Hydrolytic action of phospholipase A2 in monolayers in the phase transition region: direct observation of enzyme domain formation using fluorescence microscopy

Phospholipase A2, a ubiquitous lipolytic enzyme highly active in the hydrolysis of organized phospholipid substrates, has been characterized optically in its action against a variety of phospholipid monolayers using fluorescence microscopy. By labeling the enzyme with a fluorescent marker and introducing it into the subphase of a Langmuir film balance, the hydrolysis of lipid monolayers in their liquid-solid phase transition region could be directly observed with the assistance of an epifluorescence microscope. Visual observation of hydrolysis of different phospholipid monolayers in the phase transition region in real-time could differentiate various mechanisms of hydrolytic action against lipid solid phase domains. DPPC solid phase domains were specifically targeted by phospholipase A2 and were observed to be hydrolyzed in a manner consistent with localized packing density differences. DPPE lipid domain hydrolysis showed no such preferential phospholipase A2 response but did demonstrate a preference for solid/lipid interfaces. DMPC solid lipid domains were also hydrolyzed to create large circular areas in the monolayer cleared of solid phase lipid domains. In all cases, after critical extents of monolayer hydrolysis in the phase transition region, highly stabile, organized domains of enzyme of regular sizes and morphologies were consistently seen to form in the monolayers. Enzyme domain formation was entirely dependent upon hydrolytic activity in the monolayer phase transition region and was not witnessed otherwise.     

Real-time fluorescence polarization measurements: interaction of phospholipase A2 with a fluorescent lecithin derivative

We have modified an SLM 4800 fluorometer to allow continuous measurement of fluorescence polarization. The modifications included construction of simple mechanical and electronic accessories which allowed polarizer position to be program-controlled by an HP 9815 calculator. With these modifications we were able to follow the interaction of the fluorescent lipid analog 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminododecanoyl phosphatidylcholine (I) with Naja-naja venom phospholipase A2 (PLA2) (E.C.3.1.1.4). Upon addition of aliquots of PLA2 containing up to 85 ng protein to a cuvette containing 1-2 microM I, the total measured polarization decreased linearly with time for at least 1000 s. Concomitant analyses of equivalent incubation mixtures and analyses of the contents of the cuvette after incubation and collection of fluorescence data revealed time-dependent formation of N-4-nitrobenzo-2-oxa-1,3-diazole-aminododecanoic acid (II). The decrease in total measured polarization was accelerated by Ca2+ and inhibited by EDTA. These data suggest that PLA2 activity in Naja-naja venom can be measured rapidly at low concentrations of both enzyme (0.01 microgram protein) and substrate (1 microM). Since this technique can be used to collect polarization data over time intervals as short as 4 s, it should be possible to measure the early changes in polarization during the interaction of fluorescent probes with proteins or membranes.     

Myeloperoxidase interaction with mixed monolayers of lecithin and cholesterol]

Interaction between myeloperoxidase and mixtures of lecithin-cholesterol monolayers were investigated. Solubility of the enzyme in 1-octanol or in octanol were tested too. It is shown that myeloperoxidase demonstrates an ability to concentrate on phase limits. The enzyme well dissolves in lipid membrane phases. It is possible to assume that myeloperoxidase penetrates and accumulates in the eye lens.     

Local anesthetic inhibition of pancreatic phospholipase A2 action on lecithin monolayers.

Using quantitative data previously reported for the penetration of local anesthetics into lecithin monolayers, the effects of surface and subphase concentrations of anesthetics on the inhibition of pancreatic phospholipase A2 action on didecanoyl phosphatidylcholine monolayers was investigated. Inhibition as a function of subphase concentration of anesthetic was in the order: dibucaine greater than tetracaine greater than butacaine greater than lidocaine = procaine. Inhibition as a function of surface concentration showed no obvious correlation; procaine inhibited at a very low surface concentration, followed by lidocaine at a somewhat higher concentration, and tetracaine, butacaine and dibucaine only at rather high concentrations. Ultraviolet difference spectroscopy indicated an interaction between lidocaine and enzyme in the subphase. Fluorescence studies showed that lidocaine is a competitive inhibitor of enzyme-lipid interface interaction. It is proposed that the more surface-active anesthetics inhibit by surface effects while the less surface-active anesthetics (lidocaine and procaine) inhibit by interaction with the enzyme in the subphase, which prevents enzyme penetration at the monolayer interface.     

