Structural changes in vesicle membranes and mixed micelles of various lipid compositions after binding of different bile salts

Binding equilibria of common bile salts (BS) and different mixtures of membrane lipids were correlated with BS-induced structural changes of large unilamellar vesicles, with transition of vesicles to mixed micelles (MM), and with successive transformations of MM. At very low BS concentrations, in the outer vesicle monolayer definite BS/lipid aggregates are formed, the size and BS binding strength of which depend on the BS and lipid species involved. At increasing BS concentrations, binding to the membranes is hampered, and above a critical BS content, membrane stress due to asymmetric BS binding leads to formation of transient membrane holes, as shown by inulin release from the vesicles. Independent of the BS and lipid species, membrane solubilization starts at a ratio r = 0.3 of bound BS/lipid. Increasing phosphatidylserine, phosphatidylethanolamine, and cholesterol contents stabilize the lecithin membrane against BS to different degrees and in different ways, whereas the destabilization by sphingomyelin is probably due to the enhancement of the membrane gel-liquid transition temperature. Conjugation of the BS with glycine or taurine has a modulating effect on membrane hole formation, rather than on lipid solubilization. Diphenylhexatriene fluorescence anisotropy indicates a BS-induced drop of the internal membrane order and its restoration during membrane solubilization. At higher concentrations ursodeoxycholate induces additional condensation, whereas the other BS cause internal disorder in the MM. Above ratios r of approximately 8:1, we found a release of BS from these MM and suggest a rodlike structure for them. The results were discussed with respect to BS/membrane interactions during lipid excretion from the liver cell.     

Uptake and release of calcium by canine gastric corpus smooth muscle plasma membrane enriched fraction

Calcium movements across plasma membrane enriched vesicles isolated from canine gastric corpus smooth muscle were investigated. The ATP-dependent Ca2+ uptake increased with time up to 10 min. The uptake for the initial 2-min period was approximately linear with time. The apparent initial velocity of the ATP-dependent Ca2+ uptake increased monotonically with free Ca2+ concentration from 0.1 to 2 microM, and further increases in free Ca2+ concentration did not increase the Ca2+ uptake. The free Ca2+ dependence curve could be described with a Hill coefficient of approximately 1.0 and Km of 0.85 +/- 0.01 microM for free Ca2+ concentration. Passive Ca2+ uptake (reaction time = 1 h) also increased with increasing free Ca2+ concentrations from 0.02 to 4.0 mM. Dilution of loaded vesicles in isotonic media containing EGTA led to initial rapid loss (less than 1 min) followed by a slower release which showed simple exponential decay. The t 1/2 values of the slower Ca2+ loss from these vesicles were 16.1 +/- 0.9 min (actively loaded n = 5) and 18.4 +/- 0.9 min (passively loaded n = 3), respectively. Dilution in isotonic medium containing both EGTA and A23187 released all the sequestered Ca2+ from these loaded vesicles.     

Bile salt-induced calcium fluxes in artificial phospholipid vesicles

The ionic permeability of selected biological membranes is increased by bile salts. To examine changes in calcium permeability during the exposure of artificial membranes to bile salts, we investigated calcium uptake by unilamellar and multilamellar phospholipid vesicles. In the presence of 750 microM taurodeoxycholate, uptake of radiolabelled calcium by unilamellar vesicles increased 2.5-fold over control values. Calcium uptake by multilamellar vesicles as measured with a free calcium indicator, arsenazo III, increased 2.2- or 21-fold in the presence of 60 microM lithocholate or 3 beta-hydroxy-5-cholenoate, respectively. Results were directly influenced by experimental variables such as bile salt hydrophobicity, external calcium concentration, and the bile salt/lipid molar ratio. Observed membrane solubilization was minimal despite increased calcium permeability. Comparison of radiolabelled calcium uptake with radiolabelled sodium or radiolabelled rubidium uptake indicated that bile salt-dependent calcium uptake was 60-140-times greater than bile salt-dependent uptake of either monovalent cation. In an effort to delineate forces affecting calcium translocation, vesicles were exposed either to valinomycin, which induced an electrochemical gradient across the membrane, or to nigericin, which induced a proton gradient. Exposure to valinomycin minimally influenced bile salt-induced calcium uptake while exposure to nigericin significantly promoted uptake by 40-70%. The results suggest that bile salts promote calcium uptake by a mechanism which may be similar to those of other carboxylic ionophores.     

