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Phospholipid-Diacylglycerol Complexes Regulate Colipase Adsorption to Monolayers† Nancy K. Mizuno,‡ Janice M. Smaby,‡ Beth A. Cunningham,§ Maureen M. Momsen,‡ and Howard L. Brockman*,‡ The Hormel Institute, University of Minnesota, 801 N.E. 16th Avenue, Austin, Minnesota 55912 Received July 1, 2002 Monomolecular films of a 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and any of several nonphospholipids, like diacylglycerols and free fatty acids, inhibit the initial rate of adsorption of an amphipathic protein, colipase, more effectively than the same two-dimensional concentration of phosphatidylcholine alone, even though the non-phospholipids alone have no effect [Sugar, I. P.; Mizuno, N. K.; Momsen, M. M.; Brockman, H. L. Biophys. J. 2001, 81, 3387-3397]. Inhibition correlates with complex formation between the phosphatidylcholine and non-phospholipid constituents of the monolayer. In the present study, we have examined the formation of complexes of 1,3-dioleoylglycerol with 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine and other species more representative of the phospholipid classes more typically found in the inner leaflet of cellular plasma membranes. These were bovine liver phosphatidylinositol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, -phosphoserine, and -phosphate. Analysis of surface pressure-surface potential-molecular area isotherms showed that all phospholipid species tested were miscible with and formed complexes with 1,3-dioleoylglycerol. Complex compositions were in the range of 0.14-0.25 mole fraction of diacylglycerol. For the phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol mixtures with diacylglycerol at surface pressures near collapse, the initial rate of adsorption of colipase was near zero up to at least the complex composition. With increasing diacylglycerol, the initial adsorption rate increased rapidly to the value observed in the absence of lipid. In contrast, adsorption rates with the phosphatidic acid species increased rapidly beginning at low diacylglycerol mole fractions and were half-maximal at the complex composition. All the data could be analyzed successfully with a statistical model based on the phospholipid exhibiting an excluded area that is independent of overall lipid composition and packing. Values of the excluded areas obtained from the analysis showed an approximate 1:1 correspondence with molecular areas of the complexes, assuming no complex for phosphatidic acid. This study shows that phospholipid classes of the type typically exposed to peripheral proteins in the cytoplasm of cells can complex with non-phospholipid second messengers, like diacylglycerols, that are generated in the inner leaflet in response to ligand-receptor interaction on the cell surface. Thus, their complexation with phospholipids has the potential, as for colipase binding, to regulate the translocation of peripheral proteins to the plasma membrane.
The interaction of ligands with transmembrane receptors on the surface of cells leads to receptor aggregation and signal transduction across the plasma membrane. A common consequence of such an event is the activation of phospholipases on the cytoplasmic surface of the membrane. Some of these, for example, phospholipases A2 and C,1 generate non-phospholipid products such as free fatty acids and diacylglycerols.2,3 These molecules, termed lipid second messengers, help trigger the translocation of soluble proteins, termed peripheral or amphitrophic proteins, from the cytoplasm to the plasma membrane and, thereby, activate signaling cascades involving soluble proteins that ultimately reach the nuclear membrane. There, more lipid second messengers are generated by phospholipases.4 These events ultimately lead to the * To whom correspondence may be addressed. E-mail: hlbroc@ hi.umn.edu. † Part of the Langmuir special issue entitled The Biomolecular Interface. ‡ University of Minnesota. § Bucknell University, Lewisburg, PA. (1) Exton, J. H. Biochim. Biophys. Acta 1994, 1212, 26-42. (2) Ohanian, J.; Liu, G.; Ohanian, V.; Heagerty, A. M. Acta Physiol. Scand. 1998, 164, 533-548. (3) Payrastre, B.; Missy, K.; Giuriato, S.; Bodin, S.; Plantavid, M.; Gratacap, M.-P. Cell. Signalling 2001, 13, 377-387. (4) D’Santos, C. S.; Clarke, J. H.; Divecha, N. Biochim. Biophys. Acta 1998, 1436, 201-232.
