The interaction of cytochrome C with phospholipids. A pulse-radiolysis study.

The reactions of the hydrated electron (eaq-), produced during pulse radiolysis, have been used to study the binding of phosphatidyl choline (PC), phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), and phosphatidyl inositol (PI) vesicles with horse-heart cytochrome C. An interaction could only be detected between cytochrome C and either PS or PI. An apparent equivalence point in the binding was reached for both phospholipids at a molar ratio of phospholipid : protein of 6 : 1. At this point, the reactivity of (eaq-) towards the cytochrome C was very markedly reduced. Indeed, the rate of disappearance of (eaq-) under such conditions was the same as the rate of eaq- disappearance in triply-distilled water. The inclusion of cholesterol at a molar ratio of 1 : 1 within the phospholipid vesicles changed the stoichiometry of the interaction. Evidence that protonated epsilon-amino groups of lysine residues are involved in the interaction is presented. Possible models for the complexes formed are discussed.     

A circular dichroism study of charged polypeptides interaction with salts.

The effect of salts on the experimental circular dichroism spectra of polypeptides is presented using poly-L-lysine as the main model. Salt effects are analyzed into: (a) shielding at low (less than 0.5 M) concentrations of all salts; (b) binding to positively charged and some neutrally charged side-chains by certain anions (e.g., CCl3COO-, CF3C00-, ClO4-), with induction of helicity; (c) binding of these same anions, at high concentration, to the backbone leading toward random structure; (d) binding of high concentration of denaturing cations (La+3, Ca++, Li+) to the backbone, with La+3 and Ca++ leading to collapsed random structure (R) while Li+ tends to leave the polypeptide somewhat extended; (e) indirect interaction of salting-out salts (NaH2PO4, (NH4)2SO4, NH4F), at high concentration, leading toward complete alpha helicity, probably by competition with the polypeptide and the anion for available water. Effects of changing the temperature from 5 degrees to 50 degrees on the circular dishroism spectra of different polypeptide-salt solutions throughout the region from extended (LES) to alpha helical conformation are analyzed in terms of introduction of randomness (R) at high temperature. Applications to effects of salt on protein structures are considered.     

Synthesis of phospholipid-conjugated bile salts and interaction of bile salt-coated liposomes with cultured hepatocytes

To examine the possibility of targeting liposomes to hepatocytes via bile salts, the bile salt lithocholyltaurine was covalently linked to a phospholipid. The isomeric compounds disodium 3alpha-(2-(1,2-O-distearoyl-sn-glycero-3-phospho-2'-ethanolamidosuccinyloxy)ethoxy)-5beta-cholan-24-oyl-2'-aminoethansulfonate and disodium 3beta-(2-(1,2-O-distearoyl-sn-glycero-3-phospho-2'-ethanolamidosuccinyloxy)ethoxy-5beta-cholan-24-oyl-2'-aminoethansulfonate (DSPE-3beta-LCT) were synthesized and incorporated into liposomal membranes. Confocal laser scanning microscopy studies showed that bile salt-bearing liposomes (BSLs) attach to the surface of rat hepatocytes in culture. Studies with radioactively labeled liposomes revealed that the bile salt linked via the 3beta-conformation resulted in a higher attachment efficiency than that with the 3alpha-derivative. In the presence of BSLs corresponding to 2 mM liposomal phosphatidylcholine, uptake of 50 microM cholyltaurine (CT) into hepatocytes was reduced by approximately 40% by the 3beta-derivative and by approximately 17% by the 3alpha-derivative. When added simultaneously with the liposomes, CT up to 75 microM inhibited the binding of DSPE-3beta-LCT-bearing liposomes. By contrast, increasing concentrations reversed this inhibition and resulted in an increased bile salt-mediated binding. The same was true when CT was added 10 min before the liposomes were added. The attachment of BSLs to the surface of hepatocytes opens up promising possibilities for hepatocyte-specific drug delivery. More generally, not only substrates for cellular endocytosing receptors but also substrates for cellular carrier proteins should be suitable ligands for the cell-specific targeting of nanoscale particles such as liposomes.     