Energetics of vesicle fusion intermediates: comparison of calculations with observed effects of osmotic and curvature stresses

We reported previously the effects of both osmotic and curvature stress on fusion between poly(ethylene glycol)-aggregated vesicles. In this article, we analyze the energetics of fusion of vesicles of different curvature, paying particular attention to the effects of osmotic stress on small, highly curved vesicles of 26 nm diameter, composed of lipids with negative intrinsic curvature. Our calculations show that high positive curvature of the outer monolayer "charges" these vesicles with excess bending energy, which then releases during stalk expansion (increase of the stalk radius, r(s)) and thus "drives" fusion. Calculations based on the known mechanical properties of lipid assemblies suggest that the free energy of "void" formation as well as membrane-bending free energy dominate the evolution of a stalk to an extended transmembrane contact. The free-energy profile of stalk expansion (free energy versus r(s)) clearly shows the presence of two metastable intermediates (intermediate 1 at r(s) approximately 0 - 1.0 nm and intermediate 2 at r(s) approximately 2.5 - 3.0 nm). Applying osmotic gradients of +/-5 atm, when assuming a fixed trans-bilayer lipid mass distribution, did not significantly change the free-energy profile. However, inclusion in the model of an additional degree of freedom, the ability of lipids to move into and out of the "void", made the free-energy profile strongly dependent on the osmotic gradient. Vesicle expansion increased the energy barrier between intermediates by approximately 4 kT and the absolute value of the barrier by approximately 7 kT, whereas compression decreased it by nearly the same extent. Since these calculations, which are based on the stalk hypothesis, correctly predict the effects of both membrane curvature and osmotic stress, they support the stalk hypothesis for the mechanism of membrane fusion and suggest that both forms of stress alter the final stages, rather than the initial step, of the fusion process, as previously suggested.     

Cloning and physical characterization of chromosomal conjugative elements in streptococci.

A transport system for polyamines was studied with both intact cells and membrane vesicles of an Escherichia coli polyamine-deficient mutant. Polyamine uptake by intact cells and membrane vesicles was inhibited by various protonophores, and polyamines accumulated in membrane vesicles when D-lactate was added as an energy source or when a membrane potential was imposed artificially by the addition of valinomycin to K+-loaded vesicles. These results show that the uptake was dependent on proton motive force. Transported [14C]putrescine and [14C]spermidine were not excreted by intact cells upon the addition either of carbonyl cyanide m-chlorophenylhydrazone, A23187, and Ca2+ or of an excess amount of nonlabeled polyamine. However, they were excreted by membrane vesicles, although the degree of spermidine efflux was much lower than that of putrescine efflux. These results suggest that the apparent unidirectionality in intact cells has arisen from polyamine binding to nucleic acids, thus giving rise to a negligible free intracellular concentration of polyamines. Polyamine uptake, especially putrescine uptake, was inhibited strongly by monovalent cations. The Mg2+ ion inhibited spermidine and spermine uptake but not putrescine uptake.     

On the relation between rapid light-induced Ca2+ release and proton uptake in rod outer segment disk membranes

