Interactions of cytochrome c and [14C]-carboxymethylated cytochrome c with monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin

1. The interactions between cytochrome c (native and [(14)C]carboxymethylated) and monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin at the air/water interface was investigated by measurements of surface radioactivity, pressure and potential. 2. On a subphase of 10mm-or m-sodium chloride, penetration of cytochrome c into egg phosphatidylcholine monolayers, as measured by an increase of surface pressure, and the number of molecules penetrating, as judged by surface radioactivity, were inversely proportional to the initial pressure of the monolayer and became zero at 20dynes/cm. The constant of proportionality was increased when the cytochrome c was carboxymethylated or decreased when the phospholipid was hydrogenated, but the cut-off point remained at 20dynes/cm. 3. Penetrated cytochrome c could be removed almost entirely by compression of the phosphatidylcholine monolayer above 20dynes/cm. 4. With phosphatidic acid and cardiolipin monolayers on 10mm-sodium chloride the binding of cytochrome c was much stronger and cytochrome c penetrated into films nearing the collapse pressure (>40dynes/cm.). The penetration was partly electrostatically facilitated, since it was decreased by carrying out the reaction on a subphase of m-sodium chloride, and the relationship between the surface pressure increment and the initial film pressure moved nearer to that observed with phosphatidylcholine. 5. Surface radioactivity determinations showed that [(14)C]carboxymethylated cytochrome c was still adsorbed on phosphatidic acid and cardiolipin monolayers after the cessation of penetration. This adsorption was primarily electrostatic in nature because it could be prevented and substantially reversed by adding m-sodium chloride to the subphase and there was no similar adsorption on phosphatidylcholine films. 6. The penetration into and adsorption on the three phospholipid monolayers was examined as a function of the pH of the subphase and compared with the state of ionization of both the phospholipid and the protein, and the area occupied by the latter at an air/water interface. 7. It is concluded that the binding of cytochrome c to phospholipids can only be partially understood by a consideration of the ionic interaction between the components and that subtle conformational changes in the protein must affect the magnitude and stability of the complex. 8. If cytochrome c is associated with a phospholipid in mitochondria then cardiolipin would fulfil the characteristics of the binding most adequately.     

Stability of unimolecular films of 32P-labelled lecithin

1. The stability of monolayers of a highly unsaturated yeast lecithin labelled with (32)P has been investigated by a surface radioactivity technique. 2. Lecithin films on distilled water at all surface pressures between 6 and 48dynes/cm. were completely stable on rapid perfusion of the subphase and on addition of ionic amphipathic substances to the film. 3. Ultrasonically treated lecithin added to the subphase caused a slow loss of surface radioactivity but little pressure change. 4. The addition of proteins to the subphase caused negligible changes in the film even when conditions were favourable for electrostatic heterocoagulation and penetration. 5. Lecithin films were not hydrolysed by a strongly acid subphase at room temperature. The very low rate of hydrolysis produced by alkali was proportional to the subphase OH(-)ion concentration: the apparent activation energy and temperature coefficient (Q(10)) of the reaction were 14250 cal. and 2.37 respectively. 6. Alkaline hydrolysis of lecithin monolayers was markedly stimulated by adding methanol (10-20%, v/v) to the subphase. The addition of ionic amphipaths to the monolayer had the expected type of effect on the hydrolysis rate, but its magnitude was far less than that suggested by an application of the Poisson-Boltzmann equation for ion distribution at a charged interface (Davies & Rideal, 1963).     

