Inside-out Membrane Vesicles Research Articles

Effects of Calcium and Calmodulin on the Binding of Rat Parotid Secretion Granules to the Plasma Membrane

Since numerous studies suggest that Ca++ and calmodulin may modulate the fusion of secretion granules to the plasma membrane which takes place in exocytosis, we have examined the role of calcium and calmodulin in the binding of isolated parotid secretion granules to plasma membrane vesicles. 125I-labeled inside-out plasma membrane vesicles were incubated with secretion granules, the mixture was layered over 20% sucrose, the gradient was centrifuged, and the amount of 125I in the granule pellet was determined. Addition of Ca++ (20 nM to 10 microM) produced a concentration-dependent increase in the binding of 125I-labeled plasma membrane vesicles to the secretion granules, reaching a maximum value at 10 microM free Ca++; half-maximal binding occurred at 400 nM. Neither right-side-out parotid plasma membrane vesicles nor inside-out pancreatic islet plasma membrane vesicles bound to granules in the presence of 1 microM Ca++. Calmodulin produced a concentration-dependent increase in binding above that of Ca++ alone, and this effect was inhibited by the calmodulin antagonists, trifluoperazine and calmidazolium. Incubation of secretion granules with octadecylrhodamine B (R18)-loaded inside-out plasma membrane vesicles and 2 microM Ca++ caused de-quenching of fluorescence, indicating that the lipids in the granule membrane and the plasma membrane had intermixed. Added calmodulin increased the fluorescence two-fold above that with Ca++ alone. These results suggest that Ca++ and calmodulin may play a role in parotid gland exocytosis by modulating the interaction between the secretion granules and plasma membrane.

Excretion and Uptake of Putrescine by the PotE Protein in Escherichia coli

The structure and function of the polyamine transport protein PotE was studied. Uptake of putrescine by PotE was dependent on the membrane potential. In contrast, the putrescine-ornithine antiporter activity of PotE studied with inside-out membrane vesicles was not dependent on the membrane potential (Kashiwagi, K., Miyamoto, S., Suzuki, F., Kobayashi, H., and Igarashi, K. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4529-4533). The Km values for putrescine uptake and for putrescine-ornithine antiporter activity were 1.8 and 73 microM, respectively. Uptake of putrescine was inhibited by high concentrations of ornithine. This effect of ornithine appears to be due to putrescine-ornithine antiporter activity because it occurs only after accumulation of putrescine within cells and because ornithine causes excretion of putrescine. Thus, PotE can function not only as a putrescine-ornithine antiporter to excrete putrescine but also as a putrescine uptake protein. Both the NH2 and COOH termini of PotE were located in the cytoplasm, as determined by the activation of alkaline phosphatase and beta-galactosidase by various PotE-fusion proteins. The activities of putrescine uptake and excretion were studied using mutated PotE proteins. It was found that glutamic acid 207 was essential for both the uptake and excretion of putrescine by the PotE protein and that glutamic acids 77 and 433 were also involved in both activities. These three glutamic acids are located on the cytoplasmic side of PotE, and the function of these three residues could not be replaced by other amino acids. Putrescine transport activities did not change significantly with mutations at the other 13 glutamic acid or aspartic acid residues in PotE.

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.

Spermine modulates calcium flux through the rat erythrocyte membrane.

The uptake of 45Ca by inside-out erythrocyte membrane vesicles (IOV) was dose-dependently inhibited by spermine. A cationic substance, Mg2+, did not affect this inhibitory activity of spermine. Ca(2+)-ATPase activity, however, was not inhibited by spermine at a concentration that could inhibit the uptake of 45Ca by the IOV. The release of 45Ca from IOV was significantly accelerated by spermine. Spermine bound to the surface of the IOV and was removed by treatment with acetic acid. These results suggest that spermine stimulates the release of Ca2+ from the IOV (or uptake of Ca2+ by erythrocytes) via an effect on the outer surface of the IOV (or the inner surface of erythrocytes).

