Right-side-out Research Articles

The substitution of Arg149 with Cys fixes the melibiose transporter in an inward-open conformation

The melibiose transporter from Escherichia coli (MelB) can use the electrochemical energy of either H+, Na+ or Li+ to transport the disaccharide melibiose to the cell interior. By using spectroscopic and biochemical methods, we have analyzed the role of Arg149 by mutagenesis. According to Fourier transform infrared difference and fluorescence spectroscopy studies, R149C, R149Q and R149K all bind substrates in proteoliposomes, where the protein is disposed inside-out. Analysis of right-side-out (RSO) and inside-out (ISO) membrane vesicles showed that the functionally active R149Q and R149K mutants could bind externally added fluorescent sugar analog in both types of vesicles. In contrast, the non-transporting R149C mutant does bind the fluorescent sugar analog as well as melibiose and Na+ in ISO, but not in RSO vesicles. Therefore, the mutation of Arg149 into cysteine restrains the orientation of transporter to an inward-open conformation, with the inherent consequences of a) reducing the frequency of access of outer substrates to the binding sites, and b) impairing active transport. It is concluded that Arg149, most likely located in the inner (cytoplasmic) half of transmembrane helix 5, is critically involved in the reorientation mechanism of the substrate-binding site accessibility in MelB.

Identification of Conformationally Sensitive Amino Acids in the Na+/Dicarboxylate Symporter (SdcS)

The Na(+)/dicarboxylate symporter (SdcS) from Staphylococcus aureus is a homologue of the mammalian Na(+)/dicarboxylate cotransporters (NaDC1) from the solute carrier 13 (SLC13) family. This study examined succinate transport by SdcS heterologously expressed in Escherichia coli, using right-side-out (RSO) and inside-out (ISO) membrane vesicles. The K(m) values for succinate in RSO and ISO vesicles were similar, approximately 30 microM. The single cysteine of SdcS was replaced to produce the cysteine-less transporter, C457S, which demonstrated functional characteristics similar to those of the wild type. Single-cysteine mutants were made in SdcS-C457S at positions that are functionally important in mammalian NaDC1. Mutant N108C of SdcS was sensitive to chemical labeling by MTSET {[2-(trimethylammonium)ethyl]methanethiosulfonate} from both the cytoplasmic and extracellular sides, depending on the conformational state of the transporter, suggesting that Asn-108 may be found in the translocation pore of the protein. Mutant D329C was sensitive to MTSET in the presence of Na(+) but only from the extracellular side. Finally, mutant L436C was insensitive to MTSET, although changes in its kinetic properties indicate that this residue may be important in substrate binding. In conclusion, this work identifies Asn-108 as a key residue in the translocation pathway of the protein, accessible in different states from both sides of the membrane. Functional characterization of SdcS should provide useful structural as well as functional details about mammalian transporters from the SLC13 family.

Plasma membrane of Synechocystis PCC 6803: a heterogeneous distribution of membrane proteins

Proteomic studies carried out previously on the plasma membrane of Synechocystis have identified several peripheral and integral proteins. The distribution of these proteins along the membrane still remains obscure. In this study, the distribution of proteins along the plasma membrane of Synechocystis was carried out using subfractions, the right-side-out (RSO) and inside-out (ISO) vesicles, fractionated from a pure and specific fraction of the plasma membrane. These subfractions were analyzed and quantified for several proteins by immunoblotting. It was found that the ISO fraction contained higher quantities of preD1, D1 and PsaD, the integral proteins of photosystem I and II known to be present also in the plasma membrane. Lower amounts of peripheral vesicle inducing protein Vipp1 and nitrate/nitrite binding protein NrtA were present in the ISO compared to the RSO fraction. On the contrary, the distribution of two integral transporter proteins, SbtA and PxcA, was found equal in both fractions. Our studies clearly establish that the plasma membrane of Synechocystis has a heterogeneous composition with respect to protein distribution. The accumulation of photosynthesis-associated proteins in the ISO fraction provides evidence that the discrete regions of the plasma membrane harbor sites for biogenesis of photosystems.

