Giuseppe Colacicco

Applications of monolayer techniques to biological systems: symptoms of specific lipid-protein interactions.

Abstract Four examples of specific lipid-protein interaction are described as they are revealed by monolayer techniques. One, interaction of a purified antilactoside antibody with a lecithin monolayer containing 20 mole % fN-acyl dihydrosphingosyl lactoside resulted in blocking of the ΔΠ-mechanism for the penetration of protein into the lipid film; addition of lactose to the subphase rextored the large ΔΠ value typical of the nonspecific free penetration of γ-globulin. Two, mitochondrial structural protein (MSP) at low concentration (2 αg/ml) does not penetrate monolayers of either phosphatidyl choline or mitochondrial phospholipid, most likely for the same reason that this protein does not form a film by surface denaturation; however, a dramatic rise in film pressure took place when mitochondrial phospholipid and mitochondrial structural protein were mixed together in the bulk phase in the lipid/protein ratio 1 4 by weight. Three, in contrast, a marked absence of film pressure was observed when the apoprotein was mixed in the bulk phase with the total lipid of the lipoprotein from rat serum in a lipid/protein ratio 1 1 ; the marked loss of pressure, as compared with the pressure produced by either the lipid alone or the lipid-protein mixtures at other ratios, is explained by a model in which the lipid character is lost probably because the lipid is removed by interaction with the protein at that critical ratio. The sharp pressure changes occurred at lipid/protein ratios of known significance, namely, 1 1 for rat lipoprotein and 1 4 for the lipid-MSP complex. Four, and finally, the accessibility of phosphatidyl choline of films of high-density lipoprotein to phospholipase A from Crotalus atrox snake venom varied with different animal species; the results were interpreted as reflecting differences in the molecular organization of the native lipoproteins and particularly in the specific interfacial conformations of the apoprotein. Accordingly, the accessibility of egg lecithin in lecithin-protein films was influenced markedly (1) by the nature of the protein, namely, specific vs. nonspecific, and (2) by the order of addition of lipid and protein at the interface. The data are consistent with variations in the relative orientation of lipid and protein, in a model in which a mosaic structure is prevailing with lipid packages surrounded by protein. Differences in lipase effect among animal species and among experimental conditions are related to variations in the degree of masking of the lecithin surface by the specific configurations of the apoproteins.

Lipid monolayers: mechanisms of protein penetration with regard to membrane models.

The influence of lipid and protein on the properties of the air-water interface is analyzed with the view to formulate a mechanism of interaction of protein with lipid monolayers. The increase in surface pressure (ΔΠ) and the quantity of protein incorporated in the lipid film after injection of protein under lipid monolayers were studied as a function of both lipid structure and protein structure. With rabbit γ-globulin, the values of ΔΠ were cholesterol > phosphatidyl choline > sphingomyelin. Similar results were obtained with ribonuclease, lysozyme and serum albumin. The quantities of protein found in films of either cholesterol or phosphatidyl choline (egg lecithin) were much larger than those calculated from a geometric model in which a protein monolayer occupies the area made available by the compressed lipid. Arguments are produced against penetration based on simple mechanisms of compressibility of the lipid film. The mechanisms operating in the incorporation of protein into lipid monolayers are grouped into three categories: (a) free penetration, typical of lecithin; (b) binding-mediated penetration, typical of cholesterol and some glycosphingolipids; and (c) binding-inhibited penetration, typical of the albumin-ganglioside system and a specific lipid hapten-antibody system. A model is described in which nonspecific protein interacts with polymeric lecithin structures (surface micelles). In the sequence of events X»Y»Z, the globular protein X is activated into the expanded or extended form Y by contact with the lipid and then restructured into a compact form Z with release of water and free energy. The resulting lipid-protein assembly has a mosaic structure in which lipid and protein polar surfaces are exposed to water. Accessibility of lecithin to phospholipase A is consistent with the model and with current views on the state of protein in biological membranes; according to such views, protein is more likely structured inside the lipid milieu and not simply denatured on the lipid-water interface.