Spontaneous formation of DPPC monolayers at aqueous/vapor interfaces and the impact of charged surfactants

Surface tensiometry and vibrational sum-frequency spectroscopy were used to examine the structure and organization in phospholipid monolayers at the aqueous/vapor interface in the absence and in the presence of simple, charged surfactants. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was the phospholipid employed in these studies and surfactants included sodium dodecyl sulfate (SDS) and dodecyl trimethyl ammonium bromide (DTAB). DPPC spontaneously spreads on a pure water (pH = 5.5) surface to form monolayers as evidenced by an equilibrium spreading pressure (ESP) of 7.9 ± 2.3 mN/m and a clearly resolved vibrational spectrum. Low concentrations of surfactants inhibit the spreading of DPPC and result in significantly lower ESP values. Anionic and cationic surfactants at higher concentrations have opposite effects on monolayer organization; SDS creates well-organized monolayers while DTAB leads to poor organization of lipid molecules. Surface-specific vibrational spectra showed that high concentrations of charged surfactants (≥ 100 µM) lead to accumulation of net surface charges as evidenced by destructive and constructive interferences. Selectively deuterating surfactants results in changes in vibrational band intensities and phases enabling assignment of relative orientations of equivalent functional groups belonging to the lipid and surfactant.     

The interaction of phospholipase A2 with micellar interfaces. The role of the N-terminal region.

The localization of the previously postulated interface recognition site (IRS) in porcine pancreatic phospholipase A2, required for a specific interaction between the enzyme and organized lipid-water interfaces, was investigated by ultraviolet difference spectroscopy, by measurements of the intrinsic fluorescence of the unique Trp residue, and by protection experiments against specific tryptic hydrolysis. Using the enzymically nondegradable substrate analogues: CnH(2n+1)(0-)OOCH2CH2N+(CH3)3-(H,OH), it is shown that the rather hydrophobic N-terminal sequence of the enzyme, viz., Ala-Leu-Trp-Gln-Phe-Arg, is directly involved in the interaction with the lipid-water interface. Besides hydrophobic probably also polar interactions contribute to the binding process. At neutral or acidic pH the presence of a salt bridge between the N-terminal alpha-NH3+ group and a negatively charged side chain stablizes the interface recognition site and allows the enzyme to penetrate micellar surfaces, even in the absence of metal ion. At alkaline pH, interaction of the enzyme with micellar interfaces requires the presence of Ca2+ (Ba2+) ions.     

Characterization of the interaction of phospholipase A(2) with phosphatidylcholine-phosphatidylglycerol mixed lipids

The first requirement in the hydrolysis of phospholipid bilayers by phospholipase A(2) is the interaction of the enzyme with the bilayer surface. The catalytic ability of phospholipase A(2) has been shown to be extremely sensitive to the topology of the bilayer to which it binds and hydrolyzes. Phospholipid bilayer properties and composition such as unsaturation, charge, and the presence of reaction products are known regulators of the catalytic activity of phospholipase A(2) toward the phospholipids and influences the binding of enzyme to the membrane. We show in this paper that the effect of increased anionic lipid results in enhanced binding that can be described quantitatively in terms of a simple phenomenological model. However, the interaction with anionic lipid does not singularly dominate the thermodynamics of binding, nor can the lag phase observed in the time course of hydrolysis of large unilamellar vesicles simply be the result of limited interaction between the enzyme and the bilayer. Furthermore, we show that phospholipase A(2) from Akgistrodon piscivorus piscivorus can exist in at least two bilayer-bound states and that the absence of a fluorescence change upon mixing the enzyme with lipid bilayers does not necessarily indicate the absence of an interaction.     

