Membrane Vesicle Preparations Research Articles

Isolation of Chlamydia trachomatis and membrane vesicles derived from host and bacteria

The study of intracellular bacteria and nanometer-size membrane vesicles within infected host cells poses an important challenge as it is difficult to identify each distinct population in the context of the complex populations generated from active host-pathogen interactions. Here, suspension cultures of L929 cells infected with the prevalent obligate intracellular bacterium Chlamydia trachomatis strain F/Cal-IC-13 are utilized for the large scale preparation and isolation of natural membrane vesicles and bacterial forms. Cell lysis with nitrogen cavitation in combination with differential centrifugation, OptiPrep™ density gradient separation, and immunoenrichment using anti-chlamydial lipopolysaccharide antibodies and MagnaBind beads allows for the isolation of both productive and persistent bacterial forms, as well as membrane vesicles derived from the host and pathogen. We have evaluated these populations by electron microscopy and Western blot analysis for identification of biomarkers. In addition, purified persistent forms of C. trachomatis induced by ampicillin display adenosine-5′-triphosphate (ATP) transport activity, suggesting that ampicillin-induced persistent C. trachomatis organisms, at least in part, rely upon host ATP as an energy source. Importantly, several chlamydial cytotoxic and/or secreted proteins are demonstrated to be associated with these vesicles, supporting the idea that membrane vesicles are generated by Chlamydia as a means of carrying and delivering virulence factors necessary for pathogenesis. The ability to produce large-scale infections and generate distinct bacteria and host-derived populations for biochemical analysis, while reducing the burdens of time and cost have implications in all areas of chlamydiology. These protocols can be applied to other strains of C. trachomatis or other intracellular bacteria.

Functional Impairment of Rat Taurine Transporter by Activation of Nitrogen Oxide through Superoxide

The objective of this study was to identify the nitrogen oxide form(s) involved in the functional impairment of the rat taurine transport system. Taurine uptake activity in the rat renal brush border membrane vesicle (RBBMV) preparation or Xenopus laevis oocytes that express the rat taurine transporter was compared after the pretreatment with nitrogen oxide donors from which nitric oxide (NO) is released at different rates. The functional impairment was associated with a reduced Vmax, but did not involve an alteration in the Km, of taurine uptake in the RBBMV preparation that had been pretreated with sodium nitroprusside, a slow release nitric oxide (NO) donor. When the preparation was pretreated with S-nitroso-N-acetyl penicillamine, a rapid release NO donor, the activity of taurine uptake was unaffected. The activity was not statistically different from the control after the pretreatment with sodium nitroprusside and superoxide dismutase. Consistent with the study with RBBMV, a similar alteration in the activity of taurine uptake by NO donors was observed in oocytes expressing the transporter. Considering the fact that peroxynitrite, a highly reactive nitrogen oxide form, is formed by the reaction between NO and superoxide, the taurine transporter, and probably other transport systems as well, may be functionally impaired by peroxynitrite.

Intrinsic membrane association of the cytoplasmic tail of influenza virus M2 protein and lateral membrane sorting regulated by cholesterol binding and palmitoylation

The influenza virus transmembrane protein M2 is a proton channel, but also plays a role in the scission of nascent virus particles from the plasma membrane. An amphiphilic helix in the CT (cytoplasmic tail) of M2 is supposed to insert into the lipid bilayer, thereby inducing curvature. Palmitoylation of the helix and binding to cholesterol via putative CRAC (cholesterol recognition/interaction amino acid consensus) motifs are believed to target M2 to the edge of rafts, the viral-budding site. In the present study, we tested pre-conditions of this model, i.e. that the CT interacts with membranes, and that acylation and cholesterol binding affect targeting of M2. M2-CT, purified as a glutathione transferase fusion protein, associated with [3H]photocholesterol and with liposomes. Mutation of tyrosine residues in the CRAC motifs prevented [(3)H]photocholesterol labelling and reduced liposome binding. M2-CT fused to the yellow fluorescent protein localized to the Golgi in transfected cells; membrane targeting was dependent on CRAC and (to a lesser extent) on palmitoylation. Preparation of giant plasma membrane vesicles from cells expressing full-length M2-GFP (green fluorescent protein) showed that the protein is partly present in the raft domain. Raft targeting required palmitoylation, but not the CRAC motifs. Thus palmitoylation and cholesterol binding differentially affect the intrinsic membrane binding of the amphiphilic helix.

