Association of a cellular myosin II with anionic phospholipids and the neuronal plasma membrane

Myosin II has been observed in close proximity to the neuronal plasma membrane, suggesting the possibility that at least one isoform of neuronal myosin II may be capable of direct association. Here, we demonstrate that a significant fraction (> 30%, saturable around 90%) of brain myosin II, but not myosins from skeletal or cardiac muscle, can bind to lipid vesicles composed of the anionic phospholipid L-alpha-phosphatidyl-L-serine but not with vesicles made from the neutral phospholipid L-alpha-phosphatidylcholine. Binding to lipid vesicles made from L-alpha-phosphatidyl-L-serine is enhanced in the presence of millimolar amounts of free calcium. ATPase activity remains unimpaired after vesicle association. Myosin II was also shown to remain in tight association with purified plasma membranes, even after depletion of actin. The above observations suggest that mechanisms involving membrane-bound myosin II are required to facilitate metazoan cell motility.     

Binding of insulin analogs to partially purified insulin receptor from rat liver membrane (author's transl)

The insulin binding fraction was solubilized from rat liver membrane vesicles by triton and partially purified up to the specific binding activity of 2.1 pmol/mg. A further characteristic of the partially purified soluble receptor is a decrease in irreversible binding to 0.36% (+/- 0.28) with regard to total iodine insulin and to (+/- 1.8) in comparison to reversible binding. From Scatchard plots a high affinity binding site (KD = 5 X 10(-10)M) with low capacity (5 pmol/mg) and a low affinity binding site (KD = 3 X 10(-8) M) with high capacity (30 pmol/mg) can be seen. With carefully prepared liver membrane vesicles, the dissociation constant of the high affinity binding site from Scatchard plot is only 2 X 10(-9)M. With liver membrane vesicles, isolated for preparative purification procedure, the high affinity binding site could not be demonstrated. Displacement studies with insulin analogs were performed with [A1-D-alanine] insulin, [A1-L-alanine] insulin, [des-Gly-A1, NB1, NB29-(Msc)2]-insulin, proinsulin and [desoctapeptid B23-B30]-insulin. Results of binding measurements are presented in half-maximal iodo-insulin binding, in determination of inhibitor- and dissociation constants from Dixon-, Scatchard- and Lineweaver-Burk plots. There are equal relative binding potencies of analogs, observed with crude membrane vesicles and partially purified soluble receptor, although there is a 50-fold difference in specific binding activity. Biologically active insulins are characterized by strong binding to the high affinity binding site. The binding to the low affinity binding site is not correlated to the biological activity of the insulin analog. With insulin and biologically responsive analogs a non-linear curve in the double-reciprocal Lineweaver-Burk plot can be observed. Analogs with low biological activity show a linear dependency. Functional interactions of insulin with the receptor can be demonstrated in a high affinity binding with the first binding site of the Scatchard plot and in a non-linear hyperbolic Lineweaver-Burk plot.     

Protein-catalyzed exchange of phosphatidylcholine between sonicated liposomes and multilamellar vesicles

Phospholipid exchange protein from beef heart or beef liver does not catalyze the transfer of phosphatidylcholine from multilamellar vesicles of phosphatidylcholine. Certain combinations of phospholipids, however, do yield multilamellar vesicles that will exchange phosphatidylcholine with liposomes in the presence of exchange protein. Multilamellar vesicles of phosphatidylcholine:phosphatidylethanolamine:cardiolipin (70:25:5, mol%) can be used in place of mitochondria or erythrocyte ghosts as an improved acceptor particle in the study of liposome structure with phospholipid exchange proteins. These multilamellar vesicles act as a well-defined reservoir of unlabeled phosphatidylcholine with 7% exchangable phospholipid. When the distribution of phosphatidylcholine in liposomes is studied by the exchange protein technique, results can be influence by the choice of phospholipid acceptor particle. With mitochondria as acceptor particle, the percentage of phosphatidylcholine in the outer monolayer of a liposome appears to be 60%, whereas a value of 70% is obtained when multilamellar vesicles are the acceptor. The discrepancy can be explained by a heterogeneity in liposomes prepared by sonication. A size-dependent fusion or adsorption process occurs between liposomes and mitochondria; the very small liposomal vesicles, obtained by gel filtration, combine nearly quantitatively with the natural membrane. This phenomenon is not seen with multilamellar vesicles. Thus by using multilamellar vesicles one obtains a less biased estimate of phospholipid distribution between inner and outer layers of liposomes.     