regulation of transcription of the genes that respond to the particular signaling pathway. Diacylglycerols and other lipid second messengers can function in two different ways. Peripheral proteins can have a specific binding site for a particular lipid second messenger, as exemplified by members of the protein kinase C family.5,6 However, these and other proteins are activated by molecules such as diacylglycerols and fatty acids in more nonspecific ways that have been attributed to phase separation, perturbation of membrane curvature, or increase in fluctuations due to the spacing effects of the non-phospholipids.6-9 Such regulation has also been observed for other, nonintracellular proteins that function after translocating to phospholipid-stabilized emulsion surfaces, such as triacylglycerol lipases10 and insect apolipoproteins.11 In gel-state bilayers, calorimetric and other studies have indicated complex, that is, pseudocompound, formation (5) Hurley, J. H.; Misra, S. Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 49-79. (6) Gschwendt, M. Eur. J. Biochem. 1999, 259, 555-564. (7) Zidovetzki, R.; Lester, D. S. Biochim. Biophys. Acta 1992, 1134, 261-272. (8) Cornell, R. B.; Arnold, R. S. Chem. Phys. Lipids 1996, 81, 215227. (9) Gon˜i, F. M.; Alonso, A. Prog. Lipid Res. 1999, 38, 1-48. (10) Brockman, H. L. Biochimie 2000, 82, 987-995. (11) Narayanaswami, V.; Ryan, R. O. Biochim. Biophys. Acta 2000, 1483, 15-36.
10.1021/la026172m CCC: $25.00 © 2003 American Chemical Society Published on Web 10/29/2002
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between non-phospholipid second messengers and phospholipids. For diacylglycerols mixed with phospholipids, these are generally in the range of 30-50 mol % of diacylglycerol,7,9 but for 1,3-dioleoylglycerol (a subject of this study) mixed with 1-palmitoyl-2-oleoyl-sn-glycero3-phosphorylcholine (POPC) and its 1-steaoryl homologue (SOPC) a complex containing 25 mol % diacylglycerol was reported.12 Fluid-phase bilayers of unsaturated diacylglycerols and phospholipids are reportedly miscible without evidence of complex formation.9 However, with increasing diacylglycerol in these systems a transition from the lamellar to the cubic or hexagonal phase limits the range of observation of the lamellar phase. One way to avoid changes in curvature is to study lipids in mixtures in monomolecular films at the gas-liquid interface. This has been done for a variety of non-phospholipids and POPC in the liquid-expanded state.12,13 In the case of POPC or SOPC mixed with either 1,3- or 1,2-dioleoylglycerols, a 25 mol % diacylglycerol complex was observed,12,13 just as in the bilayer study. More recently Sugar et al.14 studied the influence of SOPC mixed with 1,2- or 1,3-diacylglycerols or other nonphospholipids on the initial rate of adsorption of colipase to monolayers. Colipase is a small, rigid, wedge-shaped amphipathic protein that facilitates the interaction of pancreatic lipase to phospholipid-rich surfaces.10 The impetus for that study was the recognition that the association of colipase with phosphatidylcholine-rich interfaces is a key event in the subsequent binding of lipase and the initiation of lipolysis. The results14 showed that in liquid-expanded monolayers, SOPC inhibited the colipase adsorption rate abruptly as its two-dimensional concentration was increased, whereas non-phospholipids had no effect. Surprisingly, however, non-phospholipids such as diacylglycerols enhanced the inhibition of colipase adsorption rate by SOPC and the rates were negligible until the non-phospholipid mole fraction exceeded that in the complex. The lipid monolayers were all liquid expanded and the lipids were miscible in all proportions, precluding any sort of phase transition/separation as an explanation. These data were successfully analyzed using a simple statistical model from which excluded phospholipid molecular areas were obtained. These were in good agreement with the collapse areas of the complexes, measured independently, indicating that complexes inhibit colipase adsorption. The results described above suggest that when diacylglycerols and other non-phospholipid lipid second messengers are generated in cellular membranes they could exist in part as complexes. In that state, they may not support the interaction of peripheral proteins with membranes to the same extent as when uncomplexed. However, the inner leaflet of the plasma membrane in cells, a major site of lipid second messenger generation, is comprised primarily of phospholipids other than phosphatidylcholines. Although data are limited, the predominant classes are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol.15 Phosphatidic acid is a minor species also found in the inner leaflet that is itself a lipid second messenger generated from other phospholipids by the action of phospholipase D.16,17 The predominance of species other (12) Cunningham, B. A.; Tsujita, T.; Brockman, H. L. Biochemistry 1989, 28, 32-40. (13) Smaby, J. M.; Brockman, H. L. Biophys. J. 1985, 48, 701-708. (14) Sugar, I. P.; Mizuno, N. K.; Momsen, M. M.; Brockman, H. L. Biophys. J. 2001, 81, 3387-3397. (15) Zachowski, A. Biochem. J. 1993, 294, 1-14. (16) Bocckino, S. B.; Exton, J. H. Handb. Lipid Res. 1996, 8, 75-123.