Alterations in the proteome of the NHERF2 knockout mouse jejunal brush border membrane vesicles

To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.

Preparation, Characterization and Stability of Rabbit Intestinal Brush Border Membrane Vesicles

Preparation, Characterization and Stability of Rabbit Intestinal Brush Border Membrane Vesicles

Enterotoxigenic Escherichia coli Elicits Immune Responses to Multiple Surface Proteins

Enterotoxigenic Escherichia coli (ETEC) causes considerable morbidity and mortality due to diarrheal illness in developing countries, particularly in young children. Despite the global importance of these heterogeneous pathogens, a broadly protective vaccine is not yet available. While much is known regarding the immunology of well-characterized virulence proteins, in particular the heat-labile toxin (LT) and colonization factors (CFs), to date, evaluation of the immune response to other antigens has been limited. However, the availability of genomic DNA sequences for ETEC strains coupled with proteomics technology affords opportunities to examine novel uncharacterized antigens that might also serve as targets for vaccine development. Analysis of whole or fractionated bacterial proteomes with convalescent-phase sera can potentially accelerate identification of secreted or surface-expressed targets that are recognized during the course of infection. Here we report results of an immunoproteomics approach to antigen discovery with ETEC strain H10407. Immunoblotting of proteins separated by two-dimensional electrophoresis (2DE) with sera from mice infected with strain H10407 or with convalescent human sera obtained following natural ETEC infections demonstrated multiple immunoreactive molecules in culture supernatant, outer membrane, and outer membrane vesicle preparations, suggesting that many antigens are recognized during the course of infection. Proteins identified by this approach included established virulence determinants, more recently identified putative virulence factors, as well as novel secreted and outer membrane proteins. Together, these studies suggest that existing and emerging proteomics technologies can provide a useful complement to ongoing approaches to ETEC vaccine development.

Lipid Transfer Mediated by a Recombinant Pro-Sterol Carrier Protein 2 for the Accurate Preparation of Asymmetrical Membrane Vesicles Requires a Narrow Vesicle Size Distribution: A Free-Flow Electrophoresis Study

We applied protein-mediated lipid transfer using recombinant His-tagged pro-sterol carrier protein 2 (pro-SCP2) to prepare asymmetrical membrane vesicles (AMV) featuring an unequal transmembrane distribution of the negative phospholipid egg-phosphatidylglycerol (EPG). Pure egg-phosphatidylcholine (EPC) vesicles were used as the acceptor and EPC:EPG 90:10 mol % vesicles as the donor populations. The changes in surface charge during EPG transfer were used to quantify the degree of asymmetry by free-flow electrophoresis (FFE). The relative deflection in FFE correlated with EPG content in the outer monolayer (x(EPG)). The initial transfer rates and first order rate constants for the transfer process were determined. The addition of pro-SCP2 at a molar protein-to-lipid ratio R(P/L) of (15-20) x 10(-5) accelerated the EPG transfer to half-times of between 2 and 3 h. Thus, the transmembrane redistribution of EPG by flip-flop, which reduces the degree of asymmetry and occurs at half-times of tens of hours, was minimized during the transfer process. We investigated the influence of membrane curvature on the transfer rate using 50 and 100 nm vesicles with very low size distribution widths (RSD of 13-17%). Transfer occurred with a 55.7% higher initial rate between the smaller vesicles. The use of equally sized acceptor and donor populations of such narrow size distributions was shown to be important for the preparation of AMV with a uniform degree of asymmetry. Under these conditions, AMV were obtained after less than 3 h by preparative FFE separation. In the case of the acceptor vesicles, EPG transfer increased x(EPG) to 3 mol %, whereas it was reduced to 6 mol % in the donor vesicles.