Presence of cyclic nucleotide-Ca2+ independent protein kinase in bovine brain coated vesicles

Coated vesicles, which are membrane vesicles enclosed by a polyhedral protein lattice, are involved in many cellular events, including intracellular membrane transport and protein secretion, in which they must be able to undergo repeated membrane fusion and fission. The icosahedral lattice of protein surrounding the core of coated vesicles is composed predominantly of clathrin, a 180,000 (180 K) molecular weight protein, and other 30K and 36K polypeptides. In native conditions, the basic subunit of the coat consists of a trimer of clathrin with probably three polypeptides of 30K and/or 36K (refs 9-11). Additional minor proteins of 100K and 55K have been reported in purified coated vesicles. We describe here the presence of cyclic nucleotide- and Ca2+-independent protein kinase activity in coated vesicles. This protein kinase phosphorylates specifically a unique 50K protein which can be co-purified with clathrin and seems to be an integral protein of coated vesicles.     

The comparison of early fluid therapy in extensive flame burns between inhalation and noninhalation injuries

Dopamine hydrochloride bearing positively charged small liposomes was prepared by sonicating the multilamellar vesicles. These vesicles were characterized for their physical attributes (shape, size, charge, drug entrapment efficiency, and drug leakage). The drug release kinetics from the liposomes were also studied and found to be Fickian-type diffusion. In vivo performance of the drug-entrapped liposomes was assessed by periodic measurement of drug- (chlorpromazine) induced catatonia in Sprague-Dowley rats. These results were compared with the plain dopamine HCl and levodopa preparations as well with the marketed formulation of levodopa containing carbidopa (Syndopa). These studies revealed that the dopamine can be effectively delivered to the brain by incorporating it into liposomes, and its degradation in circulation can also be protected. The results of liposomal formulation were found to be superior compared to plain levodopa as well as Syndopa.     

Spontaneous transfer of GM3 ganglioside between vesicles

The spontaneous transfer between membranes of GM3, a ganglioside present in a vesicular form of aggregation instead of micellar form like the majority of gangliosides in aqueous medium, has been studied. Upon incubation of GM3 in the presence of dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles at 50 degrees C, mixed GM3/DPPC vesicles are formed. The maximum amount of GM3 that can be inserted into vesicles is about 8%. The temperature dependence of the kinetics has been followed by the excimer formation technique, using the fluorescent analogue pyrenyldodecanoyl-GM3. The transfer of ganglioside from its vesicles to DPPC vesicles depends on the physicochemical characteristics of both the donor and of the acceptor vesicles and increases with the temperature (k = 0.006 0.012, 0.037 at 30, 41 and 50 degrees C, respectively), with a major break point at 41 degrees C and a minor one at 35 degrees C. These temperatures correspond to the gel- to liquid-crystalline transition of DPPC (Tm = 41.3 degrees C), and to a temperature transition displayed by GM3 ganglioside. Similar experiments performed with erythrocyte ghosts yielded a rate constant of 0.04 at 37 degrees C. For the transfer of ganglioside from DPPC (donor) to DMPC (acceptor) the rate constants were 0 at 15 degrees C (both phospholipids in the gel phase), 0.005 at 37 degrees C (donor in the gel phase, acceptor in the fluid phase) and 0.04 at 50 degrees C (both phospholipids in the fluid phase).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative internalization and recycling of different amphotericin B formulations by a macrophage-like cell line