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than phosphatidylcholine in the inner membrane raises the questions of whether other phospholipids complex with non-phospholipids and whether the putative complexes or the phospholipids themselves constitute a barrier to the adsorption of proteins to the interface. To help answer these questions, we have (a) characterized the mixing in monomolecular films of bovine liver phosphatidylinositol and the 1-palmitoyl-2-oleoyl species of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid with 1,3-dioleoylglycerol and (b) measured the composition dependence of the rate of colipase binding to these films. 1,3-Dioleoylglycerol was used as a representative non-phospholipid because it is known to complex with POPC and SOPC and to inhibit colipase adsorption in complexes with SOPC. Although this is not the naturally occurring 1,2-diacylglycerol lipid second messenger,18 it is isomerically more stable and gave results almost indistinguishable from those of 1,2-dioleoylglycerol in earlier studies. The results of this study support the idea that non-phospholipid second messengers in natural membranes may exist in both complexed and uncomplexed states and that complex formation may regulate peripheral protein adsorption following receptor binding events on the cell surface. Materials and Methods Reagents. Bovine liver phosphatidylinositol (BLPI) and the 1-palmitoyl-2-oleoyl species of phosphatidylcholine (POPC), -phosphatidylethanolamine (POPE), -phosphatidylserine (POPS), and -phosphate (POPA) were from Avanti Polar Lipids (Alabaster, AL). 1,3-Dioleoylglycerol (1,3-DO) was from Nu-Chek Prep, Inc. (Elysian, MN). The purification of water and preparation of solvents, buffer, and lipid solutions have been previously described.19 Porcine colipase used in this study was purified and converted to [14C]colipase by reductive methylation as described previously.20 The radiolabeled colipase had a specific radioactivity of 9.76 µCi/µmol. Characterization of Monomolecular Films. The automated, Langmuir type film balance used for obtaining surface pressure-potential-molecular area isotherms for pure lipids and mixtures has been recently described.21 Isotherms were collected at 24 °C under a humidified argon atmosphere on an aqueous subphase of phosphate buffered saline (10 mM potassium phosphate, pH 6.6, 0.1 M NaCl, 0.01% NaN3). Phase transition pressures and areas in surface pressure-molecular area isotherms were determined as previously described.22 Miscibility of the phospholipids with 1,3-DO was characterized using parameters determined as recently summarized (ref 23 and references therein). Measured and calculated parameters were monolayer phase and collapse transition pressures, molecular areas at selected surface pressures, moduli of compression (Cs-1), surface dipole moment (µ⊥), and area-independent dipole potential (∆V0). Complex compositions were determined from phase transition pressure-composition data as previously described.24 Even though a surface pressure-potential-molecular area isotherm was not determined precisely at the complex composition (17) Hodgkin, M. N.; Pettitt, T. R.; Martin, A.; Michell, R. H.; Pemberton, A. J.; Wakelam, M. J. O. Trends Biochem. Sci. 1998, 23, 200-204. (18) Quest, A. F. G.; Raben, D. M.; Bell, R. M. Handb. Lipid Res. 1996, 8, 1-58. (19) Momsen, M. M.; Dahim, M.; Brockman, H. L. Biochemistry 1997, 36, 10073-10081. (20) Schmit, G. D.; Momsen, M. M.; Owen, W. G.; Naylor, S.; Tomlinson, A.; Wu, G.; Stark, R. E.; Brockman, H. L. Biophys. J. 1996, 71, 3421-3429. (21) Li, X.-M.; Smaby, J. M.; Momsen, M. M.; Brockman, H. L.; Brown, R. E. Biophys. J. 2000, 78, 1921-1931. (22) Brockman, H. L.; Jones, C. M.; Schwebke, C. J.; Smaby, J. M.; Jarvis, D. E. J. Colloid Interface Sci. 1980, 78, 502-512. (23) Dahim, M.; Mizuno, N. K.; Li, X.-M.; Momsen, W. E.; Momsen, M. M.; Brockman, H. L. Biophys. J. 2002, 83, 1511-1524. (24) Smaby, J. M.; Brockman, H. L. J. Lipid Res. 1987, 28, 10781087.