Calmodulin-drug interaction. A fluorescence and flow dialysis study

Various Ca2+-antagonists and related compounds were probed for possible anti-calmodulin properties. Some of them efficiently inhibit calmodulin dependent activity (the plasma membrane Ca2+-ATPase and the cyclic nucleotide phosphodiesterase). The I50-values for the most potent inhibitors varied between 15 and 30 uM. Using fluorescence spectroscopy and flow dialysis methods the stoichiometry of the binding of some of the drugs to calmodulin has been investigated. The number of Ca2+-dependent high affinity binding sites has been studied on trypsin fragments of calmodulin. Compound 12-114 was bound with high affinity in a Ca2+-dependent way to both halves of calmodulin, compound 200-737 recognized one high affinity binding site only in the C-terminal half of the molecule, whereas compound 36-079 demanded the intact protein to be able to interact with high affinity in a Ca2+-dependent manner.     

Fluorescent probe studies of mixed micelles of phospholipids and bile salts. Effect of cholesterol incorporation

The binding of the fluorescent alkylamines, $\text{N-(2-}\text{aminoethyl}\text{)-5-}\text{dimethylamino-1-naphthalene}$ sulfonamide, $\text{N-(5-}\text{aminopentyl}\text{)-5-}\text{dimethylamino-1-naphthalene}$ sulfonamide (dansyl cadaverine) and $\text{N-(10-}\text{aminodecyl}\text{)-5-}\text{dimethylamino-1-napthalene}$ sulfonamide with phospholipid and phospholipid-deoxycholate micelles, has been shown to increase with the length of the alkyl spacer chain. The probes bind more effectively to micelles containing unsaturated phospholipids and do not interact strongly with bile salt solutions at low concentrations. Cholesterol incorporation into mixed micelles results in a quenching of probe fluorescence due to displacement of probe molecules. The enhanced rigidity of the mixed micelles on solubilizing cholesterol is established by a decrease in pyrene excimer fluorescence and by the less effective perturbation of the micellar structure by 1-anilino-8-naphthalene sulfonate. The anionic probe 1-anilino-8-naphthalene sulfonate is also displaced from the mixed micelles when cholesterol is incorporated, suggesting a dominant role for packing and hydrophobic effects in binding both positively and negatively charged probes.     

A fluorescence study of A23187 interaction with phospholipid vesicles

The fluorescence of the ionophore A23187 has been monitored in suspensions of egg yolk phosphatidylcholine (EYPC) and dipalmitoyl phosphatidylcholine (DPPC) vesicles. Both the protonated form of A23187 and the Ca²⁺ complex exhibit fluorescence enhancement when extracted into a hydrophobic environment. Measurements of fluorescence intensity versus lipid concentration were thus used to establish lower limits to the lipid/ water partition coefficients. Values obtained in this way were ? 50 ml water/mg phosphatidylcholine. Quenching of A23187 fluorescence by the spin labels 5NMS (methyl ester of 5-nitroxyl stearate), 12NMS, 16NMS, and TEMPO stearamide in EYPC and DPPC vesicles was also investigated. In EYPC all the labels yielded fairly linear Stern-Volmer plots, with TEMPO stearamide quenching about half as strong as the other probes. Quenching in DPPC was generally much stronger than in EYPC, but 12 NMS and 16NMS gave hyperbolic Stern-Volmer plots, apparently due to clustering of the labels. In all the cases the protonated form of A23187 was quenched approximately twice as efficiently as the Ca²⁺ complex, possibly due to a longer fluorescence lifetime for the former. Calculations based on measured spectral properties were performed which indicate that the Förster transfer mechanism extends the nitroxides' quenching range to ～- 10 Å.     

Hydrophobic interaction of lysophospholipids and bile salts at submicellar concentrations

A series of environment-sensitive, fluorescent-labeled N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-monoacylphosphatidylethano lamine (N-NBD-lysoPE) probes of differing acyl chain length (C12-C18) was used to demonstrate the hydrophobic interaction between lysophospholipids and two different bile salts at concentrations below their respective critical micelle concentrations (cmc's). Formation of submicellar aggregates in the presence of bile salt-phospholipid mixed micelles could facilitate lipid absorption in the intestine. To ensure the use of submicellar lysolipid concentrations in the experiments, the cmc of each fluorescent lysolipid probe was determined by concentration-dependent self-quenching. The cmc values obtained for the various N-NBD-lysoPE probes were as follows (microM): monolauroyl, greater than or equal to 40; monomyristoyl, 4; monopalmitoyl, 0.3; monostearoyl, 0.04. Probe concentrations well below their respective cmc's were used in all experiments. The fluorescence of a solution of each lysolipid probe was monitored as the concentration of bile salt was gradually increased. The increase in fluorescence was taken as a measure of the ability of the bile salt molecules to complex with the probe molecule, thereby increasing the fluorescent yield of the lysolipid probe molecule. Determination of the cmc of the bile salts in the presence of the lysolipid probe was made in parallel with the fluorescence measurement by monitoring the increase in light scattering of the solution. Both bile salts were shown to induce maximal increases in fluorescence of the N-NBD-lysoPE derivatives at concentrations of bile salt well below their respective cmc values, indicating the existence of submicellar lysolipid-bile salt aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescent derivatives of bile salts. II. Suitability of NBD-amino derivatives of bile salts for the study of biological transport.