On the accessibility of phosphatidylglycerol to periodate in Escherichia coli.

The transverse distribution of phosphatidylglycerol (PG) across the inner membrane of Escherichia coli was studied. The oxidation of PG by periodate was used to discriminate between the PG present in the cytoplasmic and in the periplasmic leaflet of the inner membrane. Applied to large unilamellar vesicles derived of an E. coli lipid extract, the periodate method demonstrated the expected symmetrical distribution of PG over the bilayer. However, both in right-side-out and in inside-out inner membrane vesicles isolated from E. coli, as well as in intact E. coli cells, the entire pool of PG was oxidized by periodate. The oxidation reaction proceeded at such a high rate that it was impossible to determine the distribution of PG across the membrane. Apparently, periodate easily permeates through the membrane. This permeation could not be inhibited or slowed down, and it was concluded that the use of periodate as oxidizer of PG is not a suitable method to determine the transverse distribution of this phospholipid in the Gram-negative bacterium E. coli. However, periodate can be used to selectively modify the total PG pool in this organism.

Topological Analysis of NhaA, a Na+/H+ Antiporter from Escherichia coli

Analysis of the hydropathic profile of the amino acid sequence of NhaA, a Na+/H+ antiporter from Escherichia coli has previously suggested the existence of 11 putative transmembrane segments (Taglicht, D., Padan, E., and Schuldiner, S. (1991) J. Biol. Chem. 266, 11289-11294). In the present work to test the location of the C terminus, right-side-out and inside-out membrane vesicles were digested with carboxypeptidase B and probed with an antibody raised against a synthetic peptide whose sequence was based on the C terminus sequence. The results demonstrate that the C terminus is facing the cell interior because it is available for digestion only from the inside. Previous evidence from an NhaA-beta-galactosidase fusion to loop 5 of NhaA indicated that this loop is also facing the cytoplasm (Karpel, R., Alon, T., Glaser, G., Schuldiner, S., and Padan, E. (1991) J. Biol. Chem. 266, 21753-21759) and therefore was not consistent with the position of the C terminus in an 11-transmembrane segment model. Therefore, the model was re-evaluated. For this purpose, 10 nhaA'-'phoA gene fusions were constructed and assayed for alkaline phosphatase activity. The results support a 12-transmembrane segment model with the N and C termini located in the cytoplasm. The evidence indicates that two very short segments, 14 and 16 amino acids long, must cross the membrane in an unknown conformation.

Se-mediated domain-domain communication in band 3 of human erythrocytes.

Na2SeO3 could affect the anion flux of Band 3 of inside-out erythrocyte membrane vesicles (IOVs). Such effect was believed to be based on the interaction of SH groups of Band 3 with Na2SeO3. This effect could be eliminated when the cytoplasmic domain of Band 3 was proteolytically removed by trypsin. This suggested that SH groups in the cytoplasmic domain were involved in such interaction. Measurement of the pH dependence of intrinsic fluorescence intensity provided evidence that conformational changes of Band 3 occurred as a consequence of interaction with selenite. KI quenching of intrinsic fluorescence of Band 3 could also show that there was a conformational change in the cytoplasmic domain of Band 3 after reaction with Na2SeO3. Such conformational change in turn could be transmitted to the membrane domain of Band 3 monitored by quenching of intrinsic fluorescence of Band 3 using hypocrellin B (HB) (a photosensitive pigment obtained from a parasitic fungus growing in Yunnan, China). It is suggested that the cytoplasmic domain of Band 3 is not necessary for its anion flux, but is essential for the regulation (e.g., by Se) of its active site located at the membrane domain, and hence, it may provide evidence of communication between the cytoplasmic domain and the membrane domain of Band 3.