Effects of ionization and counterion binding on the surface areas of phosphatidic acids in monolayers

At 24-26 degrees C, force-area isotherms show that unionized dipalmitoyl phosphatidic acid forms a solid-condensed film while unionized egg and dioleoyl phosphatidic acids form liquid-expanded films. Surface area is a characteristic feature of a specific phosphatidic acid and the purity of a phosphatidic acid preparation can be established by the surface area of the unionized phosphatidic acid (acid subphase) at 17 dynes/cm (castor oil piston). Ionized dipalmitoyl phosphatidic acid desorbs from a monolayer at a measurable rate while ionized egg and dioleoyl phosphatidic acids desorb too slowly for rate studies. The apparent surface pK(2) for dipalmitoyl phosphatidic acid, calculated from desorption rates, is 9.4. Surface areas of the phosphatidic acids expand with ionization. Solid dipalmitoyl phosphatidic acid films expand only in the pK(2) region, showing one inflection point which indicates that the K(1)/K(2) ratio is less than 100 and that, as a consequence of this ratio, the apparent surface pK(1) is greater than 7.4. Liquid egg and dioleoyl phosphatidic acid films have two inflection points, expanding in both the pK(1) and pK(2) regions. The apparent surface pK(1) and pK(2) values, calculated from inflection points in surface area data, are 3.5 and 8.0, respectively. Film expansion with phosphatidate anions is less than anticipated, showing the presence of weak transient hydrogen bonds. Expanded phosphatidate anion films are condensed by alkaline earth cations. The Ca(2+) and Ba(2+) salts of completely ionized phosphatidic acids collapse from monolayers, showing that the phosphatidate anion may function as an ionophore for the transport of alkaline earth ions.-Patil, G. S., N. J. Dorman, and D. G. Cornwell. Effects of ionization and counterion binding on the surface areas of phosphatidic acids in monolayers.     

The interaction of cytochrome c with monolayers of phosphatidylethanolamine

1. The interaction between [(14)C]carboxymethylated cytochrome c and monolayers of egg phosphatidylethanolamine at the air/water interface has been investigated by measurements of surface radioactivity, pressure and potential. 2. On adding (14)C-labelled cytochrome c to the subphase under monolayers with a surface pressure below 24dynes/cm. there was an initial surface pressure increment as the protein penetrated, followed by an adsorption that could be detected only by a continued increase in the surface radioactivity. 3. Above film pressures of 24dynes/cm. only adsorption was observed, i.e. an increment in surface radioactivity with none in surface pressure. 4. The changes in surface parameters with penetration of cytochrome c added to the subphase were indirectly proportional to the initial pressure of the monolayer. With hydrogenated phosphatidylethanolamine the constant of proportionality was increased but penetration again ceased at 24dynes/cm. 5. On compressing a phosphatidylethanolamine film containing penetrated cytochrome c to 40dynes/cm. only a proportion of the protein was ejected on a subphase of 10mm-sodium chloride, whereas on a subphase of m-sodium chloride nearly all the protein was lost. 6. With both penetration and adsorption only a small proportion of the added cytochrome c interacted with the phospholipid films, and initially the amount bound was proportional to the added protein concentration. There was no evidence of a stoicheiometric relationship between the protein and phospholipid or the build-up of multilayers. The bonded protein was not released by removing cytochrome c from the subphase. 7. The addition of m-sodium chloride to the subphase delays the rate of protein penetration into low-pressure films, but the final surface-pressure increment is not appreciably decreased. In contrast, m-sodium chloride almost completely stops adsorption on to films at all pressures. 8. When sodium chloride is added to the subphase below cytochrome c adsorbed to monolayers at high pressures, so that the final concentration is 1m, only a proportion of the protein is desorbed and this decreases as the time of the interaction increases. This indicates that adsorption is initially electrostatic, followed by the formation of non-ionic bonds. 9. Alteration of the subphase pH under a high-pressure film leads to a steady increase in adsorption from pH3 to 8.5 followed by a rapid fall to zero adsorption at pH11. 10. The penetration into phospholipid monolayers at 10dynes/cm. shows a rate that is consistent with the relative electrostatic status of the two components of the interaction as the subphase pH is varied between 3 and 10.5. The final equilibrium penetration shows a pronounced peak in the increments of surface pressure at pH9.0 although a similar peak is not observed in the surface radioactivity. This indicates that more residues of the protein are penetrating into the film at about this pH. 11. Determinations were made of the electrophoretic mobilities of phosphatidylethanolamine particles both alone and after interaction with cytochrome c. 12. The electrophoretic mobilities of cytochrome c adsorbed on lipid particles showed an isoelectric point below that of cytochrome c. This and the observations on the monolayers suggest that, with cytochrome c, protein-protein interactions are weak compared with other proteins.     