The amount of amphotericin B (AmB) associated with cultured murine macrophage-like J774 cells, after incubation with various AmB lipid formulations, was determined by absorption spectroscopy. Large, negatively charged, AmB-containing, multilamellar vesicles and small cholesteryl sulphate-AmB complexes both enhanced the amount of AmB associated with J774 cells at 37 degrees C (up to 500-fold the extracellular concentration). In contrast, AmB-containing, small, negatively charged vesicles (AmBisome), positively charged, oligolamellar vesicles and mixed micelles showed a lower association of the antibiotic with cells, compared with AmB added from a solution in dimethylsulphoxide or Fungizone. Experiments performed at 40 degrees C showed a large reduction of AmB uptake for AmB preparations and AmB added from a solution in dimethysulphoxide or Fungizone, suggesting a high percentage of internalization of the antibiotic. Experiments in the presence of cytochalasin B resulted in a decrease of AmB uptake mainly for the preparations of large diameter, suggesting that these formulations were taken up by phagocytosis. A comparative study with Chinese hamster ovary cells, a model of non-phagocytic cells, showed a reduction in the take up of AmB. This reduction was always more marked when AmB was incorporated in lipid formulations. On the other hand, accumulation of the antibiotic in J774 cells was shown to be followed by its release from the cells in an unbound form, the extent of release depending on the type of vector used. The results suggest that in some cases macrophages can be considered as reservoirs of antibiotic, releasing free AmB in the medium.     

Transmembrane Ca2+ gradient-mediated phosphatidylcholine modulating sarcoplasmic reticulum C(a2+)-ATPase

The sarcoplasmic reticulum (SR) C(a2+)-ATPase was purified and reconstituted into the sealed phospholipids vesicles with or without transmembrane Ca2+ gradient. The role of phospholipids, especially phosphatidylcholine (PC), in the modulation of C(a2+)-ATPase by transmembrane Ca2+ gradient was investigated. The results are as follows. (i) Incubated with phospholipids, the enzyme activity of the delipidated C(a2+)-ATPase is inhibited by Ca2+ and the highest inhibition is observed in the presence of PC. (ii) When there exists a transmembrane Ca2+ gradient (higher Ca2+ concentration inside vesicles, 1,000 mumol/L:50 mumol/L, similar to the physiological condition), the inhibition of C(a2+)-ATPase by transmembrane Ca2+ gradient can be only observed in the vesicles containing PC:PE, but not in those containing PS:PE or PG:PE. The highest inhibition is obtained at a 50:50 molar ratio of PC:PE (iii) By comparing the effects of PC differing in acyl chains, higher inhibition of C(a2+)-ATPase is observed in vesicles containing DPPC:PE and DOPC:PE, while no inhibition in DMPC:PE vesicles (iv) If the transmembrane Ca2+ gradient is in the inverse direction, the enzyme activity of C(a2+)-ATPase is inhibited whenever reconstituted with acidic or neutral phospholipids.     

Lipid vesicle size determines the Th1 or Th2 response to entrapped antigen

Understanding the factors that control the differential induction of Th1 and Th2 responses is a key immunologic objective with profound implications for vaccination and immunotherapy of infectious and autoimmune diseases. Using Ag formulated in lipid vesicles prepared from nonionic surfactants, we describe a novel mechanism influencing the balance of the Th1 or Th2 response. Our results indicate that inoculation of BALB/c mice with vesicles with a mean diameter > or = 225 nm preferentially induces Th1 responses, as characterized by increased titers of IgG2a in plasma and elevated IFN-gamma production by lymph node cells. However, preparation of the same quantity of Ag in vesicles with mean diameter of < or = 155 nm induces a Th2 response, as identified by IgG1 in the absence of IgG2a production and increased lymph node IL-5 production. Although large (> or = 225 nm) vesicles could induce IL-12 production, smaller vesicles (< or = 155 nm) could not. However, small vesicles did induce higher levels of IL-1beta production by macrophages than larger vesicles. The role of IL-12 in this response was confirmed in IL-12-deficient mice, whose spleen cells failed to produce IFN-gamma following in vivo priming with Ag prepared in large vesicles. Our results therefore indicate that macrophages respond to endocytosis of large or small vesicles by producing different patterns of cytokines that can subsequently direct the immune response toward a Th1 or a Th2 phenotype.     