Phospholipid diversity: Correlation with membrane–membrane fusion events

The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane–membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called “fusion proteins”. This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane–membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.

Functional characterization of cysteine residues in GlpT, the glycerol 3-phosphate transporter of Escherichia coli.

In Escherichia coli, the GlpT transporter, a member of the major facilitator superfamily, moves external glycerol 3-phosphate (G3P) into the cytoplasm in exchange for cytoplasmic phosphate. Study of intact cells showed that both GlpT and HisGlpT, a variant with an N-terminal six-histidine tag, are inhibited (50% inhibitory concentration approximately 35 microM) by the hydrophilic thiol-specific agent p-mercurichlorobenzosulfonate (PCMBS) in a substrate-protectable fashion; by contrast, two other thiol-directed probes, N-maleimidylpropionylbiocytin (MPB) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), have no effect. Use of variants in which the HisGlpT native cysteines are replaced individually by serine or glycine implicates Cys-176, on transmembrane helix 5 (TM5), as the major target for PCMBS. The inhibitor sensitivity of purified and reconstituted HisGlpT or its cysteine substitution derivatives was found to be consistent with the findings with intact cells, except that a partial response to PCMBS was found for the C176G mutant, suggesting the presence of a mixed population of both right-side-out (RSO) (resistant) and inside-out (ISO) (sensitive) orientations after reconstitution. To clarify this issue, we studied a derivative (P290C) in which the RSO molecules can be blocked independently due to an MPB-responsive cysteine in an extracellular loop. In this derivative, comparisons of variants with (P290C) and without (P290C/C176G) Cys-176 indicated that this residue shows substrate-protectable inhibition by PCMBS in the ISO orientation in proteoliposomes. Since PCMBS gains access to Cys-176 from both periplasmic and cytoplasmic surfaces of the protein (in intact cells and in a reconstituted ISO orientation, respectively) and since access is unavailable when the substrate is present, we propose that Cys-176 is located on the transport pathway and that TM5 has a role in lining this pathway.

Orientation selective immobilization of human erythrocyte membranes on ultrathin cellulose films

We report the orientation selective immobilization of human erythrocyte membranes on planar solid supports. The orientation of the immobilized membrane was identified with selective fluorescence labels. When the right-side-out (RSO) ghosts were incubated with planar glass cover slides, no adsorption or rupture of erythrocytes could be observed. To increase the interfacial attraction between cells and the surface, two types of hydrated polymer films were deposited on the glass cover slides; (a) physisorbed films of cationic polylysine, and (b) Langmuir–Blodgett (LB) films of cellulose derivatives. On polylysine films, patches of the ruptured membranes could be observed, but the surface coverage still remained poor. On the other hand, RSO ghosts were likely to coat the surface of cellulose films more continuously. The fluorescence labeling demonstrated that immobilized erythrocyte membranes selectively inverted their native orientation. Tentatively, we interpreted this larger surface coverage on the cellulose film in terms of the “wetting affinity” between the cell surface glycocalix and the polysaccharides. Such ultrathin (thickness 5–10 nm), biological polysaccharide films have a large potential to immobilize native cell membranes without denaturing their structure, membrane orientation, and functions.