The hydrolysis of phosphatidylinositol monolayers at an air/water interface by the calcium-ion-dependent phosphatidylinositol phosphodiesterase of pig brain.

1. The activity of Ca2+-dependent phosphatidylinositol phosphodiesterase (EC 3.1.4.10) of pig brain against [32P]phosphatidylinositol monolayers at an air/water interface has been measured. As the monolayer pressure was increased a sharp cut-off of enzymic hydrolysis occurred at 33 X 10(-3) N/m. 2. The addition of either phosphatidic acid, phosphatidylglycerol or oleyl alcohol increased the film pressure at which cut off occurred, as well as increasing the rate of hydrolysis at lower pressures. 3. The rate of hydrolysis, but not the cut-off pressure, was markedly increased by oleic acid and slightly increased by phosphatidylethanolamine. 4. Phosphatidylcholine, palmitoylcholine and octadecylamine decreased the cut-off pressure, as well as the enzymic activity below this pressure. 5. Stearic acid and stearyl alcohol had no effect on either the cut-off pressure or the activity. 6. All activators decreased the length of the lag phase before enzyme activity began, and phosphatidylcholine increased it. 7. These results are compared with the stimulatory and inhibitory effects of various amphiphiles observed previously with phosphatidylinositol dispersions [Irvine, Hemington & Dawson (1979) Eur. J. Biochem. 99, 525-530], and their possible relevance to the control of the phosphatidylinositol phosphodiesterase in vivo are discussed.     

Solution of fatty acids from monolayers spread at the air-water interface: identification of phase transformations and the estimation of surface charge

The contraction or decrease in area of fatty acid monolayers maintained at a constant surface pressure of 16 dynes/cm was studied as a function of fatty acid chain length, unsaturation, temperature, and the hydrogen ion concentration in the subphase. The data were consistent with the hypothesis that fatty acid solution from the monolayer into the subphase was the mechanism for film loss. Autoxidative reactions did not contribute significantly to film loss since contraction occurred with saturated fatty acid monolayers and with unsaturated fatty acid monolayers in an anaerobic environment. The decrease in area per unit time or the solution rate was inversely proportional to chain length and directly proportional to the degree of unsaturation. Arrhenius plots showed activation energies of 1.5-2.5 kcal mole(-1) for tetradecanoic, octadecenoic, and octadecadienoic acids, and 25 kcal mole(-1) for hexadecanoic acid. The solution rate from the monolayer increased in a sigmoidal fashion with an increase in subphase pH, and the apparent surface pK(a) was estimated as the point where the solution rate was half-maximum. Apparent surface pK(a) values were: hexadecanoic acid, 9.7; octadecenoic acid, 8.3; tetradecanoic acid, 7.9; and octadecadienoic acid, 8.0.     

Shielding of phospholipid monolayers from phospholipase C hydrolysis by alpha-lactalbumin adsorption

α-Lactalbumin interacts more strongly with lecithin and cardiolipin monolayers at pH 3～4 than at pH 7 to 10. At physiological pH this protein does not penetrate monolayers of DPPC and cardiolipin above pressures of 30 dynes/cm. Enzymatic hydrolysis of these monolayers by phospholipase C (Clostridium Welchii) is inhibited partially or totally when α-lactalbumin is injected in the subphase prior to the enzyme injection.     

Can mitochondria and synaptosomes of guinea-pig brain synthesize phospholipids?