Lipid monolayers: surface potential of dipalmitoyllecithin with regard to ion sorption and Ca2+ binding

Abstract In line with previous work, the surface potential of commercial preparations of dipalmitoyllecithin on aqueous support increased in the order water 2 . This effect, indicating counterion binding on acidic sites, was not observed, however, after purification of lecithin on a silicic acid column. The potentials that previous authors attributed to fixed dipoles of the lipid do not exist. The increased potentials of dipalmitoyllecithin on CaCl 2 solutions in comparison with NaCl were the effect of interaction of Ca 2+ with the fixed charges of acidic contaminants present in ordinary preparations of the otherwise neutral phospholipid. The electrical potential of the Gouy diffuse double layer found in monolayers of the neutral phospholipid is indeed produced by ionic impurities. The claim that the fixed dipoles have large potentials comparable to those of fixed charges is thus incorrect. The fallacy is caused by a fundamental error in the theory of surface potentials at the point where the formalism of the parallel plate capacitor, requiring free charges, is applied to the treatment of uncharged dipoles, which are systems of neutralized charges.

Lipid monolayers: Interaction of synthetic dihydroceramide lactosides with proteins

Abstract Appreciation of the chemical basis of specific lipid-protein interactions in biological systems requires more extensive study of nonspecific (as well as specific) interactions. Monolayers of glycosphingolipids were studied as these lipids are immunologically active and are known to form specific complexes with antibody. Rabbit γ-globulin, rabbit serum albumin, and bovine ribonuclease were injected under monolayers of synthetic N-myristoyl-, N-palmitoyl-, and N-stearoyl dihydrosphingosyl lactosides (C14-, C16-, and C18-dihydroceramide lactosides) at a definite film pressure. The interaction was followed by measuring the increase in surface pressure (π) as a function of time, of initial film pressure (πi), of temperature, and of protein concentration. The degree of interaction of these lipids with each of the proteins at 25°C. was in the order C16 > C18 ⪢ C14. In the interaction with rabbit γ-globulin, the surface pressure-temperature curve had a maximum at 25°C. for N-palmitoyl dihydroceramide lactoside and 35°C. for the stearoyl homolog. When different classes of lipids were compared, the order of interaction at 25°C. was: cholesterol > N-palmitoyl dihydroceramide lactoside > phosphatidyl ethanolamine > phosphatidyl choline. As expected, the increase in π was smaller at larger values of πi, but a maximum at low pressure (1 dyne/cm.) was found. With monolayers of N-palmitoyl dihydroceramide lactoside at an initial film pressure of 2 dynes/cm., the degree of interaction at 25°C. was in the order ribonuclease > γ-globulin > albumin. Curves relating degree of interaction to protein concentration showed discontinuities that were different for each system. The data agree with earlier studies on the relative reactivity of different classes of lipids and lend support to two concepts. First, the mechanism of penetration of a lipid film at low pressure by protein is one involving interaction between polar groups rather than simple diffusion of protein into the film. Second, the structure of water around the polar groups of both lipid monolayer and protein is an important factor in lipid-protein interaction.

pH, temperature, humidity and the dynamic force-area curve of dipalmitoyl lecithin

Both high pH at 25 degrees C and humidity at 37 degrees C prevent DPL films from attaining zero surface tension, whereas humidity at 25 degrees C and high pH at 37 degrees C do not. At 37 degrees C DPL lowered surface tension to zero when spread from organic solvent or when absorbed from aqueous 0.15 M NaCl in the surface balance in which the surface film was exposed to the room air (dry film). Upon saturation of the atmosphere with water vapor in equilibrium with the aqueous phase at 37 degrees C in a closed chamber, DPL lost the ability to produce zero surface tension, and the gamma min of the DPL film increased from zero to 22 dyne/cm. Addition of DPL in chloroform to distilled water before dispersion by sonication did not prevent the effect of the humidity. However, when the chloroform solution of DPL was added to 0.15 M NaCl before sonication, the adsorbed film produced immediately a stable gamma min of zero in a saturated atmosphere, 37 degrees C. In the absence of chloroform, with DPL adsorbed from either distilled water or 0.15 M NaCl, the effect of humidity was reversed either by removing the chamber and returning the wet film to room air or by introducing small quantities of dispersing agents such as cholesteryl palmitate. However, whereas the effect of humidifying the air was reversible indefinitely, the effect of cholesteryl palmitate (zero surface tension, wet or dry film) was irreversible. This means that there are substances or conditions that can assist DPL films in maintaining zero surface tension when such films are exposed to humidity-saturated air at 37 degrees C.

Lipid monolayers: Ionic impurities and their influence on the surface potentials of neutral phospholipids

Abstract The ionic effects attributed to lecithin and sphingomyelin are due to ionic contaminants present in ordinary preparations of these lipids. Removal of acidic impurities by silic acid chromatography produces lecithins which are neutral and do not show interaction with Ca ++ by surface potential measurements. Similarly, improved preparations of sphingomyelin show a zero charge, whereas silicic acid chromatography removes cationic impurities which otherwise confer sphingomyelin a positive charge.