Interaction of unconjugated and taurine-conjugated NBD-amino-dihydroxy-5 beta-cholan-24-oic acids bearing the fluorophor in the 3 alpha, 3 beta, 7 alpha, 7 beta, 12 alpha, or 12 beta position with albumin results in a small hypsochromic shift of the emission maximum and an increase in quantum yield, suggesting binding by hydrophobic interactions. The different unconjugated fluorescent bile salt derivatives are metabolized by intact rat liver in different ways. The unconjugated 3 beta-NBD-amino derivative is completely transformed to its taurine conjugate and secreted as such, whereas the 3 alpha-NBD-amino derivative is completely transformed to a polar fluorescent compound not identical with its taurine conjugate. The unconjugated 7 alpha- and 7 beta-NBD-amino derivatives are only partially conjugated with taurine and mainly secreted in unmetabolized form. The unconjugated 12 alpha- and 12 beta-NBD-amino derivatives are not at all transformed to their taurine conjugates, but are partially metabolized to unidentified compounds. They are predominantly secreted as the unmetabolized compounds. In contrast to the unconjugated derivatives, all taurine-conjugated fluorescent bile salt derivatives are secreted into bile unmetabolized. With the exception of the 3 alpha-compound, all synthesized taurine-conjugated fluorescent derivatives interfere with the secretion of cholyltaurine. Differential photoaffinity labeling studies using (7,7-azo-3 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2'-[2'-3H(N)]aminoethanesulfonate as a photolabile derivative revealed that in liver cells all fluorescent bile salt derivatives interact with the same polypeptides as the physiological bile salts. The hepatobiliary transport of taurine-conjugated NBD-amino bile salt derivatives is, due to hydrophobic interactions, accompanied by an increase in fluorescence intensity which is favorable for the study of biological bile salt transport by fluorescence microscopy.     

EPR study of the hydrophobic interaction of spectrin with fatty acids.

The hydrophobic interaction between spin-labelled stearic acid and spectrin was studied by electron paramagnetic resonance (EPR) and fluorescence quenching. The results are quantitatively interpreted in terms of two types of binding site on spectrin. A comparison between the results of the EPR and fluorescence experiments show the drawback of the fluorescence method in binding studies.     

The interaction of adriamycin with small unilamellar vesicle liposomes. A fluorescence study

The interaction of the antineoplastic agent adriamycin with sonicated liposomes composed of phosphatidylcholine alone and with small amounts (1-6%) of cardiolipin has been studied by fluorescence techniques. Equilibrium binding data show that the presence of cardiolipin increases the amount of drug bound to liposomes when the bilayer is below its phase transition temperature and when the ionic strength is relatively low (0.01 M). At higher ionic strength (0.15 M) and above the Tm (i.e. conditions which are closer to the physiological state) the binding of the drug to the two liposome types is nearly the same. Thus the differences in the interactions of adriamycin with cardiolipin-containing membranes, as opposed to those composed of phosphatidylcholine alone, are not due simply to increased binding but rather to an altered membrane structure when this lipid is present. Quenching of adriamycin fluorescence by iodide shows that bound drug is partially, but not completely, buried in the liposomal membrane. Both in the presence and absence of cardiolipin the bulk of the adriamycin is more accessible to the quencher below the Tm than above it; that is, a solid membrane tends to exclude the drug from deep penetration. Above the Tm, the presence of cardiolipin alters the nature of liposome-adriamycin interaction. Here the fluorescence quenching data suggest that the presence of small amounts of cardiolipin (3%) in a phosphatidylcholine matrix creates two types of binding environments for drug, one relatively exposed and the other more deeply buried in the membrane. The temperature dependence of the adriamycin fluorescence and the liposome light scattering reveal that cardiolipin alters the thermal properties of the bilayer as well as its interaction with adriamycin. At low ionic strength lateral phase separations may occur with both pure phosphatidylcholine and when 3% cardiolipin is present; under these conditions the bound adriamycin exists in two kinds of environment. It is notable that only adriamycin fluorescence reveals this phenomenon; thebulk property of liposome light scattering reports only on the overall membrane phase change. These data suggest that under certain conditions the drug binding sites in the membranes are decoupled from the bulk of the lipid bilayer.     