1. The use of ;marker' enzymes for investigating the contamination by endoplasmic reticulum of mitochondrial and synaptosomal (nerve-ending) fractions isolated from guinea-pig brain was examined. NADPH-cytochrome c reductase appeared to be satisfactory. With the synaptosomal preparation there was a non-occluded enzymic activity believed to arise from contaminating microsomes and an occluded form released by detergent, which probably was derived from some type of intraterminal smooth endoplasmic reticulum. 2. Isolated brain mitochondria, both intact and osmotically shocked, could not synthesize more labelled phosphatidylcholine from CDP-[Me-(14)C]choline or phosphoryl[Me-(14)C]choline than could be accounted for by microsomal contamination. They could synthesize only phosphatidic acid and diphosphatidylglycerol from a [(32)P]P(i) precursor and not nitrogen-containing phosphoglycerides or phosphatidylinositol. 3. The synaptosomal outer membrane and the intraterminal mitochondria could not synthesize phosphatidylcholine from CDP-[Me-(14)C]choline but the synaptic vesicles and probably the intraterminal ;endoplasmic reticulum' appeared to be capable of catalysing the incorporation of label from this substrate into their phospholipids. 4. Microsomal fractions and synaptosomes from guinea-pig brain could incorporate [Me-(14)C]choline into their phospholipids by a non-energy-requiring exchange process, which was catalysed by Ca(2+). Fractionation of the synaptosomes after such an exchange had taken place revealed that the label was predominantly in the intraterminal mitochondria and not associated with membranes containing NADPH-cytochrome c reductase. 5. On the intraperitoneal injection of [(32)P]P(i) into guinea pigs, incorporation of radioactivity into phosphatidylinositol and phosphatidic acid was much faster than into the nitrogen-containing phosphoglycerides. Mitochondria and microsomal fractions showed a roughly equivalent incorporation into individual phospholipids, and that into synaptosomes was appreciably less, whereas the phospholipids of myelin showed little (32)P incorporation up to 10h.     

Ca2+-induced lateral phase separation in phosphatidic acid/phosphatidylcholine monolayers as revealed by fluorescence microscopy

Phase separation in mixed monolayers of phosphatidylcholine (PC) and pyrene-labeled phosphatidic acid (PA) was observed by fluorescence microscopy on an air/water interface as a function of subphase Ca2+ concentration and lateral packing pressure of the film. Below 45 mN m-1 and in the absence of Ca2+ no indications of phase immiscibility were observed. Addition of 1 mM Ca2+ caused extensive phase separation, which was evident immediately after spreading of the film. Further increase in Ca2+ concentration up to 30 mM increased the pyrene excimer intensity of the separated phosphatidic acid enriched domains. In the presence of Ca2+ (1-30 mM) and at surface pressures below 10 mN m-1 phase separation was always evident. However, as surface pressure exceeded 10 mN m-1, mixing of PC and PA occurred. Upon decompression of the film, phase separation reappeared at surface pressures close to 10 mN m-1. The surface textures of the film before and after the compression and subsequent relaxation were different. Inclusion of 30 mol% cholesterol increased the number and decreased the size of the PA domains. In films containing 50 mol% cholesterol no phase separation could be detected at the resolution available.     

Interactions of acetylcholine receptor and acetylcholinesterase with lipid monolayers

The interaction of acetylcholine receptor and acetylcholinesterase with lipid monolayers was followed by measuring changes in surface pressure.When injected into the subphase of a lipid monolayer, the proteins caused increases in surface pressure from 5 to 10 dynes/cm, indicating a penetration of protein into the monolayer. At pH values below the isoelectric point of the proteins the incorporation was improved. The same was observed when Ca²⁺ (2 mM) was added.The presence of the enzyme in the mixed film could be demonstrated by using diiso[³H]propyl fluorophosphate-labelled acetylcholinesterase as well as by measuring enzyme activity. Acetylcholine receptor was shown to be present in the mixed film by using a complex made of the receptor and α-[³H]neurotoxin.     

Adsorption of glyceraldehyde 3-phosphate dehydrogenase on condensed monolayers of phospholipid.