Arg-425 of the Citrate Transporter CitP Is Responsible for High Affinity Binding of Di- and Tricarboxylates

The citrate transporter of Leuconostoc mesenteroides (CitP) catalyzes exchange of divalent anionic citrate from the medium for monovalent anionic lactate, which is an end product of citrate degradation. The exchange generates a membrane potential and thus metabolic energy for the cell. The mechanism by which CitP transports both a divalent and a monovalent substrate was the subject of this investigation. Previous studies indicated that CitP is specific for substrates containing a 2-hydroxycarboxylate motif, HO-CR(2)-COO(-). CitP has a high affinity for substrates that have a "second" carboxylate at one of the R groups, such as divalent citrate and (S)-malate (Bandell, M., and Lolkema, J. S. (1999) Biochemistry 38, 10352-10360). Monovalent anionic substrates that lack this second carboxylate were found to bind with a low affinity. In the present study we have constructed site-directed mutants, changing Arg-425 into a lysine or a cysteine residue. By using two substrates, i.e. (S)-malate and 2-hydroxyisobutyrate, the substrate specificity of the mutants was analyzed. In both mutants the affinity for divalent (S)-malate was strongly decreased, whereas the affinity for monovalent 2-hydroxyisobutyrate was not. The largest effect was seen when the arginine was changed into the neutral cysteine, which reduced the affinity for (S)-malate over 50-fold. Chemical modification of the R425C mutant with the sulfhydryl reagent 2-aminoethyl methanethiosulfonate, which restores the positive charge at position 425, dramatically reactivated the mutant transporter. The R425C and R425K mutants revealed a substrate protectable inhibition by other sulfhydryl reagents and the lysine reagent 2,4,6-trinitrobenzene sulfonate, respectively. It is concluded that Arg-425 complexes the charged carboxylate present in divalent substrates but that is absent in monovalent substrates, and thus plays an important role in the generation of the membrane potential.

The topology of phosphatidylglycerol populations is essential for sustaining photosynthetic electron flow activities in thylakoid membranes

The transmembrane distribution of phosphatidylglycerol (PG) was determined in rightside-out (RO) and inside-out vesicles (IO) obtained by fragmentation of spinach thylakoids in a Yeda press, followed by partition in an aqueous dextran-polyethyleneglycol two-phase system. Using the phospholipase A 2 from porcine pancreas to digest selectively PG molecules in the outer monolayer (exposed to the incubation medium) of the membrane, we found the molar outside/inside distribution to be 70/30±5 in RO and 40/60±3 in IO. The transmembrane distribution of PG in IO was the opposite of that in intact thylakoids (molar ratio 58/42±3). The phospholipid population which sustained most of the uncoupled photosystem II electron flow activity was localized in the inner monolayer (exposed to the thylakoid lumen) of both thylakoid and RO membranes. In contrast, the activity in IO membranes was highly dependent on the PG population located in the outer monolayer. This finding brings the first direct demonstration of the dependence of the photosynthetic electron flow activity on the integrity of the inner topological pool of PG in the thylakoid membrane.

Functional Symmetry of UhpT, the Sugar Phosphate Transporter ofEscherichia coli

UhpT, the sugar phosphate transporter of Escherichia coli, acts to exchange internal inorganic phosphate for external hexose 6-phosphate. Because of this operational asymmetry, we studied variants in which right-side-out (RSO) or inside-out (ISO) orientations could be analyzed independently to ask whether the inward- and outward-facing UhpT surfaces have different substrate specificities. To study the RSO orientation, we constructed a histidine-tagged derivative, His10K291C/K294N, in which the sole external tryptic cleavage site (Lys294) had been removed. Functional assay as well as immunoblot analysis showed that trypsin treatment of proteoliposomes containing His10K291C/K294N led to loss of about 50% of the original population, reflecting retention of only the RSO population. To study the ISO orientation, we used a His10V284C derivative, in which a newly inserted external cysteine (Cys284) conferred sensitivity to the thiol-reactive agent, 3-(N-maleimidylpropionyl)biocytin. In this case, 3-(N-maleimidylpropionyl)biocytin treatment of proteoliposomes containing His10V284C gave about a 60% loss of activity, and immunodetection of biotin showed parallel modification of an equivalent fraction of the original population. Together, such findings indicate that the UhpT RSO and ISO orientations are in about equal proportion in proteoliposomes and that a single population can be generated by exposure of these derivatives to the appropriate agent. This allowed us to study proteoliposomes with UhpT functioning in RSO orientation (His10K291C/K294N) or ISO orientation (His10V284C) with respect to the kinetics of glucose 6-phosphate transport by phosphate-loaded proteoliposomes and also the inhibitions found with 2-deoxy-glucose 6-phosphate, mannose 6-phosphate, galactose 6-phosphate, fructose 6-phosphate, and inorganic phosphate. We found no significant differences in the behavior of UhpT in its different orientations, indicating that the transporter possesses an overall functional symmetry.