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for each phospholipid-1,3-DO system, the relevant parameters for the complex could be reasonably estimated by extrapolation using values from isotherms obtained at compositions both above and below the complex composition. Analysis of isotherms was implemented using FilmFit software (Creative Tension, Austin, MN). Determination of Colipase Adsorption Rate. Experimental details for measuring the adsorption of [14C]colipase have been described previously.19,25,26 Briefly, a cylindrical Teflon trough (surface area ) 20 cm2, volume ) 18 mL) was filled with phosphate buffered saline. The temperature was held at 24 °C. Lipid films were spread from a hexane/ethanol (95:5) solution until the desired surface pressure was reached. For this study, experimental pressures were set at ∼95% of the collapse pressure of the monolayer or, if the film exhibited a liquid expanded to condensed monolayer phase transition, ∼95% of the pressure at the onset of the transition. After allowing the lipid monolayer to stabilize for 5 min, stirring (90 rpm) was started, and after 2 min, [14C]colipase solution was injected to 22.6 nM in the aqueous phase. Stirring was continued for 10 min, and then the monolayer was collected on one side of a hydrophobic filter paper disk. The amount of colipase adsorbed was determined by liquid scintillation counting. On the basis of prior studies, 90% recovery of the monolayer and 15 µL carryover of subphase onto the paper was assumed.27 At the [14C]colipase concentration used, adsorption occurred under initial rate conditions.14 Analysis of Colipase Adsorption Rate. As previously described in detail,14 it is assumed that in a phospholipiddiacylglycerol monolayer at any composition and surface pressure, each phospholipid molecule exhibits an excluded area, AEX, and that the surface at any phospholipid molecular area, APL, can be considered to be a matrix of free and excluded sites with area AEX. Note that APL is simply the total surface area at any pressure and lipid composition divided by the number of phospholipid molecules present, that is, the apparent molecular area of the phospholipid. The measured molecular area of colipase fully bound to the surface, 500 Å2, divided by AEX and rounded to the nearest integer gives the number of matrix sites, n, occupied by a bound colipase molecule. For colipase binding to be initiated, it is assumed that gm of n sites must be nonexcluded and that the fraction of such sites determines the fractional colipase adsorption rate, that is, the rate normalized to the highest rate measured at any lipid composition and packing density. Under these conditions, the fractional adsorption rate, Fa, is given by
Figure 1. Selected surface pressure-phospholipid molecular area isotherms for phospholipids mixed with 1,3-dioleoylglycerol. Mole fractions shown from left to right in each panel are 0.0, 0.2, 0.4, 0.6, and 0.8 for (A) BLPI, (B) POPS, (C) POPE, and (D) POPA.
n
Fa )
∑ P (1 - P) j
(n-j)
n!/[j!(n - j)!]
(1)
j)m
where P ()1 - AEX/APL) is the probability that a lattice site is not occupied by complex. Using this equation, AEX and m are systematically varied with integer values to determine the parameter pair that best minimizes the sum of the squares of the differences between calculated and measured values of Fa.
Results Formation of Phospholipid/1,3-DO Complexes. Earlier studies of the surface behavior of liquid-expanded monolayers of diacylphosphatidylcholines with non-phospholipids exhibited a characteristic type of behavior.13 As shown in Figure 1A, such behavior is also seen in selected surface pressure-APL isotherms for BLPI mixtures with 1,3-DO. Plotting the surface pressure as a function of the apparent molecular area of the phospholipid, APL, shows, as expected, that increasing the mole fraction of 1,3-DO increases APL. In addition, note that at lower mole fractions of 1,3-DO the collapse pressure of the monolayers is essentially constant at a value equal to that of the BLPI alone. As 1,3-DO composition is increased, however, a (25) Muderhwa, J. M.; Brockman, H. L. J. Biol. Chem. 1990, 265, 19644-19651. (26) Dahim, M.; Brockman, H. L. Biochemistry 1998, 37, 8369-8377. (27) Momsen, W. E.; Brockman, H. L. Methods Enzymol. 1997, 286, 292-305.