Efficient Insertion of Odd-numbered Transmembrane Segments of the Tetracycline Resistance Protein Requires Even-numbered Segments

Functional membrane insertion elements in the pBR322 tetracycline resistance protein were identified by comparing the ability of odd-numbered transmembrane segments and their attached periplasmic loops to insert into the membrane individually or when combined with the next even-numbered segment in the tetracycline resistance protein sequence. The efficiency with which individual odd-numbered segments and periplasmic loops inserted was probed by treating proteins truncated at the distal ends of periplasmic loops P2-P6 with carboxypeptidases and endoproteases in inside-out membrane vesicles. Insertion of odd-numbered segments and attached loops is inefficient when they occupy a C-terminal position in the protein. The C-terminal odd-numbered segment and loop sequences of 34-54% of the molecules of periplasmic loop truncation mutants could be removed by carboxypeptidase Y. In contrast, odd-numbered segments and loops insert efficiently if the next even-numbered segment in the sequence is present. In such cytoplasmic loop truncation mutants, only the cytoplasmic tail sequences of the proteins could be removed by carboxypeptidases. Remarkably, insertion of individual odd-numbered segments and loops is inefficient even though free energies for insertion of these sequences are highly favorable. The results indicate that pairs of adjacent segments, possibly "helical hairpins," are necessary for efficient membrane insertion of the tetracycline resistance protein.

Reconstitution of ATP-dependent Leukotriene C4 Transport by Co-expression of Both Half-molecules of Human Multidrug Resistance Protein in Insect Cells

Multidrug resistance protein (MRP) confers a multidrug resistance phenotype similar to that associated with overexpression of P-glycoprotein. Unlike P-glycoprotein, MRP has also been shown to be a primary active ATP-dependent transporter of conjugated organic anions. The mechanism(s) by which MRP transports these compounds and increases resistance to natural product drugs is unknown. To facilitate studies on the structure and function of MRP, we have determined whether a baculovirus expression system can be used to produce active protein. Full-length MRP as well as molecules corresponding to either the NH2- or COOH-proximal halves of the protein were expressed individually and in combination in Spodoptera frugiperda Sf21 cells. High levels of intact and half-length proteins were detected in membrane vesicles from infected cells. Although underglycosylated, the full-length protein transported leukotriene C4 (LTC4) with kinetic parameters very similar to those of MRP produced in transfected HeLa cells. Neither half-molecule was able to transport LTC4. However, a functional transporter with characteristics similar to those of intact protein could be reconstituted when both half-molecules were co-expressed. Transport of LTC4 by Sf21 membrane vesicles containing either intact or reconstituted MRP was competitively inhibited by both S-decylglutathione and 17beta-estradiol 17-(beta-D-glucuronide), with Ki values similar to those reported previously for MRP expressed in HeLa cells (Loe, D. W., Almquist, K. C., Deeley, R. G., and Cole, S. P. C. (1996) J. Biol. Chem. 271, 9675-9682; Loe, D. W., Almquist, K. C., Cole, S. P. C., and Deeley, R. G. (1996) J. Biol. Chem. 271, 9683-9689). These studies demonstrate that human MRP produced in insect cells can function as an active transporter of LTC4 and that the NH2- and COOH-proximal halves of the protein can assemble efficiently to form a transporter with functional characteristics similar to those of the intact protein.

Bacterial heavy metal resistance: new surprises.

