Inside-out Membrane Vesicles Research Articles

Export of guanosine 3',5'-cyclic monophosphate (cGMP) from human erythrocytes characterized by inside-out membrane vesicles.

The present study was undertaken to characterize the export of cGMP from human erythrocytes at 37 degrees C. Inside-out membrane vesicles were exposed to cGMP and [3H]-cGMP in the presence and absence of 2 mmol l-1 ATP. In the absence of ATP, an equilibrium was reached within 15 min for the lowest tested concentration (0.65 mumol l-1), and the amount of cGMP in the vesicles was linearly correlated to the cGMP concentrations in the incubate. These observations suggest that the ATP-independent process represents passive diffusion or non-saturated binding to membrane components. In the presence of ATP, cGMP accumulated linearly during the test period (up to 120 min) and the transport into the inside-out vesicles was dependent on both low- and high-Km transport. The kinetic parameters for the low-Km process were determined after 5 and 120 min, the Km values being 4.6 (SD 1.9) and 4.7 (SD 1.1) mumol l-1 (n = 3), respectively. The corresponding Vmax values were 400 (SD 50) and 440 (SD 70) fmol mg-1 min-1. The high-Km process was characterized by Km = 170 (SD 50) mumol-1 and Vmax = 1610 (SD 280) fmol mg-1 min-1 (n = 5). The present data demonstrate an ATP-requiring saturable transport system for cGMP in human erythrocytes.

Bacterial resistances to toxic metal ions - a review

Bacterial plasmids encode resistance systems for toxic metal ions, including Ag +, AsO 2 -, AsO 4 3-, Cd 2+, Co 2+, CrO 4 2-, Cu 2+ Hg 2+, Ni 2+, Pb 2+, Sb 3+, TeO 3 2-, Tl + and Zn 2+. The function of most resistance systems is based on the energy-dependent efflux of toxic ions. Some of the efflux systems are ATPases and others are chemiosmotic cation/proton antiporters. The Cd 2+-resistance ATPase of Gram-positive bacteria (CadA) is membrane cation pump homologous with other bacterial, animal and plant P-type ATPases. CadA has been labeled with 32P from [ α- 32p]ATP and drives ATP-dependent Cd 2+ (and Zn 2+) uptake by inside-out membrane vesicles (equivalent to efflux from whole cells). Recently, isolated genes defective in the human hereditary diseases of copper metabolism, namely Menkes syndrome and Wilson's disease, encode P-type ATPases that are more similar to bacterial CadA than to other ATPases from eukaryotes. The arsenic resistance efflux system transports arsenite [As(III)], alternatively using either a double-polypeptide (ArsA and ArsB) ATPase or a single-polypeptide (ArsB) functioning as a chemiosmotic transporter. The third gene in the arsenic resistance system, arsC, encodes an enzyme that converts intracellular arsenate [As(V)] to arsenite [As(III)], the substrate of the efflux system. The triple-polypeptide Czc (Cd 2+, Zn 2+ and Co 2+) chemiosmotic efflux pump consists of inner membrane (CzcA), outer membrane (CzcC) and membrane-spanning (CzcB) proteins that together transport cations from the cytoplasm across the periplasmic space to the outside of the cell.

Partitioning of HIV-1 Gag and Gag-related proteins to membranes.

The binding of HIV-1 Gag and Gag-related proteins to model membranes was examined using three experimental systems: (i) large unilamellar phospholipid vesicles (LUVs) and recombinant Gag purified from Escherichia coli; (ii) LUVs added to a mammalian cell extract in which Gag proteins were expressed by a coupled transcription/translation system; and (iii) inside-out plasma membrane vesicles purified from human red blood cells (RBC) and recombinant, purified Gag from E. coli. Several novel aspects of HIV-1 Gag membrane interactions were observed: (i) Gag proteins bound with high affinity to both model membranes with a negatively charged surface and to RBC membranes. (ii) Binding of the Gag precursor and mature Gag proteins exhibited different sensitivities to ionic strength indicating that the precursor directed membrane binding through interactions that were qualitatively and quantitatively distinct from those of any of its individual domains. Studies using energy transfer between tryptophan residues in the proteins and anthroyloxy-containing probes inserted in the LUVs indicated that the orientation of the precursor and of the mature proteins on the membrane surface were distinct; (iii) Gag oligomers appear to have facilitated high-affinity binding under high salt conditions, suggesting that protein-protein interactions led to formation of stronger electrostatic or new hydrophobic membrane binding determinants. Since binding studies with model membranes permit quantitative analysis, these experimental approaches may permit identification of interactions that drive Gag assembly on the membrane.