Figure 2. Partial phase diagrams for phospholipids mixed with 1,3-dioleoylglycerol. Shown are onset surface pressures for monolayer collapse or the transition to a condensed state (0) and the onset of the expulsion of bulk 1,3-DO from the monolayer (9) for mixtures of 1,3-DO with (A) BLPI, (B) POPS, (C) POPE, and (D) POPA.
phase transition appears at a surface pressure that decreases progressively until it reaches the collapse pressure of 1,3-DO alone. Above the first transition, the isotherms form an envelope connecting to the transitions of other isotherms. Figure 2A shows a diagram of phase transitions from all isotherms obtained for mixtures of BLPI and 1,3-DO. The interpretation of such data by the two-dimensional
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Table 1. Characterization of 1,3-Diacylglycerol Complexes with Phospholipids phospholipid
XC a
APL,t|X)0,b Å2
APL,t|X)XC,c Å2
Xcond d
µ⊥, mD
∆V0, mV
Cs,35-1, mN/m
BLPI POPS POPE POPA POPC
0.25 0.21 0.14 0.20 0.22
50.5 54.0 50.6 53.3 52.8
67.3 68.0 58.3 66.7 67.9
0.43 0.47 nd nd 0.47
276 415 437 445 411
59 27 103 4 135
131 134 143 148 128
a Mole fraction of 1,3-DO at the complex composition. b Phospholipid area at the onset pressure for transition to a bulk phase or condensed monolayer phase. c Phospholipid area at the complex composition evaluated at the onset pressure for transition to a bulk phase or condensed monolayer phase. d Composition of maximum deviation from ideal mixing at 20 mN/m.
phase rule derives from the work of Crisp,28,29 as reported in the earlier study of POPC mixtures with non-phospholipids.13 At low mole fractions of 1,3-DO where only one transition is observed (open symbols below 0.25 1,3DO) and in the pressure regime below the open symbols, the monolayer consists of a mixture of BLPI and a complex of BLPI with 1,3-DO. Because the surface pressure is constant in this regime, miscibility of the complex and BLPI cannot be ascertained. The transition reflects monolayer collapse pressures in this compositional region, and the value of ∼47 mN/m is consistent with the upper limit for phospholipids observed by others.30 The additional transition appearing at lower surface pressures and higher mole fractions of 1,3-DO (filled symbols above 0.25 1,3-DO) reflects the expulsion of pure 1,3-DO from the monolayer. Moreover, the variation of this transition pressure with composition indicates the miscibility of the complex with 1,3-DO in the monolayer phase. The higher transition observed in this compositional range (open symbols above 0.25 1,3-DO) arises from the collapse of the complex following expulsion of 1,3-DO in excess of that in the complex. This transition is increasingly difficult to detect as the fraction of lipid present as complex progressively decreases toward zero. Overall, the region between zero surface pressure and the first symbol at all compositions defines the pressure-composition space in which the films are monomolecular and liquid expanded with no bulk phases present. The region between the filled and open symbols above 0.25 1,3-DO is a two-phase region comprised of bulk 1,3-DO and a mixed monolayer of 1,3DO and complex. Above the open symbols, no monolayer phases are present. To determine more precisely the composition of the complex, the transition pressures in the compositional region surrounding it from Figure 2A can be plotted as a function of the negative logarithm of the composition (Figure 3A).24 The discontinuity in this plot occurs at the complex composition (XC, expressed as its mole fraction of 1,3-DO) which has a value of 0.25 mole fraction of 1,3DO (Table 1). Using isotherms of the type shown in Figure 1A obtained at 1,3-DO mole fractions near to the complex composition, the apparent area of the phospholipid in the complex at its collapse surface pressure (67.3 Å2) was also determined for use in later analysis (Table 1). Additional parameters calculated from surface pressure-potentialmolecular area isotherms to characterize the complex were its dipole moment (µ⊥), area-independent dipole potential (∆V0), and modulus of compression at 35 mN/m (Cs,35-1). The values of these parameters are given in Table 1. The miscibility of the complex with BLPI and 1,3-DO was further characterized by plotting the molecular area, specifically APL(1 - XC), as a function of 1,3-DO mole (28) Crisp, D. J. In Surface Chemistry; Butterworth: London, 1949; pp 17-22. (29) Crisp, D. J. In Surface Chemistry, Butterworth: London, 1949; pp 23-35. (30) Lee, S.; Kim, D. H.; Needham, D. Langmuir 2001, 17, 55445550.