Fluorescent derivatives of bile salts. I. Synthesis and properties of NBD-amino derivatives of bile salts.

In order to visualize bile salt transport, fluorescent bile salt derivatives were synthesized by introduction of the relatively small fluorescent 4-nitrobenzo-2-oxa-1,3-diazol (NBD)-amino group in either the 3-, 7-, or 12-position of the steroid structure, thus providing a complete set of diastereomeric derivatives, 3 alpha-NBD-amino-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 3 beta-NBD-amino-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 7 alpha-NBD-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 7 beta-NBD-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 12 alpha-NBD-amino-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid, 12 beta-NBD-amino-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid, as well as their taurine conjugates. Their optical properties with absorption maxima at about 490 nm and emission maxima at 550 nm make them suitable for fluorescent microscopic studies. Fluorescence of the NBD-derivatives is strongly dependent on polarity of the solvent, on the concentration of the bile salt derivatives, and only slightly on temperature.     

A proton-relaxation enhancement study of the interaction of manganous ions with phospholipids in aqueous dispersions.

An interaction of dipalmitoylphosphatidylcholine (PC) and phosphatidylserine (PS) with manganous ions has been investigated by measuring the effect of bound manganese upon the longitudinal relaxation rate, 1/T1, of the solvent water protons and evaluating the enhancement factor epsilon b. The observed enhancement values were used to determine the number of interacting sites per polar head group, n, and the values of association constants, KA, of manganese to PC and PS. Changes in epsilon b correlate with structural changes at the interacting site. By increasing the temperature one can see an abrupt decrease in epsilon b within the temperature interval from 40 to 50 degrees C indicating the thermal phase transition of PC as established by calorimetry, fluorescence and high-resolution NMR measurements. That an enhancement of 1/T1 of the solvent-water protons occurs at all is explained by assuming a restricted rotation of the Mn2+-aquo complex in the bound state. In addition we suppose that the rotation of the Mn2+-aquo complex is the mechanism which dominates the relaxation of the water protons in teh bulk solvent when phospholipids are present.     

Lipid interaction of tetanus neurotoxin. A calorimetric and fluorescence spectroscopy study.

The interaction of Tetanus toxin with phospholipid vesicles containing gangliosides (GD1a, GD1b or GT1b) or phosphatidic acid has been investigated at neutral or acidic pH. Change in the thermotropic properties of the vesicles occurred only after addition of the toxin at acidic pH, and led to surface binding or membrane insertion of the protein, dependent on the physical state of the membrane. Most remarkably, toxin addition at acidic pH to dipalmitoyl-phosphatidylcholine vesicles containing GT1b ganglioside, caused formation of ganglioside microdomains on the vesicle surface.     

Divalent cation binding to phospholipids: An EPR study

Summary Divalent cation association to sonicated phospholipid liposomes has been examined with electron paramagnetic spectroscopy. Spectra were obtained suggesting that, in some cases, divalent cations associated with acidic phospholipid head groups are highly mobile.Using the amplitude of its characteristic sextet signal as a measure of free Mn(H₂O) ₆ ⁺⁺ , the apparent affinities of cardiolipin and phosphatidylserine for Mn²⁺ were measured as a function of monovalent electrolyte. Monovalent cations having smaller nonhydrated radii were more effective in displacing Mn from the phospholipids. Under conditions of low divalent cation concentrations, it is shown that the Gouy-Chapman diffuse double layer theory predicts a Mn-affinity (K _A ) inversely proportional to the square of monovalent salt concentration. Although this relationship was closely obeyed for Mn binding to cardiolipin, the fall-off inK _A with added sodium chloride was slower in the cases of Mn binding to phosphatidylserine or phosphatidic acid.When phosphatidylcholine or cholesterol was incorporated into mixed vesicles along with a fixed amount of charged phospholipid, the Mn-binding strength was roughly proportional to the weight fraction of the latter. This result is consistent with: (1) a random dispersal of lipids in the bilayer, and (2) a 1:2 divalent cation-phospholipid interaction.     

The interaction of spectrin-actin and synthetic phospholipids. II. The interaction with phosphatidylserine.