Bacterial plasmids encode resistance systems for toxic metal ions including Ag+, AsO2-, AsO4(3-), Cd2+, CO2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, Sb3+, TeO3(2-), Tl+, and Zn2+. In addition to understanding of the molecular genetics and environmental roles of these resistances, studies during the last few years have provided surprises and new biochemical mechanisms. Chromosomal determinants of toxic metal resistances are known, and the distinction between plasmid resistances and those from chromosomal genes has blurred, because for some metals (notably mercury and arsenic), the plasmid and chromosomal determinants are basically the same. Other systems, such as copper transport ATPases and metallothionein cation-binding proteins, are only known from chromosomal genes. The largest group of metal resistance systems function by energy-dependent efflux of toxic ions. Some of the efflux systems are ATPases and others are chemiosmotic cation/proton antiporters. The CadA cadmium resistance ATPase of gram-positive bacteria and the CopB copper efflux system of Enterococcus hirae are homologous to P-type ATPases of animals and plants. The CadA ATPase protein has been labeled with 32P from gamma-32P-ATP and drives ATP-dependent Cd2+ uptake by inside-out membrane vesicles. Recently isolated genes defective in the human hereditary diseases of copper metabolism, Menkes syndrome and Wilson's disease, encode P-type ATPases that are more similar to the bacterial CadA and CopB ATPases than to eukaryote ATPases that pump different cations. The arsenic resistance efflux system transports arsenite, using alternatively either a two-component (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 three-component Czc (Cd2+, Zn2+, and CO2+) chemiosmotic efflux pump of soil microbes 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. Finally, the first bacterial metallothionein (which by definition is a small protein that binds metal cations by means of numerous cysteine thiolates) has been characterized in cyanobacteria.

Evidence for a selective and electroneutral K+/H+-exchange in Saccharomyces cerevisiae using plasma membrane vesicles

The existence of a K+/H+ transport system in plasma membrane vesicles from Saccharomyces cerevisiae is demonstrated using fluorimetric monitoring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purification/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside dilution/gel filtration procedure. This method produces a high percentage of tightly-sealed inside-out plasma membrane vesicles. In these vesicles, the K+/H+ transport system, which is able to catalyse both K+ influx and efflux, is mainly driven by the K+ transmembrane gradient and can function even if the plasma membrane H+-ATPase is not active. Using the anionic oxonol VI and the cationic DISC2(5) probes, it was shown that a membrane potential is not created during K+ fluxes. Such a dye response argues for the presence of a K+/H+ exchange system in S. cerevisiae plasma membrane and established the non-electrogenic character of the transport. The maximal rate of exchange is obtained at pH 6·8. This reversible transport system presents a high selectivity for K+ among other monovalent cations and a higher affinity for the K+ influx into the vesicles (exit from cells). The possible role of this K+/H+ exchange system in regulation of internal potassium concentration in S. cerevisiae is discussed.

Energetics and Mechanism of Drug Transport Mediated by the Lactococcal Multidrug Transporter LmrP

The gene encoding the secondary multidrug transporter LmrP of Lactococcus lactis was heterologously expressed in Escherichia coli. The energetics and mechanism of drug extrusion mediated by LmrP were studied in membrane vesicles of E. coli. LmrP-mediated extrusion of tetraphenyl phosphonium (TPP+) from right-side-out membrane vesicles and uptake of the fluorescent membrane probe 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) into inside-out membrane vesicles are driven by the membrane potential (Deltapsi) and the transmembrane proton gradient (DeltapH), pointing to an electrogenic drug/proton antiport mechanism. Ethidium bromide, a substrate for LmrP, inhibited the LmrP-mediated TPP+ extrusion from right-side-out membrane vesicles, showing that LmrP is capable of transporting structurally unrelated drugs. Kinetic analysis of LmrP-mediated TMA-DPH transport revealed a direct relation between the transport rate and the amount of TMA-DPH associated with the cytoplasmic leaflet of the lipid bilayer. This observation indicates that drugs are extruded from the inner leaflet of the cytoplasmic membrane into the external medium. This is the first report that shows that drug extrusion by a secondary multidrug resistance (MDR) transporter occurs by a "hydrophobic vacuum cleaner" mechanism in a similar way as was proposed for the primary lactococcal MDR transporter, LmrA.

Isolated beta-globin chains reproduce, in normal red cell membranes, the defective binding of spectrin to alpha-thalassaemic membranes.