Figure 3. Determination of complex composition for mixtures of phospholipids with 1,3-dioleoylglycerol. Shown are selected data from Figure 2 for the onset surface pressures for monolayer collapse or the transition to a condensed state (0) and the onset or the expulsion of bulk 1,3-DO from the monolayer (9) for mixtures of 1,3-DO with (A) BLPI, (B) POPS, (C) POPE, and (D) POPA. The complex composition was determined from the abscissa at the intersection of the line segments.
fraction at surface pressures of 1, 10, and 20 mN/m, as shown in Figure 4A. Comparison of experimental with ideal mixing areas of the pure lipids (solid lines) shows that there is substantial area condensation at each selected surface pressure. Plotting the fractional difference at 20 mN/m as a function of composition (Figure 5A, filled squares) and fitting the data to a second order polynomial (Figure 5A, solid line) allows estimation of the composition at which the deviation is maximal. For BLPI, the value is 0.43 at 20 mN/m (Table 1), showing that the maximal deviation from ideal mixing does not coincide with the complex composition determined from the phase diagram. Using a third, instead of a second, order polynomial moved the minimum composition to slightly lower values that were still well above the complex composition of 0.25. This condensation analysis suggests that complex formation is not simply a consequence of tight geometric packing of the lipids in the monolayer. The same type of measurement and analysis described above was performed with the other phospholipids studied, POPS, POPE, POPA, and POPC. The plots for POPS are shown as Figures 1-5B, those for POPE as Figures 1-4C and 5C (open circles), and those for POPA as Figures 1-4D and 5C (filled squares). Most of the data obtained with POPC are not shown because the results were nearly
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Figure 4. Variation of lipid molecular area with monolayer composition. Molecular areas were taken from isotherms of the type shown in Figure 1 at selected surface pressures of (9) 1, (b) 10, and (2) 20 mN/m for mixtures of 1,3-DO with (A) BLPI, (B) POPS, (C) POPE, and (D) POPA. Solid lines indicate additive values expected for ideal mixing.
Figure 5. Relative area condensation in mixtures of 1,3dioleoylglycerol with phospholipids. Fractional deviations were calculated for data of Figure 4 at 20 mN/m for mixtures of 1,3-DO with (A) BLPI, (B) POPS, (C) POPE (O) and POPA (9), and (D) POPC. Arrows indicate the composition of the complex for each set of lipids taken from Table 1.
identical to those reported earlier,13 but the parameters obtained in this study are reported in Table 1 and fractional area deviations are shown in Figure 5D. Inspection of the data (graphs and Table 1) shows similarities and differences. The surface pressure-area isotherms for POPS are like those for BLPI and show complex formation. The isotherms for POPE and POPA are more complicated in
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that they show transitions from the liquid-expanded to more condensed state(s) before ultimate collapse, particularly at low contents of 1,3-DO. These are not shown in the partial phase diagrams (Figure 2) and were not analyzed further. Despite the formation of more condensed monolayer states near collapse, the transition pressures for the onset of the liquid expanded to condensed transitions (Figure 2C,D) show that both POPE and POPA form complexes with 1,3-DO and that these are miscible with both excess phospholipid and 1,3-DO. Overall, the observed complex compositions range from 0.14 to 0.25 mole fraction of 1,3-DO, the collapse/transition areas of the pure phospholipids range from 50.5 to 54.0 Å2, and the areas of the complexes range from 58.3 to 68 Å2 at their collapse or phase transition pressures (Table 1). Both POPS and POPC show significant area condensation (Figure 5B,D). As with BLPI, the condensation is maximal at compositions clearly above that of the complex indicated by the phase diagrams, confirming that complexes are not based on condensation alone. Further supporting this conclusion is the observation that the more compact phospholipids, POPE and POPA, show little or no area condensation in mixtures with 1,3-DO (Figures 4C,D and 5C) but clearly form complexes (Figure 3C,D). The potential parameters for the complexes at collapse show no particular patterns that would suggest the origins of complex formation (Table 1). In considering these parameters, however, it should be remembered that with BLPI, POPS, and POPA the net lipid charge contributes to the values obtained under the conditions of our measurements, that is, pH 6.6 and 100 mM NaCl. Regulation of Colipase Adsorption Rate by Phospholipids. As described in the Introduction, we recently demonstrated, using mixtures of SOPC and several nonphospholipids, that complexes exhibited an excluded area larger than that of SOPC alone with respect to inhibiting the adsorption of a small amphipathic protein, colipase. In that study, colipase was used as a rigid, amphipathic diffusional projectile to probe surface organization on a nanometer length scale. In the present study, we have carried out similar experiments (Materials and Methods) with the 1,3-DO and phospholipids presently under investigation. The lipid monolayers used were initially spread at constant molecular area to a surface pressure ∼95% of the value of the collapse or expanded to condensed phase transition at each lipid composition, as determined from the phase diagrams. Colipase was injected to a concentration of 22.6 nM, and its adsorption to the monolayer was measured after 10 min. Because under similar conditions adsorption is proportional to both time and colipase concentration,14 the adsorption rate constant could be calculated. