [ 3H]DNP-SG uptakes, consistent with MRP1 being responsible for export of calcein. At concentrations achievable within cells, GSSG (70 μM) inhibited uptake whereas GSH (1 and 3 mM) enhanced uptake. We suggest that variations in both GSH and GSSG levels within cells may affect MRP1-mediated anion transport.