Recombinant outer membrane vesicles to augment antigen-specific live vaccine responses

Release of outer membrane vesicles is a common feature of Gram negative bacteria. There is growing interest in the use of these vesicles in the development of affordable vaccines. However, to exploit their full potential a convenient system to generate recombinant vesicles would be highly desirable. Here, we report the design of a versatile system for preparation of recombinant outer membrane vesicles based on an engineered autotransporter. Two model vaccine antigens of Leishmania were expressed as fusion proteins with the transporter domain of AIDA, the Escherichia coli adhesin involved in diffuse adhesion. Single subcutaneous injections of recombinant vesicles boosted vaccine antigen-specific antibody responses in mice primed with live recombinant salmonella vaccines by 6–40-fold. The results further show an expansion of only the vaccine antigen-specific antibody response indicating a great potential of this approach for prime boost vaccination strategies.

Cortisol-sensitive urea transport across the gill basolateral membrane of the gulf toadfish (Opsanus beta)

Gulf toadfish (Opsanus beta) use a unique pulsatile urea excretion mechanism that allows urea to be voided in large pulses via the periodic insertion or activation of a branchial urea transporter. The precise cellular and subcellular location of the facilitated diffusion mechanism(s) remains unclear. An in vitro basolateral membrane vesicle (BLMV) preparation was used to test the hypothesis that urea movement across the gill basolateral membrane occurs through a cortisol-sensitive carrier-mediated mechanism. Toadfish BLMVs demonstrated two components of urea uptake: a linear element at high external urea concentrations, and a phloretin-sensitive saturable constituent (K(m) = 0.24 mmol/l; V(max) = 6.95 micromol x mg protein(-1) x h(-1)) at low urea concentrations (<1 mmol/l). BLMV urea transport in toadfish was unaffected by in vitro treatment with ouabain, N-ethylmaleimide, or the absence of sodium, conditions that are known to inhibit sodium-coupled and proton-coupled urea transport in vertebrates. Transport kinetics were temperature sensitive with a Q(10) > 2, further suggestive of carrier-mediated processes. Our data provide evidence that a basolateral urea facilitated transporter accelerates the movement of urea between the plasma and gills to enable the pulsatile excretion of urea. Furthermore, in vivo infusion of cortisol caused a significant 4.3-fold reduction in BLMV urea transport capacity in lab-crowded fish, suggesting that cortisol inhibits the recruitment of urea transporters to the basolateral membrane, which may ultimately affect the size of the urea pulse event in gulf toadfish.

Activation of Protein Kinase Cζ Increases OAT1 (SLC22A6)- and OAT3 (SLC22A8)-mediated Transport

Organic anion transporters (OATs) play a pivotal role in the clearance of small organic anions by the kidney, yet little is known about how their activity is regulated. A yeast two-hybrid assay was used to identify putative OAT3-associated proteins in the kidney. Atypical protein kinase Czeta (PKCzeta) was shown to bind to OAT3. Binding was confirmed in immunoprecipitation assays. The OAT3/PKCzeta interaction was investigated in rodent renal cortical slices from fasted animals. Insulin, an upstream activator of PKCzeta, increased both OAT3-mediated uptake of estrone sulfate (ES) and PKCzeta activity. Both effects were abolished by a PKCzeta-specific pseudosubstrate inhibitor. Increased ES transport was not observed in renal slices from OAT3-null mice. Transport of the shared OAT1/OAT3 substrate, rho-aminohippurate, behaved similarly, except that stimulation was reduced, not abolished, in the OAT3-null mice. This suggested that OAT1 activity was also modified by PKCzeta, subsequently confirmed using an OAT1-specific substrate, adefovir. Inhibition of PKCzeta also blocked the increase in ES uptake seen in response to epidermal growth factor and to activation of protein kinase A. Thus, PKCzeta acted downstream of the epidermal growth factor to protein kinase A signaling pathway. Activation of transport was accompanied by an increase in V(max) and was blocked by microtubule disruption, indicating that activation may result from trafficking of OAT3 into the plasma membrane. These data demonstrate that PKCzeta activation up-regulates OAT1 and OAT3 function, and that protein-protein interactions play a central role controlling these two important renal drug transporters.