Local anaesthetic bupivacaine alters function of sarcoplasmic reticulum and sarcolemmal vesicles from rabbit masseter muscle

The effects of local anaesthetics, bupivacaine and lidocaine, on Ca2+ flux behaviour of sarcoplasmic reticulum and on sarcolemmal functions were studied in the rabbit masseter muscle. The experiments were performed on sarcoplasmic reticulum and sarcolemmal vesicles prepared at 1 to 10 days after injection of local anaesthetics or saline into masseter muscle as well as on sarcoplasmic reticulum vesicles prepared from non-treated rabbits (for assessment of the effect on in vitro incubation with local anaesthetics). Bupivacaine potently reduced the efficiency of active sarcoplasmic reticulum Ca2+ transport as evaluated by coupling ratio (Ca2+ transported/ATP hydrolyzed, in the presence of oxalate) at 3 days after the injection; there was only a slight degree of uncoupling of Ca2+ transport from ATP hydrolysis with lidocaine injection. Bupivacaine but not lidocaine, at 3 days after injection, decreased both the apparent permeability of sarcoplasmic reticulum vesicles to Ca2+, determined by measuring net efflux of Ca2+ after stopping pump-mediated fluxes, and the steady-state Ca2+ load in sarcoplasmic reticulum, but had no effect on overall turnover of the Ca2+ATPase. The effects of bupivacaine on apparent sarcoplasmic reticulum Ca2+ permeability and steady-state Ca2+ load were inhibited by a Ca2+ antagonist verapamil. The reduction of Ca2+ uptake of sarcoplasmic reticulum and the protective effect of verapamil were reproduced in unfractionated homogenates prepared at 3 days after bupivacaine injection. In vitro exposure of sarcoplasmic reticulum vesicles to bupivacaine (0.5 to 50 mM) reduced steady-state Ca2+ load in a dose-dependent manner. The observed effect elicited by bupivacaine (25 mM) was partially protected by procaine, an inhibitor of Ca2(+)-induced Ca2+ release from sarcoplasmic reticulum, or by specific closure of the sarcoplasmic reticulum Ca2+ release channel by ryanodine, suggesting the possibility that in vitro exposure of sarcoplasmic reticulum vesicles to bupivacaine may produce an increase in apparent permeability of sarcoplasmic reticulum to Ca2+. In sarcolemma, bupivacaine reduced Na+,K(+)-ATPase and Na(+)-Ca2+ exchange activities at 3 days after injection; the effects on sarcolemmal vesicles were prevented by verapamil. These results suggest that although the effects elicited by bupivacaine injection and the in vitro exposure to bupivacaine on steady-state Ca2+ load of sarcoplasmic reticulum vesicles were similar, the membrane properties of the vesicles from bupivacaine-treated masseter muscles and those from normal untreated muscles may not be the same, which indicates that pure bupivacaine effect is due partly by an effect on ryanodine- and procaine-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cholesteryl ester transfer from phospholipid vesicles to human density lipoproteins

The exchange of cholesteryl esters between different lipoproteins was reported to bae mediated by a protein present in human plasma. In this study wer have examined the movement of cholesteryl ester from unilamellar phospholipid vesicles to high density lipoprotein (HDL). Experimental conditions were establisehd so that vesicles containing egg yolk lecithin and cholesteryl oleatea (molar ratio of 86:1) could be incubated with human HDL so that neaither disruption of particles nor transfer of lipid occurred. Addition of human lipoprotein-deficient plasma to the system promoted the transfer of cholesteryl oleate, but not phospholipid, from vesicles to HDL. Cholesteryl oleate transfer was dependent upon amount of HDL or lipoproteain-deficient plasma added and occurred when either HDL2 or HDL3 were present. Addition of unesterified cholesterol to the vesicles did not influence lcholesteryl ester transfer to HDL. When phospholipid vesicles containing both cholesteryl oleate and triolein (molar ratio 86:1:1) were incubated with HDL and lipoprotein-deficient plasma, only cholesteryl oleate was transferred from the vesicles to HDL. Lipoprotein-deficient plasma derived from rabbits promoted the selective transfer of cholesteryl oleate from these visicles, but rat plasma did not cause any movement of cholesteryl oleate or triolein from vesicles to HDL. HDL containing labeled cholesteryl esters was prepared and incubated with vesicles containing unlabeled cholesteryl esters or phospholipid alone. Addition of lipoprotein-deficient plasma did not promote transfer of cholesteryl esters from HDL to vesicles, whereas transfer from HDL to low density lipoprotein was readily observed. The results indicated that a protein present in rabbit and human plasma is effective in the selective, unidirectional transport of cholesteryl esters from a phospholipid bilayer to a plasma lipoprotein.     