Alpha-thalassaemic erythrocytes develop a specific membrane skeletal defect that is manifest as a loss of normal spectrin-binding sites on the inner surface of the thalassaemic membranes. To test whether this lesion could be caused by the excess free beta-globin chains that accumulate in alpha-thalassaemic red cells, we incubated normal red cell membranes with native, haem-containing alpha or beta globin chains or with haemoglobin A. Spectrin-depleted inside-out membrane vesicles (IOVs) derived from membranes incubated with beta-globin chains bound only 9 +/- 3% as much spectrin as IOVs from control membranes incubated with bovine serum albumin. In contrast. IOVs from membranes incubated with alpha-globin chains or haemoglobin A were nearly normal (79 +/- 3% and 86 +/- 5% of controls, respectively). This differential effect of globin chains was not seen when membranes were first transformed into spectrin-depleted IOVs and then incubated with the isolated globin chains. Under these conditions, both alpha and beta globin chains reduced the spectrin-binding capacity of the IOVs by approximately 45% (alpha 46 +/- 7%, beta 43 +/- 6%) whereas haemoglobin A had no effect. Unlike IOVs, spectrin isolated from membranes exposed to alpha or beta globin chains bound normally to IOVs and to actin (in the presence of protein 4.1). These studies show that isolated beta-globin chains (but not alpha-globin chains) can produce a spectrin-binding defect in normal red cell membranes similar to that seen in alpha thalassaemia. The existence of similar defects in the membrane skeletons of red cells from other diseases with unstable beta globins suggests a common pathophysiology for the premature destruction of these cells.

Transmembrane topology of Escherichia coli H +-ATPase (ATP synthase) subunit a

Escherichia coli H +-ATPase subunit a is a hydrophobic F 0 subunit. To investigate the topology of the subunit in the membrane, we prepared site-specific polyclonal antibodies against amino-terminal (Ser-3 to Leu-16), middle loop (Lys-167 to Gln-181), and carboxyl-terminal (Thr-259 to His-271) peptide segments. Enzyme-linked immunosorbent assay revealed that these antibodies specifically reacted with subunit a of inside-out membrane vesicles, but not with that of right-side-out spheroplasts. Full reactivity appeared when spheroplasts were disrupted with Triton X-100 (0.5%) or by sonication. These results suggest that at least parts of the three peptide segments of subunit a face the cytoplasm. Based on these observations, we propose a novel transmembrane topology of subunit a.

ATP-dependent uptake of natural product cytotoxic drugs by membrane vesicles establishes MRP as a broad specificity transporter.

MRP is a recently isolated ATP-binding cassette family transporter. We previously reported transfection studies that established that MRP confers multidrug resistance [Kruh, G. D., Chan, A., Myers, K., Gaughan, K., Miki, T. & Aaronson, S. A. (1994) Cancer Res. 54, 1649-1652] and that expression of MRP is associated with enhanced cellular efflux of lipophilic cytotoxic agents [Breuninger, L. M., Paul, S., Gaughan, K., Miki, T., Chan, A., Aaronson, S. A. & Kruh, G. D. (1995) Cancer Res. 55, 5342-5347]. To examine the biochemical mechanism by which MRP confers multidrug resistance, drug uptake experiments were performed using inside-out membrane vesicles prepared from NIH 3T3 cells transfected with an MRP expression vector. ATP-dependent transport was observed for several lipophilic cytotoxic agents including daunorubicin, etoposide, and vincristine, as well as for the glutathione conjugate leukotriene C4 (LTC4). However, only marginally increased uptake was observed for vinblastine and Taxol. Drug uptake was osmotically sensitive and saturable with regard to substrate concentration, with Km values of 6.3 microM, 4.4 microM, 4.2 microM, 35 nM, and 38 microM, for daunorubicin, etoposide, vincristine, LTC4, and ATP, respectively. The broad substrate specificity of MRP was confirmed by the observation that daunorubicin transport was competitively inhibited by reduced and oxidized glutathione, the glutathione conjugates S-(p-azidophenacyl)-glutathione (APA-SG) and S-(2,4-dinitrophenyl)glutathione (DNP-SG), arsenate, and the LTD4 antagonist MK571. This study establishes that MRP pumps unaltered lipophilic cytotoxic drugs, and suggests that this activity is an important mechanism by which the transporter confers multidrug resistance. The present study also indicates that the substrate specificity of MRP is overlapping but distinct from that of P-glycoprotein, and includes both the neutral or mildly cationic natural product cytotoxic drugs and the anionic products of glutathione conjugation. The widespread expression of MRP in tissues, combined with its ability to transport both lipophilic xenobiotics and the products of phase II detoxification, indicates that the transporter represents a widespread and remarkably versatile cellular defense mechanism.

Sodium-coupled energy transduction in the newly isolated thermoalkaliphilic strain LBS3.

Strain LBS3 is a novel anaerobic thermoalkaliphilic bacterium that grows optimally at pH 9.5 and 50 degrees C. Since a high concentration of Na+ ions is required for growth, we have analyzed the primary bioenergetic mechanism of energy transduction in this organism. For this purpose, a method was devised for the isolation of right-side-out membrane vesicles that are functional for the energy-dependent uptake of solutes. A strict requirement for Na+ was observed for the uptake of several amino acids, and in the case of L-leucine, it was concluded that amino acid uptake occurs in symport with Na+ ions. Further characterization of the leucine transport system revealed that its pH and temperature optima closely match the conditions that support the growth of strain LBS3. The ATPase activity associated with inside-out membrane vesicles was found to be stimulated by both Na+ and Li+ ions. These data suggest that the primary mechanism of energy transduction in the anaerobic thermoalkaliphilic strain LBS3 is dependent on sodium cycling. The implications of this finding for the mechanism of intracellular pH regulation are discussed.

Detection of plasma membrane Ca2+-ATPase activity in mouse T lymphocytes by flow cytometry using fluo-3-loaded vesicles

The plasma membrane Ca(2+)-ATPase (PMCA) is the primary means by which many cell types pump calcium out of the cytosol following release of calcium from internal stores, returning intracellular calcium concentrations to normal levels. Traditional methods for measuring PMCA activity utilizing isotopic calcium uptake into inside-out (IO) membrane vesicles have poor specificity for PMCA activity and require large numbers of cells. A flow cytometric method has been devised that allows the measurement of calcium uptake in IO vesicles using the fluorescent calcium chelator fluo-3. IO vesicles from mouse lymphocytes were loaded with fluo-3 pentapotassium salt and analyzed by flow cytometry following treatment with buffered calcium and/or ATP. IO vesicles appeared as a subpopulation of low forward-scatter/low side-scatter events, which were distinguishable from higher side-scatter debris. Treatment of vesicles with calcium and ATP resulted in a 5-fold to 30-fold increase in IO vesicle fluo-3 fluorescence. Measurement of uptake kinetics gave K0.5 values of approximately 0.2-0.8 microM and 2 mM for calcium- and ATP-stimulated PMCA activity, respectively, which were consistent with published values obtained by other methods. Broad specificity P-type ATPase inhibitors and more narrowly specific PMCA and calmodulin inhibitors all blocked calcium uptake, whereas thapsigargin (an endoplasmic/sarcoplasmic reticulum (ER/SR-AT-Pase) inhibitor) had no effect, indicating that the assay provides a specific measure of vesicular PMCA activity. Flow cytometric analysis, therefore, may represent a useful approach for quantifying PMCA activity in mammalian cells.

Isolation of InsP4 and InsP6 binding proteins from human platelets: InsP4 promotes Ca2+ efflux from inside-out plasma membrane vesicles containing 104 kDa GAP1IP4BP protein