Aquaporin-6 is expressed along the rat gastrointestinal tract and upregulated by feeding in the small intestine

BackgroundSeveral aquaporins (a family of integral membrane proteins) have been recently identified in the mammalian gastrointestinal tract, and their involvement in the movement of fluid and small solutes has been suggested. In this direction we investigated, in some regions of the rat gastrointestinal tract, the presence and localization of aquaporin-6, given its peculiar function as an ion selective channel.ResultsRT-PCR and immunoblotting experiments showed that aquaporin-6 was expressed in all the investigated portions of the rat gastrointestinal tract. The RT-PCR experiments showed that aquaporin-6 transcript was highly expressed in small intestine and rectum, and less in stomach, caecum and colon. In addition, jejunal mRNA expression was specifically stimulated by feeding.Immunoblotting analysis showed a major band with a molecular weight of about 55 kDa corresponding to the aquaporin-6 protein dimer; this band was stronger in the stomach and large intestine than in the small intestine. Immunoblotting analysis of brush border membrane vesicle preparations showed an intense signal for aquaporin-6 protein.The results of in situ hybridization experiments demonstrate that aquaporin-6 transcript is present in the isthmus, neck and basal regions of the stomach lining, and throughout the crypt-villus axis in both small and large intestine. In the latter regions, immunohistochemistry revealed strong aquaporin-6 labelling in the apical membrane of the surface epithelial cells, while weak or no labelling was observed in the crypt cells. In the stomach, an intense staining was observed in mucous neck cells and lower signal in principal cells and some parietal cells.ConclusionThe results indicate that aquaporin-6 is distributed throughout the gastrointestinal tract. Aquaporin-6 localization at the apical pole of the superficial epithelial cells and its upregulation by feeding suggest that it may be involved in movements of water and anions through the epithelium of the villi.

Following the Path of a Twin-arginine Precursor along the TatABC Translocase of Escherichia coli

The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Consistent with previous in vivo data, we show that the model Tat substrate TorA-PhoA is translocated by the TatABC translocase of Escherichia coli inner membrane vesicles, only if the PhoA moiety was allowed to fold by disulfide bond formation. Although even unfolded TorA-PhoA was found to physically associate with the Tat translocase of the vesicles, site-specific cross-linking revealed a perturbed interaction of the signal sequence of unfolded TorA-PhoA with the TatBC receptor site. Some of the folded TorA-PhoA precursor accumulated in a partially protease-protected membrane environment, from where it could be translocated into the lumen of the vesicles upon re-installation of an H+-gradient. Translocation arrest occurred in immediate vicinity to TatA. Consistent with a neighborhood to TatA, TorA-PhoA remained protease-resistant in the presence of detergents that are known to preserve the oligomeric structures of TatA. Moreover, entry of TorA-PhoA to the protease-protected environment strictly required the presence of TatA. Collectively, our results are consistent with some degree of quality control by TatBC and a recruitment of TatA to a folded substrate that has functionally engaged the twin-arginine translocase.