Histidine 225, a Residue of the NhaA-Na+/H+ Antiporter of Escherichia coli Is Exposed and Faces the Cell Exterior

Cysteine residues were found nonessential in the mechanism of the NhaA antiporter activity of Escherichia coli. The functional C-less NhaA has provided the groundwork to study further histidine 225 of NhaA which has previously been suggested to play an important role in the activation of NhaA at alkaline pH (Rimon, A., Gerchman, Y., Olami, Y., Schuldiner, S. and Padan, E. (1995) J. Biol. Chem. 270, 26813-26817). C-less H225C was constructed and shown to possess an antiporter activity 60% of that of C-less antiporter and a pH profile similar to that of both the C-less or wild-type antiporters. Remarkably, whereas neither the wild-type nor the C-less antiporters were affected by N-ethylmaleimide, C-less H225C was inhibited by this reagent. To determine the degree of alkylation of the antiporter protein by N-ethylmaleimide, antiporter derivatives tagged at their C termini with six histidines residues were constructed. Alkylation of C-less H225C was measured by labeling of everted membrane vesicles with [14C]N-ethylmaleimide, affinity purification of the His-tagged antiporter, and determination of the radioactivity of the purified protein. This assay showed that H225C is alkylated to a much higher level than any of the native cysteinyl residues of NhaA reaching saturation at alkyl/NhaA stoichiometry of 1. The wild-type derivative showed at least 10-fold less alkylation even at higher concentrations, suggesting that H225C resides in a domain that is much more exposed to N-ethylmaleimide than the native cysteinyl residues of NhaA. Since H225C residues both in right-side out and inside-out membrane vesicles were quantitatively alkylated by N-ethylmaleimide, this assay was used to determine the accessibility of H225C to other SH reagents by titrating the H225C left free to react with N-ethylmaleimide, following exposure of the membranes to the reagents. Furthermore, since membrane-impermeant probes can react with residues in membrane-embedded protein only if accessible to the medium containing the reagent, the assay was used to determine the membrane topology of H225C. As expected for a membrane-permeant probe, p-chloromercuribenzoate reacted with H225C as efficiently as N-ethylmaleimide in both membrane orientations. Similar results were obtained with methanethiosulfonate ethylammonium supporting the recent observations that this probe is membrane-permeant. On the other hand, both membrane-impermeant reagents p-chloromercuribenzosulfonate and methanethiosulfonate ethyl-trimethyl ammonium bromide reacted with H225C 10-fold more in right-side out than in inside-out vesicles, and p-chloromercuribenzosulfonate also blocked completely the H225C in intact cells. These results strongly suggest that H225C is exposed at the periplasmic face of the membrane.

Effect of substrates and pH on the intestinal Na +/phosphate cotransporter: evidence for an intervesicular divalent phosphate allosteric regulatory site