Cellular and in vitro transport of glutathione conjugates by MRP.

MRP is a recently identified ATP-binding cassette transporter. We previously established that MRP confers resistance to a spectrum of natural product cytotoxic drugs [Kruh, G.D., (1994) Cancer Res. 54, 1649-1652], that expression of MRP is associated with enhanced drug efflux [Breuninger, L.M., (1995) Cancer Res. 55, 5342-5347], and that MRP transcript is widely expressed in human tissues and solid tumor cell lines [Kruh, G.D., (1995) J. Natl. Cancer Inst. 87, 1256-1258]. In the present study the relationship between MRP and drug glutathione S-conjugates was examined. We observed that MRP was labeled by azidophenacylglutathione (APA-SG), a photoaffinity analog of glutathione, and that inside-out membrane vesicles prepared from MRP-overexpressing HL60/ADR cells transported this compound. Transport into membrane vesicles was ATP-dependent, sensitive to osmolarity, and saturable with regard to APA-SG and ATP concentrations, with Km values of 15 and 61 microM, respectively. APA-SG transport was competitively inhibited by the natural product cytotoxic drugs daunorubicin, vincristine, and etoposide, with Ki values of 4.8, 3.8, and 5.5 microM, respectively. Oxidized glutathione, the drug-glutathione S-conjugate DNP-SG, the LTD4 antagonist MK571 and arsenate were also competitive inhibitors, with Ki values of 9.0, 23.4, 1.1, and 15.0 microM, respectively. Analysis of the fate of monochlorobimane in MRP transfectants revealed reduced intracellular concentrations of drug-glutathione S-conjugates associated with enhanced efflux and altered intracellular distribution. These results indicate that MRP can transport glutathione conjugates in vitro and in living cells and suggest the possibility that the transporter may represent a link between cellular resistance to some classes of cytotoxic drugs and glutathione-mediated mechanisms of resistance. In addition, the observation that both mildly cationic or neutral natural product cytotoxic drugs and anionic compounds such as DNP-SG, MK571, and arsenate are competitive inhibitors of MRP action suggests that the substrate specificity of the transporter is quite broad.

Ca2+-Calmodulin-Dependent Phosphorylation and Dephosphorylation of Rat Parotid Secretion Granules

In our studies on the control of fusion of secretion granules with the plasma membrane which occurs during exocytosis, we have recently found that Ca++ and calmodulin stimulated the fusion of isolated parotid secretion granules with isolated inside-out plasma membrane vesicles. We are now examining the possibility that they do so by stimulating protein phosphorylation. Secretion granules were isolated from rat parotid by differential and gradient centrifugation and incubated with [Y32P]ATP. The granules were solubilized with sodium dodecylsulfate and the proteins resolved on a 7-15% polyacrylamide gel. Calmodulin plus Ca++, but neither alone, stimulated phosphorylation of four proteins with molecular masses of 64, 58, 55 and 31 kDa, and decreased the phosphorylation of a 36 kDa protein. Trifluoperazine and calmidazolium inhibited these effects. results suggest that Ca++ and calmodulin may facilitate fusion of secretion granules with the plasma membrane by changing the phosphorylation state of one or more secretion granule proteins.

Transport of bimane-S-glutathione in human erythrocytes

Export of glutathione S-conjugate of bimane (BSG) was studied in human erythrocytes. Characteristics of the BSG transport is similar to that of dinitrophenyl-S-glutathione (DNP-SG). BSG transport has two kinetic components, one of high affinity and low capacity ( K m = 7.4 ± 0.2 μmol/ml cells, V m = 2.7 ± 0.1 nmol/min per ml RBC) and another of low affinity and high capacity ( K m = 242 ± 8 μmol/ml cells, V m = 9.6 ± 1.6 nmol/min per ml RBC). BSG export is inhibited by vanadate ( K i = 65 ± 6 μM) and fluoride ( K i = 11.4 ± 0.8 mM). Activation energy of the transport is 67 ± 7 kJ/mol. BSG transport is independent of membrane potential; its rate increases with pH in the pH range of 6–8, in line with the assumption that the anionic conjugate is cotransported with proton. BSG import to erythrocyte membrane inside-out vesicles is stimulated by ATP. Fluorimetric measurements of BSG export require low amounts of cells and may also be useful for other cell types as an alternative to studies of glutatione S-conjugate transport using radioactive substrates.