A low-density membrane fraction from human platelets contained the plasma membrane marker glycoprotein Ib (GpIb) and selective binding sites for InsP4 and InsP6. It was separated from the bulk of InsP3-receptor-containing membranes, but was heterogeneous, probably also containing surface-connected canalicular system and some lighter elements of the internal dense tubule system. After loading with calcium oxalate and re-centrifugation on Percoll gradients, this mixed fraction was subfractionated into light membranes containing all of the GpIb, high-affinity InsP4 binding sites (KD = 18 nM) and phosphate-stimulated Ca2+ transport activity. InsP4 (EC50 0.6 microM), but not InsP3 or InsP6, released up to 35% of the accumulated Ca2+ from these vesicles, which were shown to be inside-out plasma membrane vesicles by a biotinylation labelling technique and selective removal of right-side-out plasma membrane vesicles with streptavidin-agarose. Most of the InsP4, and all of the InsP6, binding was present in the much denser calcium oxalate-loaded subfractions, which were free of GpIb. InsP6 binding activity was chromatographically purified as a 116 kDa protein (KD for InsP6 = 5.9 nM), with an amino acid content and two internal peptide sequences identical to those of 116 kDa vinculin. A 104 kDa InsP4 binding protein (KD for InsP4 = 12 nM), probably identical to GAP1IP4BP described by Cullen, Hsuan, Truong, Letcher, Jackson, Dawson and Irvine [(1995) Nature (London) 376, 527-530], was also isolated. This InsP4 receptor may mediate Ca2+ influx in platelets that occurs subsequent to receptor-stimulated production of InsP3 and unloading of internal Ca2+ stores.

Isolation by preparative free-flow electrophoresis and aqueous two-phase partition from rat adipocytes of an insulin-responsive small vesicle fraction with glucose transport activity

Preparative free-flow electrophoresis and aqueous two-phase polymer partition were used to obtain a plasma membrane-enriched fraction of adipocytes isolated from epididymal fat pads of the rat together with a fraction enriched in small vesicles with plasma membrane characteristics (thick membranes, clear dark-light-dark pattern). The electrophoretic mobility of the small vesicles was much less than that of the plasma membrane consistent with an inside-out orientation whereby charged molecules normally directed to the cell surface were on the inside. When plasma membranes and the small vesicle fraction were isolated from fat cells treated or not treated with 100 μU/ml insulin and the resident proteins of the two fractions analyzed by SDS-PAGE, the two fractions exhibited characteristics responses involving specific protein bands. Insulin treatment for 2 min resulted in the loss of a 90 kDa band from the plasma membrane. At the same time, a ca. 55-kDa peptide band that was enhanced in the plasma membrane was lost from the small vesicle fraction. The latter corresponded on Western blots to the GLUT-4 glucose transporter. Thus, we suggest that the small vesicle fraction with characteristics of inside-out plasma membrane vesicles may represent the internal vesicular pool of plasma membrane subject to modulation by treatment of adipocytes with insulin.

Purification and Characterization of the Ca2+-ATPase of Flavobacterium odoratum

The P-type Ca2+-ATPase from Flavobacterium odoratum has been purified to homogeneity and characterized. Inside-out membrane vesicles were extracted with C12E8, followed by ammonium sulfate fractionation, centrifugation through two successive 32-48% glycerol gradients, and DE52 ion exchange chromatography. The purified Ca2+-ATPase consists of a single polypeptide. It migrates electrophoretically with an apparent molecular mass of 60,000 Da, consistent with the phosphorylation pattern originally reported in membrane vesicles. This single polypeptide is functional and capable of calcium-dependent vanadate-sensitive ATP hydrolysis and of forward and reverse phosphorylation. Maximum hydrolysis activity occurs at pH 8.0, with a specific activity of approximately 75 micromol of ATP hydrolyzed min-1 mg-1 protein. The purified Ca2+-ATPase has an apparent Km for calcium of 1.5 microM and for ATP of 90 microM. Vanadate strongly inhibits the activity with an IC50 of 0.6 microM. The prokaryotic Ca2+-ATPase is insensitive to the SR Ca2+-ATPase inhibitors fluorescein isothiocyanate, thapsigargin, and cyclopiazonic acid. It is rapidly phosphorylated by [gamma-32P]ATP in a calcium-dependent vanadate-inhibited manner and can be phosphorylated by Pi in both the presence and absence of calcium.