In this paper we review our experiments on the light-induced Ca2+ release and proton uptake at the rod outer segment (ROS) disk membrane using flash-spectrophotometry and the indicating dyes arsenazo III and bromcresol purple. We used three different ROS preparations in order to locate the intracellular site of Ca2+ release. The ionophore A23187 was required to communicate the Ca2+ release to the indicator located in the external medium in both ROS with an intact and with a leaky plasma membrane. A23187 was also required to observe the Ca2+ released in the interior of vesicles prepared by sonication of ROS. From this we conclude that the site of Ca2+ release is located at the luminal side of the disk membrane, whereas this Ca2+ was not transported across the disk membrane under our experimental conditions and on the time scale of our experiments (20 s). Light-induced Ca2+ release was inhibited by electrolysis in the suspension medium provided that the electrolytes gained access to the compartment where Ca2+ was released. The effectivity to inhibit Ca2+ release markedly increased from monovalent to divalent to trivalent cations. The results strongly suggest that electrolytes (cations) act by screening the electrostatic potential at the disk membrane surface due to the presence of a net fixed negative surface charge. The surface potential controls the free Ca2+ concentration at the membrane surface and, therefore, controls the amount of Ca2+ bound to the disk membrane. The kinetics of light-induced Ca2+ release and proton uptake showed a similar dependence on the structural status of the ROS. In sonicated ROS almost linear Arrhenius plots were observed for metarhodopsin II formation, Ca2+ release and proton uptake (energy of activation 150 kJ/mol). In intact ROS both Ca2+ release and proton uptake showed a nonlinear Arrhenius plot with rate constants up to 30-fold slower than metarhodopsin II formation. At temperatures above 10 degrees C a process other than metarhodopsin II formation rate limited both ligh-induced proton uptake and Ca2+ release (energy of activation 42 kJ/mol). A model is discussed in which metarhodopsin II formation triggers the uptake of proton(s) into the disk membrane lowering the surface potential. A reduction potential of the surface in turn decreases the free Ca2+ concentration at the surface thereby causing the release of part of the bound Ca2+.     

Detergent-like action of the antibiotic peptide surfactin on lipid membranes

Surfactin is a bacterial lipopeptide with powerful surfactant-like properties. High-sensitivity isothermal titration calorimetry was used to study the self association and membrane partitioning of surfactin. The critical micellar concentration (CMC), was 7.5 microM, the heat of micellization was endothermic with DeltaH(w-->m)(Su) = +4.0 kcal/mol, and the free energy of micellization DeltaG(O,w-->m)(Su) = -9.3 kcal/mol (25 degrees C; 100 mM NaCl; 10 mM TRIS, 1 mM EDTA; pH 8.5). The specific heat capacity of micellization was deduced from temperature dependence of DeltaH(w-->m)(Su) as DeltaC(w-->m)(P) = -250 +/- 10 cal/(mol.K). The data can be explained by combining the hydrophobicity of the fatty acyl chain with that of the hydrophobic amino acids. The membrane partition equilibrium was studied using small (30 nm) and large (100 nm) unilamellar POPC vesicles. At 25 degrees C, the partition coefficient, K, was (2.2 +/- 0.2) x 10(4) M(-1) for large vesicles leading to a free energy of DeltaG(O, w-->b)(Su) = -8.3 kcal/mol. The partition enthalpy was again endothermic, with DeltaH(w-->b)(Su) = 9 +/- 1 kcal/mol. The strong preference of surfactin for micelle formation over membrane insertion explains the high membrane-destabilizing activity of the peptide. For surfactin and a variety of non-ionic detergents, the surfactant-to-lipid ratio, inducing membrane solubilization, R(sat)(b), can be predicted by the simple relationship R(sat)(b) approximately K. CMC.     

Role of leaflet asymmetry in the permeability of model biological membranes to protons, solutes, and gases.