Facile Preparation of Inside-Out Plasma Membrane Vesicles from Tumor Cells for Functional Studies of Pharmacologically Relevant Translocating ATPases

A reasonably facile and effective procedure is described for the preparation of inside-out plasma membrane vesicles from tumor cells. The method incorporates nitrogen cavitation, optimized with respect to the applied N2 pressure, in the absence of added divalent cations followed by differential centrifugation and discontinuous, sucrose gradient centrifugation. With the three tumor cell types utilized, multidrug-resistant (MEL/VCR-6) and parental (MEL/O) murine erythroleukemia cells and methotrexate-resistant (L1210/R24) L1210 leukemia cells, yields were in the range of 8-12 mg of plasma membrane vesicles/1010 cells at a purity of 87-94% with average inside-out sidedness among preparations varying from 65 to 93% depending upon the cell type. Inside-out plasma membrane vesicles so derived were capable of sustaining ATP-dependent transport inward of two common antitumor cytotoxic agents, vinblastine and methotrexate. The former was demonstrated with inside-out vesicles from only P-glycoprotein-overexpressing, multidrug-resistant MEL/VCR-6 cells, while the latter was readily demonstrated in inside-out vesicles from all three cell types.

Zn2+-Mediated Domain-Domain Communication in Human Erythrocyte Band 31

Zn2+ could inhibit the anion transport activity of spectrin-stripped inside-out human erythrocyte membrane vesicles (IOVs). Removal of the cytoplasmic domain from Band 3 by trypsin could eliminate Zn2+ inhibition. The location of a Zn(2+)-binding site was confirmed by atomic absorbance spectrometry. The results of time-resolved fluorescence and intrinsic fluorescence quenching by KI and hypocrellin B (a photosensitive pigment obtained from a parasitic fungus growing in Yunnan, China) showed that the cytoplasmic domain is necessary for the Zn(2+)-induced conformational changes of the whole molecule as well as the membrane domain of Band 3. It is suggested that Zn2+ induced a conformational change in the cytoplasmic domain of Band 3, which in turn was transmitted to the membrane domain, resulting in an inhibition of activity of Band 3. Such long-range conformational changes may imply that the cytoplasmic domain is poised to function as a cytosolic arm in order to modulate the structure of the membrane domain of Band 3.

Effect of NO 3−, CI-and DIDS on H +-ATPase of plasma Membrane Vesicles Isolated from Corn Roots

Summary The effect of nitrate, chloride and 4, 4' -diisothiocyano-2, 2'-disulfonic stilbene acid (DIDS) on H + -ATPase of plasma membrane vesicles isolated from corn roots was investigated in order to acquire information on mechanism(s) of anion transport. The initial rate of ATP-dependent intravesicular H + accumulation in inside-out plasma membrane vesicles was stimulated both in the presence of NO 3 − and Cl − . However, the kinetics of stimulation were different, NO 3 − showing a clear saturable profile and Cla complex apparently biphasic pattern with no saturation even at concentrations as high as 200 mM. The stimulation was not related to a direct effect of the anions on H + -ATPase as indicated by the lack of effect on the phosphohydrolyzing activity measured in the presence of Brij 58 or Gramicidin D. DIDS at 1-2 μM strongly inhibited the anion-stimulated H + accumulation without a simultaneous effect on passive membrane permeability or on phosphate hydrolysis, which was almost unaffected even at a DIDS concentration of 20 μM. The results suggest that transport of NO 3 − and Cl − , in a direction that would correspond in vivo to the efflux from the cell, is mediated by proteinaceous structure(s) possessing different kinetic properties with respect to these anions.

Association State of Human Red Blood Cell Band 3 and its Interaction With Ankyrin

We have studied the association state of band 3, the anion channel and predominant transmembrane protein of the human red blood cell, and the anomalous stoichiometry and dynamics of its interaction with ankyrin, which acts as a link to the spectrin of the membrane skeletal network. Band 3 exists in benign nonionic detergent solutions as a dimer. Tetramer is formed irreversibly in the course of manipulations, particularly in ion-exchange chromatography. The dimer in solution binds ankyrin without self-associating. In ankyrin-free inside-out membrane vesicles and when incorporated into phosphatidylcholine liposomes, only some 10% to 15% of band 3 chains bind ankyrin at saturation. Moreover, in liposomes this was independent of protein:lipid ratio between 1:2 and 1:40. The bound fraction of band 3 remains with the detergent-extracted membrane cytoskeleton, but is released if the ankyrin has been cleaved with chymotrypsin before detergent treatment; thus, the attachment to the membrane cytoskeleton is entirely through ankyrin and not through other constituents such as protein 4.1. The ratio of band 3 to ankyrin in this complex implies that it consists of two chains of band 3 and one chain of ankyrin, at least after detergent extraction. The bound and free populations of band 3 exchange freely in the membrane. In the artificial liposome membrane binding of ankyrin to band 3 dimers cause association of the band 3 into higher aggregates, as seen in freeze-fracture electron microscopy. Successive manipulations of the red blood cell membrane, which are involved in the preparation of ghosts, of inside-out vesicles, and of inside-out vesicles stripped of peripheral proteins are accompanied by progressive aggregation of intramembrane particles, as judged by freeze-fracture electron microscopy. Thus the intramembrane particles are evidently stabilized in the intact cell by the peripheral protein network and the cytosolic milieu. Aggregation may be expected to limit the number of functional ankyrin binding sites. However, although extraneous ankyrin binds to the unoccupied binding site on the spectrin tetramers in intact ghost membranes, little or no ankyrin can bind to the unoccupied band 3 dimers in situ, perhaps by reason of occlusion of binding sites by the membrane skeletal network.

Functional Modulation of Multidrug Resistance-related P-glycoprotein by Ca2+-Calmodulin

Studies with inside-out plasma membrane vesicles from multidrug-resistant (MDR 3) murine erythroleukemia (MEL/VCR-6) cells have provided evidence for down-modulation of P-glycoprotein (P-gp) function by Ca(2+)-calmodulin (CLM). These studies showed that CLM in the presence or absence of Ca2+ had no effect on binding of [3H]vinblastine (VBL) by P-gp in inside-out plasma membrane vesicles. However, profound inhibition of ATP-dependent [3H]VBL efflux by these vesicles was demonstrated by the addition of subnanomolar concentrations of CLM (IC50 = 0.15 +/- 0.02 nM). The addition of 1 microM Ca2+ reduced the inhibition of [3H]VBL efflux by CLM, shifting the concentration required for inhibition to the nM range (IC50 = 2.55 +/- 0.35 nM). The inhibition of as 0.01 mM Ca2+, and no inhibition occurred with concentrations greater than 0.2 mM Ca2+. Binding of CLM, itself, to P-gp was demonstrated in two ways. The P-gp content of detergent-solubilized plasma membrane from MEL/VCR-6 cells could be appreciably depleted by treating this material with CLM-Sepharose beads as shown by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting with anti-P-gp antibody (C219) before and after CLM-Sepharose treatment. Also, depletion of P-gp from solution by CLM was less in the presence of 1 mM Ca2+. Blotting of P-gp after SDS-PAGE of plasma membrane from MEL/VCR-6 cells was also obtained using 125I-CLM as a probe. These results strongly suggest that the MDR 3 homolog of P-gp is a CLM-binding protein and that direct interaction of Ca(2+)-CLM with P-gp, while not affecting its binding of [3H]VBL, down-modulates the translocation of this agent in the presence of ATP.