The adsorption of [14C] alkylated glyceraldehyde 3-phosphate dehydrogenase from rabbit muscle to condensed monolayers of phosphatidic acid was investigated under a variety of conditions. 2. The rate constant for association at 20 degrees C depended on ionic strength. At I/2=60mM the rate constant was 0.39min-1. At I/2=260mM it decreased to 0.27min-1. 3. The apparent association constant (Kass.) for adsorption at I/2=60mM was 1.06 X 10(6)M-1 and was strongly influenced by subphase changes in pH and ionic strength. Measurements of Kass. at 20 degrees and 5 degrees C gave a value for the apparent enthalpy change on adsorption of -33kJ-mol-1. Calculations of the apparent change in free energy and apparent entropy change for the adsorption process gave values of -34kJ-mol-1 and +2J-K-1-mol-1 respectively. 4. Decreasing the amount of phosphatidic acid in the monolayer by replacement with phosphatidylcholine caused the shape of the adsorption isotherm to change from apparent hyperbolic to sigmoid. Subphase changes in pH or ionic strength did not affect the shape of the adsorption isotherm. However, adsorption of enzyme on monolayers of 100% phosphatidic acid in the presence of 1mM-CaCl2 was sigmoid in nature. 5. It is concluded that glyceraldehyde 3-phosphate dehydrogenase binds to condensed charged monolayers by multiple electrostatic interactions. At low concentrations of phosphatidic acid in the monolayer or in the presence of Ca2+, this occurs in a two-step process and depends on lateral diffusion of phosphatidic acid for strong binding to take place.     

Lipid monolayers: interactions with the apoprotein of high density plasma lipoprotein

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.     

An analysis of the interaction of protein with lipid monolayers at the air/water interface

1. Measurements have been made of the interaction of cytochrome c, bovine serum albumin and synthetic oxytocin with low-pressure (2dyn/cm) monolayers of stearic acid, phosphatidylcholine and phosphatidylethanolamine. 2. [(14)C]Carboxymethylation of the cytochrome c and albumin followed by surface-radioactivity determinations have shown that only a proportion of the protein added to the subphase is bound to the monolayers and that initially the degree of binding is dependent on the protein concentration. The binding is irreversible in the sense that the adsorbed protein cannot be removed by transferring the film containing the interacted protein to a fresh subphase containing no protein. 3. Three successive types of interaction can usually be recognized. (a) Initially, whole molecules of protein penetrate the lipid film and occupy the same area as those of the protein spread at the air/water interface. (b) Above certain film pressures a part of each protein molecule, probably hydrophobic side chains, penetrates the film. The change in surface pressure per unit of bound protein is much smaller than in (a). (c) At higher film pressures, adsorption without penetration occurs. With cytochrome c this is initially dependent on a favourable electrostatic interaction.     

The penetration of serum albumin into phospholipid monolayers of different fatty acid chain length and interfacial charge

1. The highest surface pressure of phosphatidylcholine monolayers allowing penetration of delipidated serum albumin decreased in the order dibehenoyl>distearoyl>dipalmitoyl=dimyristoyl. This pressure was not related to the area occupied or to the space available between the phospholipid molecules at the interface. 2. Penetration of albumin into yeast phosphatidylcholine monolayers was increased by adding a small percentage of long-chain anions (phosphatidic acid, dicetylphosphoric acid) to the film but only when the protein was below its isoelectric point (i.e. positively charged). 3. Stearylamine added to phosphatidylcholine monolayers had no effect on albumin penetration even when the protein was oppositely charged to that of the phospholipid/water interface. 4. The results are discussed in relation to the activation of certain phospholipases by anionic amphipathic substances.     