Bilayer asymmetry in the apical membrane may be important to the barrier function exhibited by epithelia in the stomach, kidney, and bladder. Previously, we showed that reduced fluidity of a single bilayer leaflet reduced water permeability of the bilayer, and in this study we examine the effect of bilayer asymmetry on permeation of nonelectrolytes, gases, and protons. Bilayer asymmetry was induced in dipalmitoylphosphatidylcholine liposomes by rigidifying the outer leaflet with the rare earth metal, praseodymium (Pr3+). Rigidification was demonstrated by fluorescence anisotropy over a range of temperatures from 24 to 50 degrees C. Pr3+-treatment reduced membrane fluidity at temperatures above 40 degrees C (the phase-transition temperature). Increased fluidity exhibited by dipalmitoylphosphatidylcholine liposomes at 40 degrees C occurred at temperatures 1-3 degrees C higher in Pr3+-treated liposomes, and for both control and Pr3+-treated liposomes permeability coefficients were approximately two orders of magnitude higher at 48 degrees than at 24 degrees C. Reduced fluidity of one leaflet correlated with significantly reduced permeabilities to urea, glycerol, formamide, acetamide, and NH3. Proton permeability of dipalmitoylphosphatidylcholine liposomes was only fourfold higher at 48 degrees than at 24 degrees C, indicating a weak dependence on membrane fluidity, and this increase was abolished by Pr3+. CO2 permeability was unaffected by temperature. We conclude: (a) that decreasing membrane fluidity in a single leaflet is sufficient to reduce overall membrane permeability to solutes and NH3, suggesting that leaflets in a bilayer offer independent resistances to permeation, (b) bilayer asymmetry is a mechanism by which barrier epithelia can reduce permeability, and (c) CO(2) permeation through membranes occurs by a mechanism that is not dependent on fluidity.     

Solubilizing effects caused by the nonionic surfactant dodecylmaltoside in phosphatidylcholine liposomes.

The interaction of the nonionic surfactant dodecylmaltoside (DM) with phosphatidylcholine liposomes was investigated. Permeability alterations were detected as a change in 5(6)-carboxyfluorescein released from the interior of vesicles and bilayer solubilization as a decrease in the static light scattered by liposome suspensions. This surfactant showed higher capacity to saturate and solubilize PC liposomes and greater affinity with these structures than those reported for the octyl glucoside. At subsolubilizing level an initial maximum in the bilayer/water partitioning (K) followed by an abrupt decrease of this parameter occurred as the effective molar ratio of surfactant to phospholipid in bilayers (Re) rose. However, at solubilizing level a direct dependence was established between both parameters. A direct correlation took place in the initial interaction steps (Re up to 0.28) between the growth of vesicles, their fluidity, and Re. A similar direct dependence was established during solubilization (Re range from 0.9 to 1.7) between the decrease in both the surfactant-PC aggregate size, the light scattering of the system, and Re (composition of aggregates). The fact that the free DM concentration at subsolubilizing and solubilizing levels showed values lower than and similar to its critical micelle concentration indicates that permeability alterations and solubilization were determined, respectively, by the action of surfactant monomer and by the formation of mixed micelles.     

On the regulatory role of dipeptidyl peptidase IV (=CD=adenosine deaminase complexing protein) on adenosine deaminase activity

The molecular mechanism controlling the variable activity of the malignancy marker adenosine deaminase (ADA) is enigmatic. ADA activity was found to be modulated by the membrane-bound adenosine deaminase complexing protein (CP=DPPIV=CD26). The role of lipid-protein interactions in this modulation was sought. While direct solubilization of ADA in vesicles resulted in loss of ADA activity, the binding of ADA to CP reconstituted in vesicles restored the specific activity. The activity of ADA, free or bound to CP in solution, resulted in continuous linear Arrhenius plots. However, ADA bound to reconstituted CP exhibited two breaks associated with approximately 30% increased activity, at 25 and 13 degrees C, yielding three lines with similar apparent activation energies (E(a)). Continuum solvent model calculations of the free energy of transfer of the transmembrane helix of CP from the aqueous phase into membranes of various widths show that the most favorable orientations of the helix above and below the main phase transition may be different. We suggest that the 20% change in the thickness of the bilayer below and above the main phase transition may modify the orientation of CP in the membrane, thereby affecting substrate accessibility of ADA. This could account for ADA's reduced activity associated with increased membrane fluidity in transformed vs. normal fibroblasts.     