Effects of prostaglandins E2 and F2α on lecithin biosynthesis by cultured lung cells

Transformed cells from human lung carcinoma (Line A549), resembling type II pneumocytes, were cultured in monolayer at 37 degrees C and incubated for five hours with 3H-choline and 14C-palmitate in the presence of various concentrations of prostaglandins (PGS) E2 and F2alpha. In the control (no PG) the level of % palmitate incorporation was 13.5 x as high as that of choline, after taking isotope dilution into account. Between the concentrations studied, 0.1 and 10 muM, both prostaglandins stimulated markedly the incorporation of both precursors, though choline up to 3 x better than palmitate. This was indicated by a change in the palmitate/choline incorporation ratio from 13.5 to as low as 4.2. At the lowest PG concentration, 0.1 muM, PGE2 was much more effective than PGF2alpha in stimulating the incorporation of both precursors.

Lipid monolayers: Surface viscosity of dipalmitoyl lecithin in relation to surface potential and ion binding☆

Abstract The surface potential-surface pressure curve of dipalmitoyl lecithin on 150 m M NaCl at 25°C is identical with that on 75 m M CaCl 2 over the entire film pressure region; at high film pressure, however, the surface viscosity on CaC1 2 is markedly greater than on NaCl. We propose a model of molecular organization in which a lattice is formed from the coalescence of the water structured around the lecithin polar groups in the hydrophilic region below the II interface ; such a lattice is responsible for the surface viscosity but does not influence the surface potential. The greater viscosity effect of CaCl 2 over that of NaCl is probably determined by the influence that the electrolyte exerts on the water structured around the polar surface of the lipid and thus on the extent and strength of the coalescence, and it may not be due to binding of Ca 2+ onto the ionic groups of lecithin.

Lamb Fetal Pulmonary Fluid. II. Fate of Phosphatidylcholine

Extract: Using the radio-iodinated human serum albumin ([131I]-RISA) dilution method to measure lamb fetal pulmonary fluid (FPF) volume, we followed the disappearance of protein complexed, 14C-labeled phosphatidylcholine ([14C]PC) during the first 90 min after its injection into FPF. The FPF samples were analyzed for total lipid 14C activity and for distribution of 14C in PC, other phospholipids (PL), fatty acids (FA), and neutral lipids (NL). For most sampling periods ascending aortic (AAo) and right atrial (RA) blood samples were obtained simultaneously with FPF and serum was analyzed for total lipid 14C activity and for distribution of 14C in total PL, FA, and NL. These studies indicate that (1) PC is cleared rapidly from FPF with an estimated half-time of 15–57 min; (2) FPF-PC may be metabolized to lyso-PC and FA within the fluid itself; and (3) FA derived from FPF-PC enter the pulmonary circulation, thus establishing a pulmonary arteriovenous FA gradient. The possible sites at which PC may be cleared from FPF are considered.Speculation: The novel possibility is suggested that FPF contains appropriate enzymes (phospholinase(s) for deacvlation of and also that PC-degradative enzymes are active at the surface of the alveolar epithelial cells. By comparison with results of others regarding the half-life of PC in the air-lung, it appears that PC clearance outside the cell (i.e., after secretion) occupies a relatively short period in the turnover of the molecule. Since the products of PC degradation appear in arterial blood as FA primarily, we may consider FPF as a possible source of serum FA.

Effect of serum albumin on dynamic force-area curve of dipalmitoyl lecithin.

In line with previous findings at 25 degrees C, solutions of serum albumin in the subphase stabilized the surface activity of DPL spread films at 25 degrees C as well as 37 degrees C. In contrast, films adsorbed from mixtures of DPL and albumin exhibitied a marked inhibitory action of the albumin on DPL activity. The inhibitory effect increased with the relative protein concentration but, with albumin/DPL ratios smaller than 2, the DPL activity was regained gradually with cycling. With larger albumin/DPE negative effect of albumin was counteracted by higher temperatures (37 degrees C vs 25 degress C) and modest cholesterol concentrations; with greater cholesterol concentrations the known inhibitory effect of cholesterol prevailed. The inhibitory effect of albumin was potentiated by humidity; saturation of the atmosphere with water vapor at 37 degrees C abolished the DPL character of DPL-RSA mixtures and prevented its return (zero surface tension) upon reversal of the atmosphere from saturated water vapor to dry air. The data are important in the interpretation of the surface activity of pulmonary washings and other pulmonary extracts.