Anatomo-clinical conference: bronchiolo-alveolar cancer and confusion

Rapid membrane recycling in nerve terminals is required to maintain rapid synaptic transmission. Following the fusion of synaptic vesicles with synaptic plasma membranes, recycling can occur via clathrin-coated vesicles (CCVs) [1-3]. The fate of these vesicles is uncertain: they could simply uncoat and acquire other proteins from the cytosol to regenerate synaptic vesicles or they may fuse with endosomal structures from which synaptic vesicles could then bud. We have purified both CCVs and synaptic vesicles from rat brain, and measured the ability of these vesicle fractions to take up the excitatory neurotransmitter glutamic acid. We found that the normalized levels of glutamate uptake by the two types of vesicle were very similar. For each vesicle fraction, uptake required ATP and Cl- and could be fully inhibited by the specific vacuolar proton pump (v-ATPase) inhibitor concanamycin. We suggest that this ability to refill vesicles with neurotransmitter at the earliest intermediate on the recycling pathway - the CCV - may allow uncoated vesicles to immediately enter the releasable pool without sacrificing the quantal nature of neurotransmitter release.     

Insulin-mediated targeting of phosphatidylinositol 3-kinase to GLUT4-containing vesicles

Phosphatidylinositol (PI) 3-kinase is hypothesized to be a signaling element in the acute redistribution of intracellular GLUT4 glucose transporters to the plasma membrane in response to insulin. However, some receptors activate PI 3-kinase without causing GLUT4 translocation, suggesting specific cellular localization may be critical to this PI 3-kinase function. Consistent with this idea, complexes containing PI 3-kinase bound to insulin receptor substrate 1 (IRS-1) in 3T3-L1 adipocytes are associated with intracellular membranes (Heller-Harrison, R., Morin, M. and Czech, M. (1995) J. Biol. Chem. 270, 24442-24450). We report here that in response to insulin, activated complexes of IRS-1.PI 3-kinase can be immunoprecipitated with anti-IRS-1 antibody from detergent extracts of immunoadsorbed GLUT4-containing vesicles prepared from 3T3-L1 adipocytes. The targeting of PI 3-kinase to rat adipocyte GLUT4-containing vesicles using vesicles prepared by sucrose velocity gradient ultracentrifugation was also demonstrated. Insulin treatment caused a 2.3-fold increase in immunoreactive p85 protein in these GLUT4-containing vesicles while anti-p85 immunoprecipitates of PI 3-kinase activity in GLUT4-containing vesicle extracts increased to a similar extent. HPLC analysis of the GLUT4 vesicle-associated PI 3-kinase activity showed insulin-mediated increases in PI 3-P, PI 3,4-P2, and PI 3,4,5-P3 when PI, PI 4-P, and PI 4,5-P2 were used as substrates. Our data demonstrate that insulin directs the association of PI 3-kinase with GLUT4-containing vesicles in 3T3-L1 and rat adipocytes, consistent with the hypothesis that PI 3-kinase is involved in the insulin-regulated movement of GLUT4 to the plasma membrane.     

Packing constraints and electrostatic surface potentials determine transmembrane asymmetry of phosphatidylethanol

The energetic determinants of the distribution of anionic phospholipids across a phosphatidylcholine (PtdCho) bilayer with different packing constraints in the two leaflets were studied, using (13)CH2-ethyl-labeled phosphatidylethanol (PtdEth) as a (13)C NMR membrane probe. PtdEth is unique in exhibiting a split (13)CH2-ethyl resonance in sonicated vesicles, the two components originating from the inner and outer leaflets, thus permitting the determination of the PtdEth concentration in each leaflet. Small and large unilamellar PtdEth-PtdCho vesicles were prepared in solutions of different ionic strengths. A quantitative expression for the transbilayer distribution of PtdEth, based on the balance between steric and electrostatic factors, was derived. The transbilayer difference in packing constraints was obtained from the magnitude of the PtdEth signal splitting. The electrostatic contribution could be satisfactorily described by the transmembrane difference in Gouy-Chapman surface potentials. At low (0.1-0.25%) PtdEth levels and high (up to 500 mM) salt concentrations, PtdEth had a marked fivefold preference for the inner leaflet, presumably because of its small headgroup, which favors tighter packing. At higher PtdEth content (4.8-9.1%) and low salt concentrations, where electrostatic repulsion becomes a dominant factor, the asymmetry was markedly reduced and an almost even distribution across the bilayer was obtained. In less curved, large vesicles, where packing constraints in the two leaflets are approximately the same, the PtdEth distribution was almost symmetrical. This study is the first quantitative analysis of the balance between steric and electrostatic factors that determines the equilibrium transbilayer distribution of charged membrane constituents.     

Developmental changes in synaptic formation in the hypothalamic arcuate nucleus of female rats

The hypothalamic arcuate nucleus (ARCN) of female rats at 5, 20, 45 and 90 days of age was examined ultrastructurally. Axodendritic and axosomatic synapses were counted in 18,000 mum2 area of the ARCN in each brain. Axodendritic and axosomatic synapses in the ARCN of day 5 rats were very small in number. Axon terminals contained small spherical vesicles (SSVs, 40-60 nm in diameter). Occasionally large granular vesicles (LGVs, 75-130 nm in diameter) were found to coexist with SSVs in the endings. Pre- and postsynaptic membranes were thin. The ARCN at this age exhibited a large extracellular space which decreased with advancing age. In day 20 rats, axodendritic and axosomatic synapses increased in number up to about one-half of those of day 45 or day 90 animals. Synaptic vesicles increased in number and mitochondria were frequently encountered in the axon terminals. Pre- and postsynaptic membranes became thicker than those of day 5 rats. Further increase in the number of axodendritic and axosomatic synapses in the ARCN of day 45 rats was observed, and there were no significant difference in the morphology and incidence of synapses between day 45 and day 90 rats. Synaptic vesicles were numerous and pre- and postsynaptic membranes were thick. In tissue incubated with 5-hydroxydopamine (5-OH-DA) before fixation, small granular vesicles (SGVs, about 50 nm in diameter) which were labeled with 5-OH-DA were detected in a certain number of endings in all material taken from each age group, but the incidence of synapses containing SGVs was usually low. From these results, it can be proposed that an increase in the number of synapses in the ARCN is correlated wihh functional maturation of the ARC neurons.     

Altered visual field asymmetry for lexical decision as a result of concurrent presentation of music fragments of different emotional valences

OBJECTIVES: To investigate the effect of membrane lipid composition on the susceptibility to bile salt damage and protection. DESIGN: Artificial model cholesterol/phospholipid (c/p) membranes (vesicles) with a varying cholesterol (0-15 mM) and phospholipid content (3-30 mM), and with a c/p ratio ranging up to 1.70, were prepared by sonication. We examined the effect of incubation with increasing concentrations of either tauroursodeoxycholate (TUDC), taurocholate (TC) or taurodeoxycholate (TDC) alone, or with proportionally varying mixtures of TUDC and TDC. METHOD: Vesicle integrity was assessed by the change in optical absorbance at 340 nm. RESULTS: Absorption of the bile salt-vesicle mixture decreased, with increasing bile salt concentration and hydrophobicity: TUDC less than TC less than TDC. Moreover, bile salt-induced damage also depended on membrane composition: vesicles containing more than 5 mM cholesterol and with a c/p ratio greater than 0.8 were less likely to be solubilized by 30 mM bile salt. Similarly, only in cholesterol-rich vesicles (c/p > 0.5) was a protective effect of TUDC against membrane disruption by TDC revealed upon incubation with various TUDC/TDC mixtures. CONCLUSION: Apart from the bile salt concentration and hydrophobicity, the cholesterol content of vesicles is pivotal, both in the bile salt-induced solubilization of cholesterol/phospholipid vesicles and in the potency of TUDC to prevent this.     

Separation and characterization of Na+,K(+)-ATPase containing vesicles

Na+,K(+)-ATPase was reconstituted in vesicles prepared by a dialysis method. Ion-exchange chromatography was used to obtain well characterized fractions from the inhomogeneous vesicle preparation. Lipid and protein content was determined by optical methods during the elution process. It was possible to separate fractions with distinct enzymatic and transport activities. A protocol was set up, which allowed to calculate the average number of 5-IAF labeled ion pumps per vesicle in the different fractions. The dependence of the number of protein molecules per vesicle was studied as function of the initial protein concentration added to the lipid solution before dialysis. The transport activity disappears completely at very low protein concentrations (3.3 micrograms protein per mg lipid). This observation is in favor of the proposal discussed in the literature, that the heterodimer (alpha beta)2 is the transport-active form of the Na+,K(+)-ATPase. The presented method can be applied to all reconstituted vesicle preparations in which the proteins can be labeled quantitatively with a fluorescence dye.     