Intervesicular divalent phosphate-induced inhibition of the intestinal brush-border membrane Na +/phosphate cotransporter was examined using Na +-dependent phosphate uptake, substrate-induced tryptophan fluorescence quenching, and the apparent p K a values for substrate-induced conformational changes. In right-side-out (RSO) reconstituted proteoliposomes, only monovalent phosphate inhibited Na +-dependent phosphate uptake in the absence of pre-equilibration. Addition of divalent phosphate to inside-out (ISO) proteoliposomes resulted in 80 ± 5% inhibition of Na +-dependent phosphate uptake in the absence of pre-equilibration. The nature of divalent phosphate-induced inhibition of cotransporter function was examined using cotransporter partial reaction assays based on substrate-induced conformational changes reported as changes in tryptophan fluorescence. Na + but not K + induced a quenching of tryptophan fluorescence with a K 0.5 of 25 mM and an apparent Hill coefficient of 1.8. Monovalent phosphate (difluorophosphate) induced a further quenching of tryptophan fluorescence with a K 0.5 of 53 μM. Divalent phosphate (monofluorophosphate) had no effect on tryptophan fluorescence, but inhibited the difluorophosphate-induced quenching of tryptophan fluorescence. The Na + to Na + + divalent phosphate (monofluorophosphate) conformation and the Na + to Na + + monovalent phosphate (difluorophosphate) conformations were compared using tryptophan quench reagents. These transitions had different apparent p K a values and different phenylglyoxal sensitivities consistent with monovalent phosphate and divalent phosphate interacting with the cotransporter at separate sites.

Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles—I. Binding and transport

To gain insight into the mechanisms governing cellular uptake of glucocorticoids, we studied the binding and membrane transport of corticosterone (B) on a highly purified plasma membrane fraction from rat liver that was homogenized using a gentle, isotonic procedure. The fraction was mostly in the form of right-side out and osmotically active vesicles that were free of intracellular glucocorticoid receptors (GCR), transcortin (CBG) and ATP. Our uptake and binding studies carried out at 22°C with [ 3H]B in physiological concentrations resulted in the following findings: (1) unlabeled B competed with [ 3H]B for uptake by the membrane vesicles; half-maximal competition of specific uptake was achieved with a 10- to 11-fold molar excess of unlabeled B. (2) [ 3H]B uptake was a saturable process of unusual kinetic (multiple sigmoidity); modified Scatchard plots revealed three significantly different apparent K d -values of 1.3, 4.7 and 17.3 nM, corresponding to free B in the blood of non-stressed rats (4–16 nM). (3) Osmotic shrinkage of the vesicles led to a linear decrease in specific uptake, while non-specific uptake was independent of vesicle volume. Passive diffusion of [ 3H]B took place in leaky, but not in intact, vesicles. Reversible binding to, and mediated transport through, the membrane were interdependent parts of a strongly linked process. B was accumulated inside the vesicle up a concentration gradient by an active that followed first-order kinetics ( K t : 3.9 nM)l; for its statistically reliable mathematical formulation and kinetic analysis, a replot was developed that revealed that relative accumulation increased with decreasing external hormone concentration. (4) Comparative binding studies disclosed that the apparent K d -values (86.5 ± 7.3 and 77.0 ± 14.3 nM, respectively) of the [ 3H]B interactions with CBG and GCR did not differ ( P > 0.3). These findings permit the conclusion that a plasma membrane-inserted carrier for B, effectively operating at physiological concentrations in the blood, is involved in a functional and regulatory manner in the biological action of glucocorticoids.

Escherichia coli alpha-ketoglutarate permease is a constitutively expressed proton symporter.

Escherichia coli kgtP which maps at 56.5 min codes for alpha-ketoglutarate permease (KgtP). This protein, expressed from the cloned gene using the T7 polymerase system and [35S]methionine labeling, fractionated with cell membranes. Right-side-out (RSO) membrane vesicles prepared from a kgtP negative mutant strain did not transport alpha-ketoglutarate, but RSO vesicles from the same strain expressing KgtP from a transforming plasmid transported alpha-ketoglutarate effectively as measured by uptake of the 14C-labeled substrate. E. coli JC7623 strain grown in M9 minimal medium with glucose, glycerol, or alpha-ketoglutarate as carbon source contained a 1.3-kilobase RNA which hybridized to nick-translated kgtP probe. In addition, strain MC1061 cultures grown under these same conditions were all capable of transporting alpha-ketoglutarate, demonstrating that KgtP is constitutively expressed. The Km and Vmax of KgtP assayed in strain MC1061 vesicles were 13-46 microM and 8 nmol/min/mg protein, respectively. Uncouplers that permeabilized the membrane to protons inhibited alpha-ketoglutarate transport into energized vesicles, and the addition of alpha-ketoglutarate to vesicle suspensions under non-energized conditions resulted in an increase in pH. These results indicate that KgtP is an alpha-ketoglutarate-proton symporter.

The use of monoclonal antibodies to study the structure and function of cytochrome f

Monoclonal antibodies (MAbs) were prepared against native cytochrome f (cyt f) isolated from turnip leaves. The two MAbs obtained, designated MAb-JB2 and MAb-ED4, were Western blot positive to purified turnip cytochrome f and also reacted with inside-out (ISO) but not right-side-out (RSO) spinach thylakoid membranes. MAb-ED4 reacted with a covalent adduct formed by crosslinking cyt f and plastocyanin (PC), whereas MAb-JB2 did not. In contrast, MAb-JB2 reacted with the isolated cyt b 6 f complex but MAb-ED4 did not. These results indicate that MAb-JB2 binds to cyt f at or near the PC binding site on f, whereas MAb-ED4 binds to a portion of cyt f which is not exposed in the cyt b 6 f complex. The location of the epitopes in the primary sequence of cyt f was determined by trypsin hydrolysis, HPLC separation of tryptic peptides, and ELISA identification of the purified peptides. The molecular weights of the purified peptides, determined by gel exclusion chromatography, were found to be 5040 and 3130 Da for MAb-JB2 and MAb-ED4, respectively. Amino acid sequencing showed that the first eight amino acids of the MAb-ED4 positive peptide were LDQPLTSN. These results suggest that the 3130-Da peptide has 28 amino acids extending from Leu 223 to Arg 250. This peptide is located on the N-terminal (lumen) side of the postulated membrane-spanning sequence. The first eight amino acids of the MAb-JB2-positive peptide were NILVIGPV. This sequence and the peptide molecular weight indicate that the epitope for MAb-JB2 is located within a 44-amino acid peptide extending from Asn 111 to Arg 154.

Energetics of active transport in inverted membrane vesicles of Escherichia coli

Inside-out (ISO) membrane vesicles isolated from Escherichia coli are inverted (interior positive and acidic) with respect to right-side-out (RSO) membranes (interior negative and alkaline). As the membranes are coupled with an electron donor (D-lactate) as the energy source, ISO membranes exhibit a proton gradient (ΔpH) of 46.4 mV and an electrical potential (ΔΨ) of 63.3 mV, as measured by a flow dialysis method. This gives a proton electrochemical potential (Δ μ H+) of 110 mV across the membranes. The energetics of the formation of Δ μ H+ across ISO membranes were investigated by microcalorimetry in combination with oxygen uptake measurement. The results were compared with those previously obtained with RSO membranes. In these two types of membranes, the energetic parameters in the formation of Δ μ H+ (the enthalpy (Δ H m), the free energy (Δ G m) and the entropy (Δ S m) changes) exhibit similar positive signs. The observations suggest that the characteristics of the created Δ μ H+ across these membranes are analogous, regardless of the polarities across the membranes. The efficiency in the free energy transfer from the energy source (D-lactate oxidation reaction) to the creation of Δ μ H+ is essentially the same in ISO and RSO membranes, while the enthalpy transfer is much lower in ISO than in RSO membranes. The discrepancy in the enthalphy transfer is associated with the intrinsic difference between ISO and RSO membranes in the direction of proton movement. The employed thermochemical method can not only confirm the formation of Δ μ H+ across ISO membranes, but also reveals a significant difference in the value of ΔH m between ISO and RSO membranes, due to an opposite polarity across the membranes. This work provides an understanding of the role that membrane polarity plays in the energetics of active transport across membranes.

Folate (pteroylglutamate) uptake in human red blood cells, erythroid precursors and KB cells at high extracellular folate concentrations. Evidence against a role for specific folate-binding and transport proteins

Membrane-associated folate (pteroylglutamate, PteGlu)-binding proteins (FBPs) play an important role as PteGlu-transport proteins in malignant and normal human cells. Since high extracellular folate (PteGlu) concentrations (EFC) profoundly influenced uptake and toxicity of the anti-PteGlu methotrexate in malignant KB cells, we studied human cells to determine additional mechanisms for PteGlu uptake when the EFC was varied. At low EFC (less than 10 nM), the predominant mechanism for folate uptake in mature erythrocytes was through binding to externally oriented FBPs which were quantitatively insignificant (4-6 orders of magnitude lower) and of no apparent physiological relevance when compared with KB cells. However, the predominant mechanism of PteGlu accumulation at high EFC [10-250 nM] in intact erythrocytes and sealed right-side-out (RSO) ghosts was not FBP-mediated and non-specific. This conclusion was based on the findings that radiolabelled PteGlu uptake: (i) continued even in the presence of a 1000-fold excess of unlabelled PteGlu and was linear and not saturable up to 250 nM; (ii) was two-fold higher at pH 4.5 than 7.5; (iii) was less than 2-fold increased at 37 degrees C compared with 4 degrees C; and (iv) was unaffected after trypsin-mediated proteolysis of greater than 75% FBPs. The [3H]PteGlu and 125I-PteGlu (histamine derivative) accumulated intracellularly through the non-specific PteGlu-uptake mechanism was unaltered biochemically and in a soluble compartment. Raising the EFC 500-fold higher than controls during erythropoiesis in vitro resulted in reversal of the expected anti-(placental folate-receptor)-antiserum-induced megaloblastic changes in orthochromatic normoblasts derived from burst-forming unit-erythroid colonies. Furthermore, at EFC greater than 0.1 microM, KB-cell accumulation of [3H]PteGlu was also predominantly through a mechanism that did not involve specific FBPs. Thus, at high EFC, a major component of PteGlu transport in human cells is not mediated through FBPs and is likely to be a passive diffusion process.

Effect of divalent cation chelation on dihydropyridine binding in isolated cardiac sarcolemma vesicles

The effect of divalent cation chelation on specific nitrendipine and ouabain binding has been determined in a highly enriched sarcolemma preparation isolated from canine ventricle. Maximal high-affinity nitrendipine binding measured in the absence of added calcium or magnesium was 997 ± 103fmol/mg protein. Nitrendipine binding in the presence of EDTA significantly decreased to 419 ± 42fmol/mg protein ( P < 0.001) which equates to 42.0% of control. The simultaneous presence of EDTA and A23187 in the binding buffer resulted in a decrease in nitrendipine binding to below detectable levels. These results suggest that divalent cations trapped within vesicles can support high affinity nitrendipine binding. Evaluation of dihydropyridine binding at various pH values suggested that the loss of binding below pH 7.0 and above pH 8.0 may result indirectly from a change in divalent cation binding rather than a direct effect on dihydropyridine binding per se. The maximal binding of ouabain determined in the presence of magnesium and inorganic phosphate averaged 340 ± 7.4pmol/mg protein. Pre-treatment of the preparation with sodium dodecyl sulfate (SDS) in order to express binding in sealed inside-out (IO) vesicles, increased ouabain binding to 471 ± 27pmol/mg protein. Thus, these preparations averaged 27.8% sealed IO vesicles. Addition of EDTA in the absence of magnesium in the binding buffer reduced ouabain binding to 204 ± 7.7 and 11.7 ± 3.5pmol/mg protein in control and SDS-treated preparations, respectively. These findings suggest that this sarcolemma preparation consists of 43.6% sealed right-side-out (RO) vesicles which contain sufficient endogenous divalent cation trapped in the intravesicular space, to support ouabain binding. The correspondence between the percentage of ouabain binding that remains in the presence of EDTA and the percentage of nitrendipine binding observed under the same conditions is consistent with the hypothesis that divalent cations support nitrendipine binding by interaction with a site or sites accessible only from the cytoplasmic membrane surface and that nitrendipine and ouabain binding sites occur in the same vesicles (i.e., the nitrendipine binding site is of sarcolemma origin).

Latency of Plasma Membrane H+-ATPase in Vesicles Isolated by Aqueous Phase Partitioning

The properties of the plasma membrane H(+)-ATPase and the cause of its latency have been studied using a highly purified plasma membrane fraction from oat (Avena sativa L., cv Victory) roots, prepared by aqueous two-phase partitioning. The ATPase has a maximum specific activity (at 37 degrees C) in excess of 4 micromoles inorganic phosphate per milligram protein per minute in the presence of nondenaturing surfactants. It is inhibited by more than 90% by vanadate, is specific for ATP, has a pH optimum of 6.5, and is stimulated more than 4-fold by 50 millimolar K(+) in the presence of low levels of the nondenaturing surfactants Triton X-100 and lysolecithin. This ;latent' activity is usually explained as being a result of the inability of ATP to reach the ATPase in right-side out, sealed vesicles, until they are disrupted by surfactants. Consistent with this idea, trypsin digestion significantly inhibited the ATPase only in the presence of the surfactants. Electron spin resonance spectroscopy volume measurements confirmed that surfactant-free vesicles were mostly sealed to molecules similar to ATP. However, the Triton to protein ratio required to disrupt vesicle integrity completely is 10-fold less than that needed to promote maximum ATPase activity. We propose that plasma membrane ATPase activation is due not solely to vesicle disruption and accessibility of ATP to the ATPase but to the surfactants activating the ATPase by altering the lipid environment in its vicinity or by removing an inhibitory subunit.

Separation and characteristics of inside-out and right side-out vesicles from a rat cardiac sarcolemma preparation

1. 1. Purified cardiac sarcolemma (SL) vesicles are highly suitable to study various Ca 2+-transport systems present in the SL. 2. 2. We describe in this paper the separation of the Inside-Out (IO) and Right side-Out (RO) oriented vesicle subpopulations from a purified rat heart SL preparation. 3. 3. The isolated subfractions were characterized with respect to the number of beta-adrenergic binding sites and the Ca 2+-uptake and (Ca 2+-Mg 2+)-ATPase activities. 4. 4. It was found that the Ca 2+-uptake and the (Ca 2+-Mg 2+)-ATPase activities reside in the IO fraction and are virtually absent in the RO fraction, confirming that the active Ca 2+-uptake represents the outward directed sarcolemmal Ca 2+-flux.

Sodium transport in basolateral membrane vesicles from rat enterocytes

Basolateral membranes purified from rat jejunal enterocytes and enriched 14 times in (Na, K)-ATPase, are present as unsealed and right side out (RSO) or inside out (IO) vesicles in the ratio 2:2:1, as determined by detergent activation of ATPase activity. Entrance of 1 mM Na into basolateral membrane vesicles was measured in the presence and in the absence of 5 mM ATP by a rapid filtration technique, under different experimental conditions. Carrier-mediated Na transport across the basolateral membrane can be trans-stimulated and cis-inhibited by K and further stimulated by ATP (activation of the Na pump). The ATP effect can be suppressed by vanadate and strophanthidin and enhanced by bleomycin (19% increase), which positively also acts on (Na, K)-ATPase activity (16% increase). In addition to the Na pump this study demonstrates the existence of a carrier-mediated Na transport trans-stimulated by K. There appears to be no cotransport of Na-K.