A comparison of the behavior of cholesterol and selected derivatives in mixed sterol-phospholipid Langmuir monolayers: a fluorescence microscopy study

Eukaryotic cells require sterols to achieve normal structure and function of their plasma membranes, and deviations from normal sterol composition can perturb these features and compromise cellular and organism viability. The Smith-Lemli-Opitz syndrome (SLOS) is a hereditary metabolic disease involving cholesterol (CHOL) deficiency and abnormal accumulation of the CHOL precursor, 7-dehydrocholesterol (7DHC). In this study, the interactions of CHOL and the related sterols desmosterol (DES) and 7DHC with l-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers were compared. Pressure-area isotherms and fluorescence microscopy were used to study DPPC monolayers containing 0, 10, 20, or 30 mol% sterol. Similar behavior was noted for CHOL- and DES-containing DPPC monolayers with both techniques. However, while 7DHC gave isotherms similar to those obtained with the other sterols, microscopy indicated limited domain formation with DPPC, indicating that 7DHC packs somewhat differently in DPPC membranes compared to CHOL and DES. These results are discussed in relation to SLOS pathobiology.     

Inhibition of phospholipase A2 by cis-unsaturated fatty acids: evidence for the binding of fatty acid to enzyme.

Calcium-dependent phospholipases A2 are markedly inhibited in vitro by cis-unsaturated fatty acids (CUFAs) and to a much lesser extent by trans-unsaturated or saturated fatty acids. Thus, CUFAs may function as endogenous suppressors of lipolysis. To better understand the mechanism of inhibition, kinetic analysis, fluorescence spectroscopy and gel permeation chromatography were employed to demonstrate that CUFAs interact with a highly purified Ca(2+)-dependent phospholipase A2 from Naja mossambica mossambica venom. Arachidonate inhibited hydrolysis of both [1-14C]oleate-labelled, autoclaved Escherichia coli and [1-14C]linoleate-labelled phosphatidylethanolamine in an apparent competitive manner. When subjected to gel permeation chromatography, [3H]arachidonate, but not [3H]palmitate, comigrated with the enzyme. Arachidonic and other CUFAs increased the fluorescence intensity of the enzyme almost 2-fold in a dose-dependent fashion (50 microM = 180% of control); methyl arachidonate was without effect. Saturated fatty acids had only a modest effect on enzyme fluorescence (50 microM = 122% of control). Concentrations of arachidonate that inhibited in vitro enzymatic activity by almost 80% did not alter binding of phospholipase A2 to the E. coli substrate. Collectively, these data demonstrate that, while CUFAs selectively bind to the enzyme, they do not influence phospholipase A2-substrate interaction. Inhibition of in vitro phospholipase A2 activity by CUFAs may be mediated by the formation of an enzymatically inactive enzyme-substrate-inhibitor complex.     

Fluorescence, polarized fluorescence, and Brewster angle microscopy of palmitic acid and lung surfactant protein B monolayers

Fluorescence, polarized fluorescence, and Brewster angle microscopy reveal that human lung surfactant protein SP-B and its amino terminus (SP-B[1-25]) alter the phase behavior of palmitic acid monolayers by inhibiting the formation of condensed phases and creating a new fluid protein-rich phase. This fluid phase forms a network that separates condensed phase domains at coexistence and persists to high surface pressures. The network changes the monolayer collapse mechanism from heterogeneous nucleation/growth and fracturing processes to a more homogeneous process through isolating individual condensed phase domains. This results in higher surface pressures at collapse, and monolayers easier to respread on expansion, factors essential to the in vivo function of lung surfactant. The network is stabilized by a low-line tension between the coexisting phases, as confirmed by the observation of extended linear domains, or "stripe" phases, and a Gouy-Chapman analysis of protein-containing monolayers. Comparison of isotherm data and observed morphologies of monolayers containing SP-B(1-25) with those containing the full SP-B sequence show that the shortened peptide retains most of the native activity of the full-length protein, which may lead to cheaper and more effective synthetic replacement formulations.     

Mixed monolayers involving DPPC, DODAB and oleic acid and their interaction with nicotinic acid at the air-water interface

The behaviour of binary mixtures involving dipalmitoylphosphatidylcholine (DPPC), dioctadecyldimethylammonium bromide (DODAB) and oleic acid (OA) was investigated at the air-water interface by surface pressure-area (pi-A) measurements and by Brewster angle microscopy (BAM). Thermodynamic analysis indicates for the system DPPC/DODAB miscibility with strong negative deviations from the ideal behaviour, from low to high surface pressures over all the composition range. For systems DODAB/OA and DPPC/OA, thermodynamic analysis and BAM observation indicate miscibility from low to intermediate surface pressures, and phase separation in a limited range of composition at high surface pressures. The interaction of nicotinic acid (NA) with pure lipids and with selected compositions of mixed systems was investigated. Significant positive deviations of pi-A isotherms in the presence of NA indicate attractive interactions between NA and the polar groups of DPPC and DODAB. NA easily penetrates in expanded regimes while it tends to be segregated from condensed regimes in mixed monolayers.     