Structural preferences among folate compounds and their analogues for ATPase-mediated efflux by inside-out plasma membrane vesicles derived from L1210 cells

Our prior studies with inside-out plasma membrane vesicles from L1210 cells (Schlemmer SR and Sirotnak FM, J Biol Chem 267: 14746–14752, 1992) identified an outwardly directed, translocating ATPase as mediating the majority of methotrexate (MTX) efflux in these cells. In the current studies, we examined by competitive inhibition with [ 3H]MTX as permeant some of the structural features that determine preferences among folate compounds and their analogues as permeants for this ATPase. The results show that folate compounds are preferred over simple quinazolines (5,8-dideaza-pteridines), and IL5-CH 3-folateH 4, and probably other 5-substituted folates are preferred over folic acid. In the latter regard, the observed equivalence in preference to IL5-CH 3-folateH 4 of the 4-oxa-pyridopyrimidine, lometrexol (DDATHF), probably relates to its close similarity to folateH 4. The results also suggest that the 4-position in the case of folate analogues, but not in the case of the quinazoline analogues, is an important determinant with 4-amino preferred over 4-oxa. They also suggest that the N10 position on the bridge region in both series of compounds, and probably for the pyridopyrimidine lometrexol, is not an important determinant. In contrast to results seen with the simple quinazolines, the 2-CH 3 desamino quinazoline ZEN D1694, modified as well by a 2-benzyl to thienyl replacement on the side chain, was highly preferred. The same relative differences seen among some of these analogues as inhibitors of [ 3H]MTX efflux in inside-out vesicles were documented for their effectiveness as permeants for ATP-dependent efflux in intact L1210 cells.

The Role of Plasma Membrane Oxidoreductase Activity in Proton Transport

Summary Inside-out plasma membrane vesicles were obtained after partition of the freeze/thawed right-side out vesicles from cucumber roots in a two phase system. A sidedness of the plasma membrane vesicles was determined by the latency of vanadate-sensitive ATPase and trypsin digestion of the enzyme. In experiments with ferricyanide-loaded vesicles a low transplasma membrane electron transport with NADH as an electron donor was generated. However, introduction of Triton X-100 into the reaction medium increased the rate of NADH oxidation several times. During incubation with ATP of both unloaded vesicles and vesicles loaded with ferricyanide, the decrease in acridine orange absorbance was observed. DCCD and sodium orthovanadate added to the reaction mixture diminished distinctly the changes of acridine absorbance. These results indicate that in vesicles the ATP-dependent H + pump is active. Incubation of the ferricyanide-loaded vesicles with NADH caused a small increase of acridine absorbance that was deteriorated in the presence of quinacrine. The contribution of the plasma membrane NADH-ferricyanide oxidoreductase in the H + transport is discussed.

The cobalt, zinc, and cadmium efflux system CzcABC from Alcaligenes eutrophus functions as a cation-proton antiporter in Escherichia coli

The function of the CzcABC protein complex, which mediates resistance to Co2+, Zn2+, and Cd2+ in Alcaligenes eutrophus by cation efflux, was investigated by using everted membrane vesicles of Escherichia coli and an acridine orange fluorescence quenching assay. Since metal cation uptake could not be measured with inside-out membrane vesicles prepared from A. eutrophus and since available E. coli strains did not express the Czc-mediated resistance to cobalt, zinc, and cadmium salts, mutants of E. coli which exhibited a Czc-dependent increase in heavy metal resistance were isolated. E. coli mutant strain EC351 constitutively accumulated Co2+, Zn2+, and Cd2+. In the presence of Czc, net uptake of these heavy metal cations was reduced to the wild-type level. Inside-out vesicles prepared from E. coli EC351 cells displayed a Czc-dependent uptake of Co2+, Zn2+, and Cd2+ and a cation-triggered acridine orange fluorescence increase. The czc-encoded protein complex CzcABC was shown to be a zinc-proton antiporter.

Copper and Silver Transport by CopB-ATPase in Membrane Vesicles of Enterococcus hirae

The P-type ATPase, CopB, of Enterococcus hirae is required for the copper resistance displayed by this organism and thus was postulated to be a copper pump. Using 64Cu+ and 110mAg+, we here show ATP-driven copper and silver accumulation catalyzed by CopB in native inside-out membrane vesicles of E. hirae. CopB ATPase exhibited an apparent Km for Cu+ and Ag+ of 1 microM and for ATP of 10 microM. Transport was maximal at pH 6 and had an apparent Vmax of 0.07 nmol.min-1.mg-1 for both copper and silver transport. Vanadate displayed a biphasic effect on transport: maximal inhibition was observed at 40 microM vanadate for copper transport and 60 microM for silver transport, respectively. At higher vanadate concentrations, these inhibitions were reversed. The CopB ATPase of E. hirae is thus a pump for the extrusion of monovalent copper and silver ions, with copper probably being the natural substrate.