Effects of nonionic surfactants on the solubilization and mineralization of phenanthrene in soil-water systems

The solubilization and mineralization of (14)C-phenanthrene in soil-water systems was examined with several commercially available surface-active agents, viz., an alkyl ethoxylate C(12)E(4); two alkylphenol ethoxylate surfactants: C(8)PE(9.5) and C(9)PE(10.5); two sorbitan ethoxylate surfactants: the sorbitan monolaurate (Tween 20) and the sorbitan monooleate (Tween 80); two pairs of nonionic ethoxylate surfactant mixtures: C(12)E(4)/C(12)E(23) at a 1:1 ratio, and C(12-15)E(3)/C(12-15)E(9) at a 1:3 ratio; and two surfactants possessing relatively high critical micelle concentration (CMC) values and low aggregation numbers: CHAPS and octyglucoside. Surface tension experiments were performed to evaluate surfactant sorption onto soil and the surfactant doses required to attain the CMC in the soil-water systems. Surfactant solubilization of (14)C-phenanthrene commenced with the onset of micellization. The addition of surface-active agents was observed not to be beneficial to the microbial mineralization of phenanthrene in the soil-water systems and, for supra-CMC surfactant doses, phenanthrene mineralization was completely inhibited for all the surfactants tested. A comparison of solubilization, surface tension, and mineralization data confirms that the inhibitory effect on microbial degradation of phenanthrene is related to the CMC of the surfactant in the presence of soil. Additional tests demonstrated the recovery of mineralization upon dilution of surfactant concentration to sub-CMC levels, and a relatively high exit rate for phenanthrene from micelles. These tests suggest that the inhibitory effect is probably related to a reversible physiological surfactant micelle-bacteria interaction, possibly through partial complexing or release of membrane material with disrupting membrane lamellar structure. This study indicates that nonionic surfactant solubilization of sorbed hydrophobic organic compounds from soil may not be beneficial for the concomitant enhancement of soil bioremediation. Additional work is needed to address physicochemical processes for bioavailability enhancement, and effects of solubilizing agents on microorganisms for remediation and treatment of hydrophobic organic compounds and nonaqueous phase liquids. (c) 1992 John Wiley & Sons Inc.     

myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes.

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.     

Applying flow cytometry to identify the modes of action of membrane-active peptides in a label-free and high-throughput fashion

Membrane-active peptides (MAPs) have several potential therapeutic uses, including as antimicrobial drugs. Many traditional methods used to evaluate the membrane interactions of MAPs have limited applicability. Low-throughput methods, such as microscopy, provide detailed information but often rely on fluorophore-labeled MAPs, and high-throughput assays, such as the calcein release assay, cannot assess the mechanism behind the disruption of vesicular-based lipid membranes. Here we present a flow cytometric assay that provides detailed information about the peptide-lipid membrane interactions on single artificial lipid vesicles while being high-throughput (1000–2000 vesicles/s) and based on label-free MAPs. We synthesized and investigated six MAPs with different modes of action to evaluate the versatility of the assay. The assay is based on the flow cytometric readouts from artificial lipid vesicles, including the fluorescence from membrane-anchored and core-encapsulated fluorophores, and the vesicle concentration. From these parameters, we were able to distinguish between MAPs that induce vesicle solubilization, permeation (pores/membrane distortion), and aggregation or fusion. Our flow cytometry findings have been verified by traditional methods, including the calcein release assay, dynamic light scattering, and fluorescence microscopy on giant unilamellar vesicles. We envision that the presented flow cytometric assay can be used for various types of peptide-lipid membrane studies, e.g. to identify new antibiotics. Moreover, the assay can easily be expanded to derive additional valuable information.     

Characterization of Ca2+ uptake in plasma membrane vesicles isolated from guinea pig ileum smooth muscle

We have characterized ATP-dependent Ca2+ transport into highly purified plasma membrane fraction isolated from guinea pig ileum smooth muscle. The membrane fraction contained inside-out sealed vesicles and was enriched 30-40-fold in 5'-nucleotidase and phosphodiesterase I activity as compared to post nuclear supernatant. Plasma membrane vesicles showed high rate (76 nmol/mg/min) and high capacity for ATP dependent Ca2+ transport which was inhibited by addition of Ca2+ ionophore A23187. The inhibitors of mitochondrial Ca2+ transport, i.e., sodium azide, oligomycin and ruthenium red did not inhibit ATP-dependent Ca2+ uptake into plasma membrane vesicles. The energy dependent Ca2+ uptake into plasma membranes showed very high specificity for ATP as energy source and other nucleotide triphosphates were ineffective in supporting Ca2+ transport. Phosphate was significantly better as Ca2+ trapping anion to potentiate ATP-dependent Ca2+ uptake into plasma membrane fraction as compared to oxalate. Orthovanadate, an inhibitor of cell membrane (Ca2+-Mg2+)-ATPase activity, completely inhibited ATP-dependent Ca2+ transport and the Ki was approximately 0.6 microM. ATP-dependent Ca2+ transport and formation of alkali labile phosphorylated intermediate of (Ca2+-Mg2+)-ATPase increased with increasing concentrations of free Ca2+ in the incubation mixture and the Km value for Ca2+ was approximately 0.6-0.7 microM for both the reactions.     