Transmembrane movement of phosphatidylcholine in mitochondrial outer membrane vesicles

One of the steps in the import of phosphatidylcholine (PC) in mitochondria is transmembrane movement across the outer membrane. This process was investigated in vitro using isolated mitochondrial outer membrane vesicles (OMV) from rat liver. 14C-Labeled PC was introduced into the OMV from small unilamellar vesicles by a PC-specific transfer protein (PCTP). The membrane topology of the newly introduced PC was determined from its accessibility to phospholipase A2. Under conditions where the OMV stay intact, externally added phospholipase A2 is able to hydrolyze up to 50% of both the introduced [14C]PC and the endogenous PC. Pool size calculations showed that close to 100% of the PC in the OMV can be exchanged by PCTP. A back-exchange experiment revealed that the introduction of the labeled PC is reversible. The results demonstrate that newly introduced PC molecules readily equilibrate over both leaflets of the OMV membrane. The kinetics of the PCTP-mediated exchange process indicate that the t1/2 of the transmembrane movement at 30 degrees C is 2 min or less.     

Hydration and stability of sulfatide-containing phosphatidylethanolamine small unilamellar vesicles

The effect of sulfatide on membrane hydration of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) small unilamellar vesicles (SUVs) was investigated using steady-state and time-resolved fluorescence spectroscopy. The degree of hydration in the headgroup region of the bilayer lipids was assessed with the fluorescence lifetime of N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan olamine along with the ratio of its fluorescence intensities measured in samples prepared either in D2O- or in H2O-based buffers. Similarly, hydration of acyl chains near the headgroup region and that close to the bilayer center were studied using 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene and 1-palmitoyl-2-[2-[4-(6-phenyl-trans-1,3, 5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine as probes. Increasing sulfatide concentration up to 30 mol% resulted in an increase in surface hydration and a decrease in interchain hydration. These were correlated with an increase in bilayer stability of the DOPE/sulfatide SUVs. Moreover, variation of pH was found to affect the hydration and stability of the bilayer vesicles. No further change in headgroup hydration and interchain hydration near the bilayer center was observed at sulfatide concentrations >/=30 mol%. At such high sulfatide concentrations, bilayer hydration and stability were no longer pH-sensitive. The effects of sulfatide on hydration and stability of DOPE bilayer vesicles are discussed by taking into account the electrostatic and geometrical properties of the sulfated galactosyl headgroups.     

Ionophore-mediated uptake of ciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmembrane ion gradients

A new method, based on the ion-translocating properties of the ionophores nigericin and A23187, is described for loading large unilamellar vesicles (LUVs) with the drugs vincristine and ciprofloxacin. LUVs composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (55:45 mol/mol) or sphingomyelin (SPM)/Chol (55:45 mol/mol) exhibiting a transmembrane salt gradient (for example, internal solution 300 mM MnSO4 or K2SO4; external solution 300 mM sucrose) are incubated in the presence of drug and, for experiments involving divalent cations, the chelator EDTA. The addition of ionophore couples the outward movement of the entrapped cation to the inward movement of protons, thus acidifying the vesicle interior. External drugs that are weak bases can be taken up in response to this induced transmembrane pH gradient. It is shown that both nigericin and A23187 facilitate the rapid uptake of vincristine and ciprofloxacin, with entrapment levels approaching 100% and excellent retention in vitro. Following drug loading, the ionophores can be removed by gel exclusion chromatography, dialysis, or treatment with biobeads. In vitro leakage assays (addition of 50% mouse serum) and in vivo pharmacokinetic studies (in mice) reveal that the A23187/Mn2+ system exhibits superior drug retention over the nigericin/K+ system, and compares favorably with vesicles loaded by the standard DeltapH or amine methods. The unique features of this methodology and possible benefits are discussed.