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Using the radio-iodinated human serum albumin ([131-I]-RISA) dilution method to measure lamb fetal pulmonary fluid (FPF) volume, we followed the disappearance of protein-complexed, 14-C-labeled phosphatidylcholine ([14-C]PC) during the first 90 min after its injection into FPF. The FPF samples were analyzed for total lipid 14-C activity and for distribution of 14-C 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 14-C activity and for distribution of 14-C 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 PFP-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.

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.

Monolayer techniques were used to study the interactions of various lipids (cholesterol, lysophosphatidyl choline, phosphatidal ethanolamine, phosphatidyl choline, sphingomyelin, stearic acid, and lipids extracted from plasma high density lipoproteins and very low density lipoprotein) with the lipid-free protein subunit of rat plasma high density lipoprotein and with rat plasma albumin. The proteins were injected under the lipid monolayer at fixed area, and the increase in surface pressure (decrease in surface tension) was measured as a function of time. With all lipids, both the rate and magnitude of this increase were greater with the apolipoprotein than with albumin. The degree of film penetration of pure lipid films (at an initial film pressure of 15 dynes/cm) by the two proteins followed the same order: cholesterol > phosphatidal ethanolamine > phosphatidyl choline > stearic acid > sphingomyelin > lysophosphatidyl choline. Other variables studied were protein concentration, initial film pressure, and pH. Two distinctive properties of the apolipoprotein were the penetration of lipid films at pressures above the collapse pressure of the protein, and the formation of a film even at low salt concentration. High surface activity and strong interaction of HDL-protein with lipid monolayers may be associated with the flexibility of the protein molecule due to absence of disulfide bridges. The unusual surface activity of HDL-protein may be intimately related to the mechanism of formation of the lipoprotein.

To study mechanisms whereby lytic proteins disrupt biomembranes, artificial phospholipid spherules (liposomes) were exposed to melittin, a cationic peptide in which sequences of hydrophobic (Positions 1 to 20) and hydrophilic (Positions 21 to 26) amino acids are unequally distributed. At concentrations above 10-6 m, melittin, which is the major toxin of bee venom, released marker anions (CrO4=) or glucose from the model structures. Release of marker ions was equivalent whether liposomes were prepared with a net negative (dicetyl phosphate) or net positive (stearylamine) charge. Nor was the presence of cholesterol in the model membranes necessary for the action of melittin. Spherules with net negative charges competed approximately 10 times more effectively for melittin's lytic activity on erythrocytes than did spherules with positive charges. Studies with lipid monolayers indicated that melittin had a remarkable affinity for the air-water and more so for the lipid-water interface, for it spread as a film readily from water and penetrated lipid monolayers avidly, irrespective of the surface charge of the lipid film. Negatively stained preparations of liposomes in the electron microscope showed that melittin-treated spherules lost the integrity of their concentric lamellae, which became beaded, frayed, and finally fragmented. These studies indicate that the lytic effects of melittin upon biological membranes may be due to a similar reaction with structural phospholipids. Since ionic interactions between the cationic peptide and charged lamellae were not crucial for lysis, and since melittin has an extraordinary affinity for lipid membranes, it is suggested that the surface activity of melittin and convenient apolar associations between hydrophobic portions of melittin and the acyl chains of phospholipid could account for disruption of the spherules.

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.

The activity of phospholipase A on phosphatidyl choline and phosphatidal choline spread as monolayers on phosphate buffers containing snake venom (Crotalus atrox or Naja naja) was studied by measuring the fall of surface potential as a function of time, pH, film pressure, temperature, and concentrations of phosphate and venom. At 25 degrees C, pH 7.0, and 0.2 micrograms of venom per ml, optimal activity was observed with both venoms on both substrates at 12 dynes/cm film pressure on 0.04 m phosphate. Under these conditions, the pH optimum for C. atrox was broad (6.6-7.4) and that for N. naja was sharp (8.0) for the action on phosphatidyl choline, whereas both venoms had a sharp optimum at pH 8.0 in their action on phosphatidal choline. The optimal temperature with phosphatidyl choline was 27.5 degrees C for N. naja and 40 degrees C for C. atrox. In line with studies of phospholipase A activity in bulk phase in ether, phosphatidal choline was attacked much more slowly than phosphatidyl choline by C. atrox. Under conditions where both venoms had equal activity on phosphatidyl choline, C. atrox was only half as active as N. naja on phosphatidal choline. The studies suggest that the linkage of the hydrophobic chains in glycerophosphatides may affect their interaction with proteins.