The interaction of phospholipase A with lipids. I. Structure of the bimolecular leaflet of a lecithin analogue substance, 1-oleoyl-2-n-hexadecyl-2-deoxy-glycero-3-phosphorylcholine

Oriented multilayer systems were prepared with a chemical analogue of lecithin, 1-oleoyl-2-n-hexadecyl-2-deoxyglycero-3-phosphorylcholine and studied by X-ray diffraction. Diffraction patterns were recorded with specimens equilibrated in atmospheres of varying relative humidity. Two distinct types, the high and the low humidity diffraction patterns, were observed. At high humidities the multilayers show normal swelling behaviour which is used in the analysis. A bilayer structure with headgroup peaks and a hydrocarbon region is derived from the Fourier synthesis. At low humidity a bilayer in a state of higher order is observed. Two peaks are resolved in the headgroup region. Chain segments in the hydrocarbon region are packed in a lattice at low humidity.     

Conserved and variable segments of the snake phospholipase A2 amino acid domain]

Homologous amino acid sequences of phospholipases A2 of snakes belonging to families Elapidae, Viperidae and Colubridae were considered in order to study the location of conservative and variable regions. To identify significant conservative and variable regions a comparison between two groups of aligned sequences of snake phospholipases A2 was successfully applied. The phospholipases A2 sequences were divided into two groups (taxons) according to the phylogenetic tree reconstructed from the pair distance matrix. Results of the comparison were plotted to facilitate the identification of significant conservative and variable regions. It was shown, that the results of the comparison between two phylogenetic groups of snake phospholipases A2 didn't depend much on the number of each group representatives, and the location of conservative and variable regions didn't significantly change if one of the groups was represented by the single sequence. It should be mentioned, that the more the phylogenetic difference between groups of phospholipases A2 the more was the number of significant conservative and variable regions. The knowledge of the number and location of conservative and variable regions and their dependence on phylogenetic relations between the compared taxons can be used to predict the synthetic peptide structure to obtain antibodies of various specificity. These antibodies may have either a wide range of cross-reactivity against all of phospholipases A2 or a limited range of cross-reactivity against phospholipases A2 of one taxon.     

Molecular packing of cord factor and its interaction with phosphatidylinositol in mixed monolayers.

Cord factor (trehalose 6,6'-dimycolate, CF) is a glycolipid located in the outer mycobacterial cell wall that is implicated in the pathogenesis of mycobacteria. Furthermore, CF is a convenient model for studying mycolic acid residues, the major lipid constituents of the mycobacterial cell wall that are believed to form a barrier against drug penetration. The surface properties of CF and its interactions with phosphatidylinositol (PI) have been investigated using the monolayer technique. During compression/expansion/recompression cycles, CF monolayers switch from a loosely packed to a more tightly packed structure. The change in surface properties suggests a molecular rearrangement, perhaps involving interdigitation of long and short chains of the CF molecules. In CF-PI monolayers, maximal lateral packing density occurs between 0.5 and 0.7 mole fraction CF, which is close to the relative composition of mycolic acid residues and shorter-chain lipids in the mycobacterial cell wall. Low concentrations of CF increase the order in PI monolayers, consistent with CF toxicity involving rigidification of cell membranes.     

A continuous fluorescence displacement assay for the measurement of phospholipase A2 and other lipases that release long-chain fatty acids.

1. A new continuous fluorescence assay for phospholipase A2 is described which involves the displacement of the highly fluorescent fatty-acid probe 11-(dansylamino)undecanoic acid from rat liver fatty-acid-binding protein by long-chain fatty acids released as a result of phospholipase A2-catalysed hydrolysis of phospholipids. The initial rate of decrease in fluorescence is linearly related to enzyme activity. 2. The assay will detect enzyme activity down to about 10 pmol/min per ml and gives a linear response up to about 10 nmol/min per ml. 3. The assay will work with all phospholipids that have been tested including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol. Substrates carrying a net negative charge showed the highest rates of hydrolysis. 4. The assay will work, in principle, with an enzyme catalysing the release of long-chain fatty acids from a fatty-acylated substrate. This has been confirmed with pancreatic lipase and cholesterol esterase.     