Sonicated vesicles of phosphatidylserine and phosphatidylserine/phosphatidylcholine mixtures were recombined with spectrin-actin from human erythrocyte ghosts. Morphological properties and physicochemical characteristics of the recombinates were studied with freeze etch electron microscopy, 31P NMR and differential scanning calorimetry. Sonicated dimyristoyl phosphatidylserine vesicles show a decrease in enthalpy change of the lipid phase transition upon addition of spectrin-actin. These vesicles collapse and fuse, into multilamellar structures in the presence of spectrin-actin, as demonstrated by freeze fracturing and NMR. Spectrin-actin cannot prevent the salt formation between phosphatidylserine and Ca2+, all phosphatidylserine is withdrawn from the lipid phase transition. In contrast a protection against the action of Mg2+ could be observed. Mixed bilayers of dimyristoyl phosphatidylserine/dimyristoyl phosphatidylcholine show phase separations at molar ratios above 1/1 (van Dijck, P.W.M., de Kruijff, B., Verkleij, A.J., van Deenen, L.L.M. and de Gier, J. (1978) Biochim. Biophys. Acta 512, 84--96). These phase spearations can be prevented by spectrin-actin. Ca2+-induced lateral phase separations in cocrystallizing phosphatidylserine/phosphatidylcholine mixtures, can be reduced by spectrin-actin. Formation of the Ca2+-phosphatidylserine salt, occurring in addition to lateral phase separation when mixtures contain more than 30 mol % phosphatidylserine, cannot be prevented by spectrin-actin.     

Modification of phospholipids in erythrocyte membranes by phospholipase D. A fluorescence and ESR spectroscopic study

The conversion of more than 65% of the phospholipids in human erythrocyte membranes to phosphatidyl-methanol and phosphatidic acid by incubation with phospholipase D and methanol increased the dissociation constant of the fluorescence probe ANS compared to untreated membranes, but did not affect the number of binding sites and the limiting fluorescence enhancement at maximal binding (Imax). On the contrary, the cationic fluorescence probe dansylcadaverin showed additional binding sites without a change in Kd and an increase of Imax upon incubation with phospholipase D treated erythrocyte membranes compared to incubations of membranes with the original phospholipid pattern. The characteristic temperature-dependence of the quenching of the membrane protein fluorescence by a membrane-bound nitroxide-labeled stearic acid was not influenced by the modification of the phospholipids. A slight reduction of the order parameter, S, determined by ESR-spectroscopy with the same nitroxide spin-labeled fatty acid incorporated into modified membranes compared to controls was found at 40 degrees C, but not at 25 degrees C. The results were interpreted as an indication of membrane domains that retained their physical properties and lipid composition during the incubation with phospholipase D.     

A differential scanning calorimetry study of the interaction of alpha-tocopherol with mixtures of phospholipids

When alpha-tocopherol was included in multibilayer vesicles of dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine it induced a broadening of the main transition and a displacement of this transition to lower temperatures, as seen by differential scanning calorimetry. This effect was quantitatively more important in the samples of distearoylphosphatidylcholine than in those of the other phosphatidylcholines. Alpha-Tocopherol when present in equimolar mixtures of dimyristoylphosphatidylcholine and diastearoylphosphatidylcholine, which show monotectic behaviour, preferentially partitions in the most fluid phase. The effect of alpha-tocopherol on the phase transition of dilauroylphosphatidylethanolamine and dipalmitoylphosphatidylethanolamine is qualitatively different of that observed on phosphatidylcholines, and several peaks are observed in the calorimetric profile, probably indicating the formation of separated phases with different contents in alpha-tocopherol. The effect was more apparent in dipalmitoylphosphatidylethanolamine than in dilauroylphosphatidylethanolamine. The inclusion of alpha-tocopherol in equimolar mixtures of dilauroylphosphatidylethanolamine and dipalmitoylphosphatidylcholine, which show cocrystallization, only produced a broadening of the phase transition and a shift to lower temperatures. However, in the case of equimolar mixtures of dipalmitoylphosphatidylcholine which also show cocrystallization, the effect was to cause lateral phase separation with the formation of different mixtures of phospholipids and alpha-tocopherol. Alpha-Tocopherol was also included in equimolar mixtures of phosphatidylethanolamine and phosphatidylcholine showing monotectic behaviour, and in this case alpha-tocopherol preferentially partitioned in the most fluid phase, independently of whether this was composed mainly of phosphatidylcholine or of phosphatidylethanolamine.