Analysis of the source of heterogeneity in the osmotic response of plant membrane vesicles

Plasma membrane vesicles have been widely employed to understand the biophysics of water movements, especially when active aquaporins are present. In general, water permeability coefficients in these preparations outcome from the analysis of the osmotic response of the vesicles by means of light scattering. As from now, this is possible by following a theoretical approach that assumes that scattered light follows a single exponential function and that this behavior is the consequence of vesicle volume changes due to an osmotic challenge. However, some experimental data do not necessarily fit to single exponentials but to double ones. It is argued that the observed double exponential behavior has two possible causes: different vesicle population in terms of permeability or in terms of size distribution. As classical models cannot identify this source of heterogeneity, a mathematical modeling approach was developed based on phenomenological equations of water transport. In the three comparative models presented here, it was assumed that water moves according to an osmotic mechanism across the vesicles, and there is no solute movement across them. Interestingly, when tested in a well described plasma membrane vesicle preparation, the application of these models indicates that the source of heterogeneity in the osmotic response is vesicles having different permeability, clearly discarding the variable size effect. In conclusion, the mathematical approach presented here allows to identify the source of heterogeneity; this information being of particular interest, especially when studying gating mechanisms triggered in water channel activity.

Utilization of membrane vesicle preparations to study drug–ABC transporter interactions

Background: The last 15 years have marked an expansion in our understanding of how ABC transporters modulate the pharmacokinetic properties of drugs. Assays based on different membrane preparations were one of the first methods developed to study ABC transporters. Later, they turned out to be valuable tools to gain insight into the nature of drug–ABC transporter interactions. Objectives: Membranes prepared from different sources have been used and characterized; based on the biochemical characteristics of the transport process, a number of different assay types have been developed. Methods: This review focuses on the current experiences on how different membrane-based assays can be utilized in pharmaceutical R&D. Sources of membrane preparations, available assay types and correlation studies between different in-vitro and in-vivo methods are discussed. Results/conclusion: Membrane-based assays are valuable tools in drug discovery to characterize drug–ABC transporter interactions.

A novel method of ligand peptidomics to identify peptide ligands binding to AQP2-expressing plasma membranes and intracellular vesicles of rat kidney

Aquaporin-2 (AQP2), the vasopressin-regulated water channel in collecting duct principal cells, plays a key role in the regulation of body water balance. We aimed to isolate high-affinity peptide ligands that bind to immunoisolated AQP2-expressing plasma membrane (PM) or intracellular vesicle (ICV) preparations from rat kidney by the in vitro phage display technique. Immunoblotting revealed that AQP2 was exclusively expressed in the immunoisolated AQP2 membrane fractions (PM and ICV), compared with the nonimmunoisolated or preimmune IgG pulldown rat kidney samples. Moreover, AQP1 or H+-ATPase (B1 subunit) expression was minimal in the immunoisolated AQP2 membrane fractions, indicating the specificity of AQP2 membrane isolation. A phage peptide library based on T7 415-1b phage vector displaying CX7C was constructed. After three rounds of biopanning, seven phage clones of high frequency were selected, which showed high affinity to the AQP2-containing PM or ICV fractions compared with a nonrecombinant T7 insertless phage clone. In contrast, these phage clones showed lower affinity to H+-ATPase-containing fractions. Fluorescein-conjugated peptide labeling was associated with intracellular compartment and PM of primary cultured inner medullary collecting duct cells, relative to absent or very weak labeling with fluorescein-conjugated control peptide. Library analyses demonstrated proteins that had motifs homologous to the peptide ligands, albeit with a high probability of a random match due to short peptide sequences. In summary, we applied the in vitro phage display technique to identify high-affinity peptide ligands to AQP2-expressing membranes. Library analyses identified proteins having homologous motifs, which need to be examined for involvement in AQP2 trafficking and regulation.