Specific interaction of concanavalin a with glycolipid monolayers

The effect of ¹³¹I-labelled concanavalin A on the surface pressure and surface radioactivity of monolayers formed from phospholipids and from natural and synthetic glycolipids has been studied. The lectin binds to and penetrates dipalmitoyl phosphatidylcholine monolayers at a surface pressure of 15 dynes/cm and this interaction is inhibited by the presence of α-methyl mannose int he subphase. At surface pressures of 25 dynes/cm or higher, concanavalin A will interact with monoglucosyl diglyceride or diglucosyl diglyceride from Acholeplasma laidlawii and with synthetic glycolipids containing $\text{2}\text{or}\text{3 α1 → 4-}\text{linked}$ D-glucose residues in the headgroup, but not with phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, or with the ganglioside II³NeuAc-GgOse₄-Cer. The binding to the glycolipid sugar group and penetration of the hydrocarbon region seem to occur simultaneously, as the time courses for the development of surface pressure and surface radioactivity coincide.     

Activation of protein kinase C in lipid monolayers

The potential of lipid monolayers spread at an air-water interface was investigated as a well defined membrane model able to support protein kinase C (PKC) association and activation. PKC association to a mixed phospholipid film (phosphatidylcholine, phosphatidylserine) could be detected by an increase of the monolayer surface pressure. This association was strikingly dependent upon the presence of submicromolar concentrations of Ca2+. The effect of Ca2+ resulted in an increase of the PKC penetration into the lipid core at a given permissive surface pressure as well as in a marked increase of the critical surface pressure (29-38 dynes/cm) above which the enzyme was excluded from the membrane. Inclusion of diacylglycerol or tetradecanoate phorbol acetate (TPA) did not modify the PKC-monolayer association in a detectable manner. PKC associated to the lipid layer exhibited the expected catalytic property and was fully activated when diacylglycerol or TPA was included in the membrane. PKC activity was highly dependent upon the surface pressure of the lipid monolayer, being optimal between 30 and 35 dynes/cm. Study of the compression isotherm of various diacylglycerol structures revealed that all potent PKC agonists exhibited an expanded liquid phase behavior with collapse pressure below 40 dynes/cm, in contrast to weak activators which showed condensed isotherms with high collapse pressure (approximately equal to 60 dynes/cm). These observations showed that the lipid monolayer system is well adapted to the study of the molecular mechanisms involved in the regulation of PKC activity at a model membrane interface. They are in line with the suggestion of a major role of Ca2+ in the association (translocation) of PKC to membrane in living cell and suggest that diacylglycerol (and TPA) might activate membrane-associated PKC through local change in the surrounding lipid phase organization.     

Phospholipase A2 activity on mixed lipid monolayers: Inhibition and activation of phospholipases A2 from porcine pancreas and Crotalus adamanteus by anionic and neutral amphiphiles

The effects of anionic and neutral amphiphiles on porcine pancreatic and Crotalus adamanteus phospholipases A2 were studied in a monolayer system as a function of surface pressure. The insoluble amphiphile, dicetyl phosphate (DCP), inhibited the hydrolysis of didecanoylphosphatidylcholine (DDPC) by both enzymes below their normal cutoff pressures with pure DDPC. DCP, however, enhanced enzyme penetration and thus activated the pancreatic enzyme above its normal cutoff pressure. The soluble surfactants, 3,5-dibromo- and 3,5-diiodosalicyclate, acetyl salicylate, and salicylic acid, had similar effects. 1,2-Didecanoin inhibited the hydrolysis of DDPC below the normal cutoff pressures and increased the cutoff pressures for both enzymes. Zwitterionic detergents, N-dodecyl- and N-tetradecyl-N,N-dimethyl-3-aminopropanesulfonate, were found to be potent inhibitors of the pancreatic enzyme on DDPC monolayers. Relative substrate specificities for both enzymes were determined as a function of surface pressure with phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid. Pancreatic phospholipase A2 was more active and penetrated to higher pressures with the anionic phospholipids, while the venom enzyme was more active with phosphatidylcholine.     

Hydrophobic membrane protein from chromatophores of Rhodospirillum rubrum. Structural and spectroscopic studies of monolayers and multilayers.