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.

Staphylococcal α-toxin spreads readily as a film on aqueous media; the film contains 3 and 30 times as much protein as in films of mitochondrial structural protein and in a protein monolayer, respetively. The toxin forms a film also by adsorption from the hypophase. The ability of α-toxin both to spread and adsorb at the air-water interface is increased markedly by 6 M urea. Penetration of α-toxin into lipid monolayers is influenced greatly by the chemical structure of the lipid; the ΔΠ values are in the order cholesterol > phosphatidyl choline > phosphatidyl inositol > sphingomyelin > ganglioside. In line with the known binding of gangliosides by histones, penetration of the basic toxin into the air-water interface is inhibited by ganglioside as well as by sulfatide monolayers. Inasmuch as film penetration (ΔΠ) is consistent with anchoring of the protein at the interface, binding of the cationic toxin under the negatively charged monolayers of phosphatidyl inositol, sulfatide, or ganglioside explains the low ΔΠ values observed with these lipids and the known ability of this protein to coat the host membrane. Penetration of α-toxin into lecithin monolayers increases as the protein concentration in the hypophase becomes greater, as the initial pressure of the lipid film becomes smaller, as pH or temperature of hypophase increases.

The quantities of the N -terminal amino acids of the proteins investigated were increased twofold to tenfold and more by action of sodium dodecyl sulfate in acid; serine and threonine were affected most. The limited action of trypsin caused an increase of the quantities of all the N -terminals of porcine γ-globulin. In all the cases investigated, although the quantities changed, no new N -terminal appeared beside the ones present in the untreated protein. The submolar quantities of the N -terminals of the proteins under study were not impurities; the increased quantities of N -terminals derived not from the uncovering of masked N -terminals because of denaturation, but more likely from a specific hydrolysis, which was catalyzed by trypsin and by the acidic alkyl sulfate. It is suggested that the N -terminals of a number of proteins may be the products of autolysis or hydrolysis (of particularly labile peptide bonds) which took place at various times during the physiological and laboratory history of the protein molecule.

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.

Centrifugation techniques that reportedly yield a specific surface active lipoprotein from pulmonary extracts were applied to selected fractions with the view to see if they contained serum type lipoproteins. The fractions were investigated with the aid of qualitative and quantitative methods. These included disc electrophoresis with specific staining for lipoprotein, and determination of phospholipid and protein content of the various Centrifugation fractions. Ultracentrifugation and disc electrophoresis, which revealed consistently purified serum lipoproteins as well as the lipoproteins in intact serum, did not show any such lipoprotein in pulmonary washings or lung homogenates. We cannot exclude however the presence of a special type of lipoprotein or lipid-protein aggregate which breaks into lipid and protein during Ultracentrifugation or disc electrophoresis.

STUDIES on the action of enzymes on lipids and proteins of model membranes are aimed at identifying the relative position and orientation of the hydrophilic groups of the various membrane constituents1,2 so that models of the molecular organization and surface topography of membrane structures can be built.

A method is described for the rapid isolation of phosphatidyl inositol from soybean phosphatides (Asolectin). The product is obtained pure as the crystalline sodium salt.

Structural proteins in biological membranes are bimodal and ideally possess a unique surface activity that should be amenable to study by monolayer techniques. Proteins so studied, however, display, of necessity, a surface activity that reflects the laboratory history of the protein. The Folch-Lees proteolipid apoprotein was isolated from calf-brain myelin in both an organic solvent (CHCl 3 -CH 3 OH-CH 3 COOH; 50:50:1) and water. The apoprotein did not spread as a film from water, but did spread readily from the organic solvent into films that had high saturation pressures. The partitioning of the apoprotein from organic solvent to water was found to be reversible, as was the gain and loss of surface activity. Infrared spectroscopy demonstrated that the preponderant structure in films of the apoprotein was not that of extended polypeptide chains. The results suggested that the preferred structures at the interface are bimodal coils of alpha helix. This structure can sustain the high film pressures observed, whereas beta keratin structures would build films with high surface viscosity and very low surface pressure.