Ontogeny up-regulates renal Na +/Cl −/creatine transporter in rat

Creatine plays a role in energy storage and transport/shuttle of high-energy phosphate in heart, brain, retina, testis and skeletal muscle. These tissues take creatine from the plasma via a 2Na +/1Cl −/1creatine cotransporter (CRT). We have previously demonstrated that renal apical membrane presents a 2Na +/1Cl −/1creatine cotransport activity. The goal of this study was to determine whether this transporter is ontogenically regulated. Na +/Cl −/creatine transport activity was evaluated by measuring [ 14C]-creatine uptake into renal brush-border membrane vesicles. CRT mRNA expression was measured by Northern and real-time PCR assays. E20 foetuses, newborn, suckling, weaning and adult (2- and 8-month-old) Wistar rats were used. The results revealed that neither the vesicular volume, the binding of creatine to the brush-border membrane vesicles, nor the purity of the brush-border membrane vesicle preparations was affected by maturation. Fetal and neonatal kidneys contained a creatine transporter that was qualitatively indistinguishable from that in the adult: it was concentrative, Na +- and Cl −-dependent, electrogenic and inhibited by guanidinopropionic acid. Maturation increased this transport activity by increasing the maximal rate of transport ( V max) without significantly changing the apparent K m. Northern analysis revealed two transcripts for CRT of 2.7 kb and 4.2 kb in all the ages tested. Northern and real-time PCR assays showed that, as seen with NaCl-dependent creatine transport activity, maturation increased CRT mRNA expression. This study reports for the first time that: (i) an apical renal Na +/Cl −/creatine cotransporter is already active in rat foetuses and (ii) development regulates Na +/Cl −/creatine cotransport activity by increasing the density and/or turnover of the transporters.

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.

Deregulation of the SecYEG Translocation Channel upon Removal of the Plug Domain

Previous studies have shown that the SecY plug is displaced from the center of the SecYEG channel during polypeptide translocation. The structural and functional consequences of the deletion of the plug are now examined. Both in vivo and in vitro observations indicate that the plug domain is not essential to the function of the translocon. In fact, deletion of the plug confers to the cell and to the membranes a Prl-like phenotype: reduced proton-motive force dependence of translocation, increased membrane insertion of SecA, diminished requirement for functional leader peptide, and weakened SecYEG subunit association. Although the plug domain does not seem essential, locking the plug in the center of the channel inactivates the translocon. Thus, the SecY plug is important to regulate the activity of the channel and to confer specificity to the translocation reaction. We propose that the plug contributes to the gating mechanism of the channel by maintaining the structure of the SecYEG complex in a compact closed state.

Borrelia burgdorferi BBA74, a Periplasmic Protein Associated with the Outer Membrane, Lacks Porin-Like Properties

The outer membrane of Borrelia burgdorferi, the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a Borrelia burgdorferi plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% alpha-helix with little beta-sheet structure. BBA74 in intact B. burgdorferi cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins.

In-vitro transport characteristics discriminate wild-type ABCB1 (MDR1) from ALA893SER and ALA893THR polymorphisms

Genetic variation of the human ABCB1 (P-glycoprotein; MDR1 gene product) efflux transporter is strongly suggested as a determinant factor governing the pharmacokinetics of diverse drugs and xenobiotics. Despite various efforts to associate polymorphisms in ABCB1 to actual clinical effect and transport function, information is still inconsistent or even controversial. Using membrane vesicle preparation from ABCB1-expressing HighFive insect cells, we report here that saturation kinectic parameters of the frequently occurring ABCB1 triallelic variants 893Ser (exon 21, 2677T) and 893Thr (2677A) were considerably different from wild-type 893Ala (2677G), despite similar protein expression levels. Of importance were significant differences in transport capacities between the tested 893Ala/Ser/Thr variants. In comparison with 893Ala, maximal transport rates for vincristine of 893Ser and 893Thr increased 50% and three-fold, respectively. Cis-inhibition by digoxin, didanosine or fexofenadine was least pronounced in 893Ser, whereas no genotype differences could be observed using verapamil. These results suggest an influence of ABCB1-893 triallelic variants on transport function and drug-drug interaction, which might be most pronounced in 893Thr. Furthermore, some of the mechanisms of 2677G/T/A-based haplotype-associated alterations in ABCB1 activity may have been unveiled.