Comparison of the interaction of tomatine with mixed monolayers containing phospholipid, egg sphingomyelin, and sterols

The interaction of the glycoalkaloid tomatine with monolayers of a phospholipid (dimyristoylphosphatidylcholine, DMPC), and sphingolipid (egg sphingomyelin), and cholesterol is compared. Using measurements of the surface pressure response as a function of the subphase concentration of tomatine, interfacial binding constants are estimated for mixed monolayers of DMPC and cholesterol and for those of egg sphingomyelin and cholesterol of mole ratio 7:3. The binding constants obtained suggest a stronger interaction of tomatine with DMPC and cholesterol mixed monolayers, reflecting easier displacement of cholesterol from its interaction with DMPC than from its interaction with egg sphingomyelin. Mixtures of tomatine and cholesterol are found to spread directly at the water-air interface and form stable monolayers, suggesting that cholesterol holds tomatine at the interface despite the absence of observed monolayer behavior for tomatine alone. The interaction of tomatine with DMPC and cholesterol monolayers is found to exhibit a pH dependence in agreement with previously reported results for its interaction with liposomes; in particular, the interaction is much less at pH 5 than at pH 7 or pH 9. It is found that while tomatine interacts strongly with monolayers containing sitosterol, it does not interact with monolayers containing sitosterol glucoside. The response of monolayers of varying composition of DMPC and cholesterol to tomatine is also examined. Brewster angle microscopy (BAM) reveals further evidence for formation of suspected islands of tomatine + cholesterol complexes upon interaction with mixed monolayers of lipid and sterol.     

Comparative studies of lipase and phospholipase A2 acting on substrate monolayers.

The kinetic aspects of lipolysis by pancreatic lipase and phospholipase A2 from different sources have been compared using monomolecular films of short chain lipids as the substrates. Phosphatidylcholine monolayers, in contrast to phosphatidylethanolamine and phosphatidylglycerol monolayers, were resistant to hydrolysis by pancreatic lipase. The induction time, measured during pre-steady state conditions, increased abruptly for a given value of the surface pressure. This appears to be due to a degree of lipid packing above which the enzyme no longer can penetrate the lipid film. The existence of an optimum in the velocity versus surface pressure profile is the result of at least two counterbalancing factors. As the surface pressure increases, the amount of enzyme present in the interface decreases, whereas the minimal specific activity of the enzyme increases. From this study with monolayers we can conclude that activity of lipolytic enzymes used as tools for probing biological membranes will be greatly influenced by the physiochemical nature of the membrane-water interface. Thus, studies such as this one which can measure the penetrating ability of various lipolytic enzymes can be useful in deriving a better understanding of biological membrane structure.     

Formation of stable polypeptide monolayers at interfaces: controlling molecular conformation and orientation.

The molecular self-organization and structural properties of peptide assemblies at different interfaces, using either amphipathic or hydrophobic polypeptide helices, is described. The two peptides under investigation form stable monolayers on the water surface under the conservation of their molecular conformation, as studied by circular dichroism and polarization-modulation Fourier transform infrared (FTIR) spectroscopy. Using surface plasmon resonance and reflection-absorption FTIR, we show that such molecular layers can be transferred unaltered to solid substrates. Most importantly, the molecular orientation of the hydrophobic helices on solid supports such as gold can be controlled by choosing a particular procedure for the layer formation. The helices were oriented parallel to the interface in Langmuir-Blodgett monolayers, and perpendicular to the interface in self-assembled monolayers. Our reflection-absorption FTIR measurements have delivered for the first time direct experimental evidence for the molecular conformation and orientation of pure peptide monolayers. Suitable reference spectra of polypeptides with defined conformation and orientation are necessary to use this technique for the determination of the molecular orientation of peptides in monomolecular films. We have solved the problem for alpha-helical polypeptides by using bacteriorhodopsin as a reference in combination with synthetic alpha-helices of defined interfacial orientation. The present study shows the possibility of constructing immobilized peptide monolayers with predefined macroscopic properties and molecular structure by choosing the proper polypeptide amino acid sequence, the technique used for layer formation, and the supporting surface properties.