A hydrophobic, lipid- and pigment-free polypeptide from the chromatophore membrane of Rhodospirillum rubrum was spread from chloroform/methanol, pyridine and formic acid solutions at an air-water interface. Surface pressure versus area isotherms of the monolayers formed at the interface were partially dependent upon the spreading solvent used. From the surface area at 20 dynes/cm compression, an average molecular area of 12.9 nm2/molecule was calculated for a polypeptide monolayer spread from chloroform/methanol. Multilayers built up on germanium plates at different surface pressures were subjected to attenuated total reflection infrared spectroscopy. In all cases the amide I and II absorption bands were typical of alpha-helical and random conformations. Electron microscopy of transferred monolayers replicated by rotary platinum shadowing revealed domains of regular texture in specimens prepared at 20 dynes/cm. Such domains were virtually absent in specimens prepared at 10 and 30 dynes/cm. Light optical diffractometry of the ordered arrays yielded a smallest repetitive area of 13.5 nm2 which agrees well with the molecular area obtained from the monolayer surface. Although no drastic changes in secondary structure were detected in the course of this study, some conformational changes are indicated by solvent-dependent differences in the surface pressure versus area isotherms.     

Biosynthesis of phosphatidylcholine by enzyme preparations from spinach leaves

The enzymic incorporation of choline-1,2-(14)C from CDP-choline-1,2-(14)C into phosphatidylcholine by spinach leaf preparations was characterized. The enzyme catalyzing the incorporation, choline phosphotransferase, had a pH optimum of about 8.0 and required either Mn(2+) or Mg(2+) as cofactor. The saturation concentration of Mn(2+) was 0.3 mm and that for Mg(2+) was 13 mm. The K(m) for CDP-choline was 10 micro m. The choline phosphotransferase was inhibited by sulfhydryl reagents. The enzyme was inactivated at 30 degrees C, but this inactivation could be prevented by dithiothreitol and Mn(2+). Preincubation of the enzyme with Mn(2+) prevented inhibition by sulfhydryl reagents. The incorporation of diglyceride-U-(14)C into phosphatidylcholine was also studied. The enzyme did not show any diglyceride specificity when exogenous diglyceride was added, indicating that fatty acid distribution in phosphatidylcholine of spinach is not controlled by choline phosphotransferase.     

Hepatic lipase hydrolysis of lipid monolayers. Regulation by apolipoproteins

A monolayer technique was used to study the substrate specificity of hepatic lipase (HL) and the effect of surface pressure and apolipoproteins on hydrolysis of lipid monolayers by this enzyme. HL hydrolyzed readily phosphatidylethanolamine monolayers. Pure trioctanoylglycerol was found to be a poor substrate but when progressively diluted with nonhydrolyzable 1,2-didodecanoylphosphatidylcholine hydrolysis of triacylglycerol by HL reached maximum at a molar ratio of 1:1 triacylglycerol to phosphatidylcholine. The activation of triacylglycerol hydrolysis was not due to altered penetration of HL. The surface pressure optimum of HL for the hydrolysis of phosphatidylethanolamine monolayers was broad between 12.5 and 25 mN/m. When apolipoprotein E was injected beneath the monolayer of phosphatidylethanolamine prior to enzyme addition, a 3-fold activation of HL was observed at surface pressures equal to or below 15 mN/m. Below surface pressures of 20 mN/m apolipoprotein E did not affect the penetration of HL into the lipid-water interface. Apolipoprotein E slightly activated the hydrolysis of triacylglycerol by HL at 10 mN/m. At a high surface pressure of 25 mN/m all apolipoproteins tested (apolipoproteins A-I, A-II, C-I, C-II, C-III, and E) inhibited the penetration into and HL activity on phosphatidylethanolamine At 18.5 mN/m all apolipoproteins except apolipoprotein E inhibited the hydrolysis of triacylglycerol in the triacylglycerol:phosphatidylcholine mixed film. Based on these results we present a hypothesis that phospholipid present in apolipoprotein E-rich high density lipoprotein-1 and triacylglycerol in intermediate density lipoprotein would be preferred substrates for HL.