Evidence for a Cl-Stimulated MgATPase Proton Pump in Oat Root Membranes

The possibility that plant membrane-bound MgATPases may act as electrogenic proton pumps has been investigated. Using an oat (Avena sativa L. cv. Victory) root membrane preparation which is partially enriched in tightly sealed vesicles, we have shown that MgATP stimulates the uptake of the membrane-permeable anion [(14)C]SCN(-) by the vesicles; this indicates that an electrical potential (interior positive) is generated across the membrane. Both Cl(-) ions and the proton ionophore trifluoromethoxy(carbonyl-cyanide)phenylhydrazone inhibit the MgATP-driven [(14)C]SCN(-) uptake, presumably by collapsing the MgATP-generated membrane potential. The uptake of the pH gradient probe [(14)C]imidazole into the vesicles is also greatly stimulated by MgATP, indicating the presence of a transmembrane proton gradient (interior acid). MgATP-driven [(14)C]imidazole uptake is temperature sensitive, Cl(-)-stimulated, substrate specific for MgATP, sensitive to the MgATPase inhibitors vanadate and N,N'-dicyclohexylcarbodiimide, and completely eliminated by trifluoromethoxy(carboxyl-cyanide)phenylhydrazone. The mitochondrial ATPase inhibitor oligomycin has little effect on the MgATPase activity and on the MgATP-dependent [(14)C]SCN(-) and [(14)C]imidazole uptake. These data indicate that a class of oat root membrane-bound MgATPases, stimulated primarily by Cl ions, is capable of using the free energy of ATP-hydrolysis to generate an apparent electrochemical proton gradient in vitro.     

Surfactant peptides stimulate uptake of phosphatidylcholine by isolated cells

To determine whether small hydrophobic surfactant peptides (SP-B and SP-C) participate in recycling of pulmonary surfactant phospholipid, we determined the effect of these peptides on transfer of 3H- or 14C-labelled phosphatidylcholine from liposomes to isolated rat alveolar Type II cells and Chinese hamster lung fibroblasts. Both natural and synthetic SP-B and SP-C markedly stimulated phosphatidylcholine transfer to alveolar Type II cells and Chinese hamster lung fibroblasts in a dose- and time-dependent fashion. Effects of the peptides on phospholipid uptake were dose-dependent, but not saturable and occurred at both 4 and 37 degrees C. Uptake of labelled phospholipid into a lamellar body fraction prepared from Type II cells was augmented in the presence of SP-B. Neither SP-B nor SP-C augmented exchange of labelled plasma membrane phosphatidylcholine from isolated Type II cells or enhanced the release of surfactant phospholipid when compared to liposomes without SP-B or SP-C. Addition of native bovine SP-B and SP-C to the phospholipid vesicles perturbed the size and structure of the vesicles as determined by electron microscopy. To determine the structural elements responsible for the effect of the peptides on phospholipid uptake, fragments of SP-B were synthesized by solid-phase protein synthesis and their effects on phospholipid uptake assessed in Type II epithelial cells. SP-B (1-60) stimulated phospholipid uptake 7-fold. A smaller fragment of SP-B (15-60) was less active and the SP-B peptide (40-60) failed to augment phospholipid uptake significantly. Like SP-B and SP-C, surfactant-associated protein (SP-A) enhanced phospholipid uptake by Type II cells. However, SP-A failed to significantly stimulate phosphatidylcholine uptake by Chinese hamster lung fibroblasts. These studies demonstrate the independent activity of surfactant proteins SP-B and SP-C on the uptake of phospholipid by Type II epithelial cells and Chinese hamster lung fibroblasts in vitro.     

Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane

We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side.

In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21 °C.

In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds.

Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds.

The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.     

Vesicle to micelle transitions of egg phosphatidylcholine liposomes induced by nonionic surfactants, poly(oxyethylene) cetyl ethers.

Vesicle to micelle transitions of sonicated liposomes of egg yolk phosphatidylcholine (EPC) induced by a homologous series of nonionic surfactants, poly(oxyethylene) cetyl ethers [POE(n) cetyl ether], were investigated by using the method of turbidity titrations. The turbidities of the mixed dispersions of sonicated vesicles and surfactant were systematically measured as a function of the surfactant added for a wide range of lipid concentrations (from 0.51 to 6.35 mM EPC). From the titration curves, two threshold points representing onset and complete solubilization of liposomal membranes were determined as a probe for the effect of the length of ethylene oxide (EO) moiety on the phase behavior of ternary system of POE(n) cetyl ethers-EPC-excess water. Patterns of turbidity curves and the surfactant concentrations at two threshold points as well as widths of region between two transitions, where lamellar sheets and mixed micelles may coexist, mainly depended on the length of EO head group. With changing the lengths, solubilization of liposomes and phase diagram showed optimal behavior. That is, in the middle range of EO numbers, it resulted in narrowest coexistence region between onset and complete solubilization. Assuming the equilibrium partitioning model, critical effective molar ratios of surfactant to lipid, Rsat, free surfactant concentrations, Dw, and the partition coefficient of surfactant between bilayer and aqueous phase, K, in surfactant-saturated liposomes were quantitatively determined as a function of EO number. Effective ratios, Rsol, and free surfactant concentration in mixed micelles were also determined. In addition, the effects of CMC and HLB of surfactants on the solubilization of liposome were discussed.     

The mechanism of detergent solubilization of liposomes and protein-containing membranes.

The present study explores intermediate stages in detergent solubilization of liposomes and Ca2+-ATPase membranes by sodium dodecyl sulfate (SDS) and medium-sized ( approximately C12) nonionic detergents. In all cases detergent partitioning in the membranes precedes cooperative binding and solubilization, which is facilitated by exposure to detergent micelles. Nonionic detergents predominantly interact with the lipid component of Ca2+-ATPase membranes below the CMC (critical micellar concentration), whereas SDS extracts Ca2+-ATPase before solubilization of lipid. At the transition to cooperative binding, n-dodecyl octaethylene glycol monoether (C12E8), Triton X-100, and dodecyldimethylamine oxide induce fusion of small unilamellar liposomes to larger vesicles before solubilization. Solubilization of Ca2+-ATPase membranes is accompanied by membrane fragmentation and aggregation rather than vesicle fusion. Detergents with strongly hydrophilic heads (SDS and beta-D-dodecylmaltoside) only very slowly solubilize liposomal membranes and do not cause liposome fusion. These properties are correlated with a slow bilayer flip-flop. Our data suggest that detergent solubilization proceeds by a combination of 1) a transbilayer attack, following flip-flop of detergent molecules across the lipid bilayer, and 2) extraction of membrane components directly by detergent micelles. The present study should help in the design of efficient solubilization protocols, accomplishing the often delicate balance between preserving functional properties of detergent sensitive membrane proteins and minimizing secondary aggregation and lipid content.     

Solubilization of sarcoplasmic reticulum membranes by sodium dodecylsulphate. A Fourier-transform infrared spectroscopic study

In order to improve our understanding of membrane protein solubilization by sodium dodecylsulphate, sarcoplasmic reticulum vesicles have been treated with this surfactant at different detergent: protein mole ratios. Effects on Ca2(+)-ATPase activity, membrane protein solubilization, and protein conformation have been independently monitored, and correlations among the various parameters have been observed. The thermal denaturation of sarcoplasmic reticulum proteins in the presence of sodium dodecylsulphate has also been characterized spectroscopically.