A cell-free protein translocation system prepared entirely from a Gram-positive organism

A cell-free protein translocation system derived exclusively from a Gram-positive bacterium is described here for the first time. Highly efficient in vitro synthesis of plasmid encoded preprolipase of Staphylococcus hyicus is accomplished by coupled transcription/translation using either a cytosolic extract of S. carnosus alone or in combination with T7-RNA-polymerase. Addition of inside-out cytoplasmic membrane vesicles of S. carnosus leads to the partial conversion (processing) of preprolipase to prolipase. In addition, as shown in a protease protection assay, a significant part of preprolipase plus prolipase is translocated in vitro into the lumen of the vesicles. Translocation of preprolipase into the membrane vesicles requires the proton-motive force and the S. carnosus SecA protein.

Phosphorylation site mutants of the mannitol transport protein enzyme IImtl of Escherichia coli: studies on the interaction between the mannitol translocating C-domain and the phosphorylation site on the energy-coupling B-domain.

Mannitol binding and translocation catalyzed by the C domain of the Escherichia coli mannitol transport protein enzyme IImtl is influenced by domain B. This interaction was studied by monitoring the effects of mutating the B domain phosphorylation site, C384, on the kinetics of mannitol binding to the C domain. The dissociation constants for mannitol to the C384 mutants in inside-out membrane vesicles varied from 45 nM for the wild-type enzyme to 306 nM for the mutants. The rate constants pertinent to the binding equilibrium were also altered by the mutations. The association rate of mannitol to the cytoplasmic binding site in the mutants was accelerated for all mutants. The exchange rate of bound mannitol on the wild-type enzyme was shown to be pH dependent with a pKa of approximately 8 and increasing rates at higher pH. This rate was increased for all the mutants, but the pKas differed for the various mutants. The exchange rate for binding to the isolated IICmtl, however, was not pH dependent and exhibited a low rate. Exchange measured at 4 degrees C showed that, of the two steps, binding and occlusion, involved in binding to wild-type EIImtl in inside-out vesicles, only one could be detected for the C384E and C384L mutants. This suggests that the mutations increased the rate of the occlusion step so that it was no longer separable from the initial binding step or that the mutations eliminated the occlusion step altogether. The change in the mannitol binding kinetics of the C domain indicates that the B and C domains of EIImtl influence each other's conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cooperative binding of lactose and the phosphorylated phosphocarrier protein HPr(Ser-P) to the lactose/H+ symport permease of Lactobacillus brevis.

Lactobacillus brevis accumulates lactose and nonmetabolizable lactose analogues via sugar/H+ symport, but addition of glucose to the extracellular medium results in rapid efflux of the free sugar from the cells due to the uncoupling of sugar transport from proton transport. By using vesicles of L. brevis cells, we recently showed that these regulatory/effects could be attributed to the metabolite-activated ATP-dependent protein kinase-catalyzed phosphorylation of serine-46 in the phosphocarrier protein HPr [HPr(Ser-P)] of the phosphotransferase system and that a mutant form of HPr with the serine-46-->aspartate replacement ([S46D]HPr) is apparently locked in the seryl phosphorylated conformation. We here demonstrate that [S46D]HPr binds directly to inside-out membrane vesicles of L. brevis that contain the lactose permease. Sugar substrates of the permease markedly and specifically stimulate binding of [S46D]HPr to the membranes while certain transport inhibitors such as N-ethylmaleimide block binding. The pH dependency for binding follows that for transport. Wild-type HPr and the [S46A]HPr mutant protein did not appreciably compete with [S46D]HPr for binding to the permease. These results provide evidence for the direct interaction of HPr(Ser-P) with an allosteric site on the lactose/proton symporter of L. brevis for the purpose of regulating sugar accumulation in response to the metabolic needs of the cell.

Preparation and Characterization of Sealed Inside-Out Peribacteroid Membrane Vesicles from Pisum sativum L. and Glycine max L. Root Nodules by Aqueous Polymer Two-Phase Partitioning

Summary A method is described for the preparation of sealed inside-out peribacteroid membrane (PBM) vesicles from pea (Pisum sativum L.) and soybean (Glycine max L.) root nodules by aqueous polymer two-phase partitioning. At optimal conditions about one-fourth of the total membrane protein from osmotically shocked symbiosomes is recovered as inside-out PBM vesicles. This corresponds to a yield of 25 to 40 μg of PBM protein · g-1 nodule. The sidedness of tight vesicles is determined by measuring the activities of marker enzymes for the cytoplasmic surface (ATPase (EC 3.6.1.3) and 1,3-β-glucan synthase (GS II; EC 2.4.1.34)) with and without detergent. The difference between enzyme activity with and without detergent designates latency, and based on these results it is estimated that the preparations contain 70 to 80 % sealed inside-out PBM vesicles. The specific activity of GS II and ATPase in pea nodule PBM is compared with that of the plasma membrane from uninfected pea roots. The GS II activity of PBM constitutes 10 % of that in pea root plasma membrane whereas ATPase activity of PBM amounts to 70 % of that in pea root plasma membrane. The pea PBM ATPase resembles the plasma membrane-type ATPase on the basis of insensitivity to nitrate, stimulation by NH4+ and sensitivity to vanadate. The results reveal that high purity preparations of inside-out sealed PBM vesicles are obtained without contamination from bacteroid or plasma membrane by the present procedure. Thus, preparations suitable for studies on transport and signal transduction mechanisms across the PBM from the bacteroid side are now available.

Functional studies of P-glycoprotein in inside-out plasma membrane vesicles derived from murine erythroleukemia cells overexpressing MDR 3. Properties and kinetics of the interaction of vinblastine with P-glycoprotein and evidence for its active mediated transport.

Active [3H]vinblastine (VBL) transport (efflux) was documented for inside-out plasma membrane vesicles from murine erythroleukemia cells (MEL/VCR-6) resistant to vinca alkaloids and overexpressing MDR 3 P-glycoprotein (P-gp) 80-fold. Uptake of [3H]VBL at 37 degrees C by these inside-out vesicles, but not rightside-out vesicles or inside-out vesicles from wild-type cells, was obtained in the form of a rapid, initial phase (0-1 min) and a slower, later phase (> 1 min). The rapidity of each phase correlated with relative P-gp content among different MEL/VCR cell lines. The initial MDR-specific phase was temperature- and pH-dependent (optimum at pH 7), osmotically insensitive, and did not require ATP. The second MDR-specific phase was temperature-dependent, osmotically sensitive, and strictly dependent upon the presence of ATP (Km = 0.37 +/- 0.04 mM). Although other triphosphate nucleotides were partially effective in replacing ATP, the nonhydrolyzable analogue ATP gamma S (adenosine 5'-O-(thiotriphosphate)) was ineffective. This time course appears to represent tandem binding of [3H]VBL by P-gp and its mediated transport, with the latter process representing the rate-limiting step. In support of this conclusion, both binding and transport were inhibited by verapamil, quinidine, and reserpine, all known to be inhibitors of photoaffinity labeling of P-gp, but only transport was inhibited by C219 anti-P-gp antibody or orthovanadate. Although the rate of transport of [3H]VBL was 7-7.5-fold lower than the rate of binding (Vmax = 104 +/- 15 pmol/min/mg protein, Kon = 1.5 - 2 x 10(5) mol-1 s-1) to P-gp, each phase exhibited saturation kinetics and values for apparent Km and KD for each process were approximately the same (215 +/- 35 and 195 +/- 30 nM). Intravesicular accumulation of [3H]VBL was almost completely eliminated by high concentrations of nonradioactive VBL, suggesting that simple diffusion does not contribute appreciably to total accumulation of [3H]VBL in this vesicle system. This could be at least partially explained by the fact that these inside-out vesicles under the conditions employed did not maintain a P-gp mediated pH gradient. However, ATP-dependent, intravesicular accumulation of osmotically sensitive [3H]VBL occurred against a substantial permeant concentration gradient in both a time- and concentration-dependent manner consistent with an active, saturable process.