MUCH is known about the action of urea on proteins; the mechanism, however, is not understood. The long-accepted hydrogen-bond mechanism, once deemed quantitatively unsatisfactory1 but never tried seriously, is shown here to be erroneous on various theoretical and experimental grounds2,3. The alternatives in terms of the hydrophobic bond theories4–6 are stimulating but not fully consistent with the evidence: they are too arbitrary in that they do not take into account some important properties of urea and ignore the significant facts1,7,8 which are known about the action of urea and which cannot be in the domain of the hydrophobic forces alone. In all the previous studies no attention was paid to urea itself and to the polar properties which are obvious in the structure of the urea molecule and presumably fundamental to its mode of action. This communication aims at postulating and describing the urea cation, a zwitterion with a positive head charge.

A PREVIOUS report1 indicated that porcine γ-globulin contained at least 6 N-terminals, which, however, were not identified and measured. This communication deals with the results and interpretation of more recent work2–6.

Under conditions in which neither ribonuclease nor lysozyme formed a film by either spreading or adsorption at the air-water interface, the surface activity of staphylococcala-toxin and streptolysin S in the absence of lipid was greatly enhanced by the presence of 6 M urea in the hypophase. The influence of urea on the anchoring of the protein was quantitatively similar to that of a lecithin film. Admixture of up to 50 mole % cholesterol to egg lecithin preserved the lecithin character of the lipid monolayer in the penetration ofa-toxin, suggesting probably binding of cholesterol to phosphatidyl choline in the presence of excess lecithin. Penetration ofa-toxin and streptolysin S into the air-water interface was inhibited by lipid monolayers containing gangliosides. The inhibition, however, was removed when the subphase contained 6 M urea. The resulting penetration curve ofa-toxin was identical with the penetration curve of the protein into the lecithin film in the absence or in the presence of urea. In general, the denaturing agent causes the protein to acquire a hydrated conformation, from which restructuring in the low dielectric constant of air or lipid is favored and brings about results comparable with those effected by the lipid. Pre-incubation ofa-toxin with gangliosides or sulfatides caused a marked decrease in film penetration of the toxin into lecithin monolayers. Unlikea-toxin, streptolysin S was extremely sluggish in penetration of the air-water interface and of several lipid monolayers. Protein concentration, a complement of either lipid or membrane protein, and specific molecular organizations of membranes must be taken into account in all cases; they, in particular, may be the basis for the enhanced cytolytic activity of streptolysin S in vivo.

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.

A method is described for the rapid isolation of phosphatidyl inositol from soybean phosphatides (Asolectin). The product is obtained pure as the crystalline sodium salt.

The activity of phospholipase A on phosphatidyl choline and phosphatidal choline spread as monolayers on phosphate buffers containing snake venom (Crotalus atrox or Naja naja) was studied by measuring the fall of surface potential as a function of time, pH, film pressure, temperature, and concentrations of phosphate and venom. At 25 degrees C, pH 7.0, and 0.2 micrograms of venom per ml, optimal activity was observed with both venoms on both substrates at 12 dynes/cm film pressure on 0.04 m phosphate. Under these conditions, the pH optimum for C. atrox was broad (6.6-7.4) and that for N. naja was sharp (8.0) for the action on phosphatidyl choline, whereas both venoms had a sharp optimum at pH 8.0 in their action on phosphatidal choline. The optimal temperature with phosphatidyl choline was 27.5 degrees C for N. naja and 40 degrees C for C. atrox. In line with studies of phospholipase A activity in bulk phase in ether, phosphatidal choline was attacked much more slowly than phosphatidyl choline by C. atrox. Under conditions where both venoms had equal activity on phosphatidyl choline, C. atrox was only half as active as N. naja on phosphatidal choline. The studies suggest that the linkage of the hydrophobic chains in glycerophosphatides may affect their interaction with proteins.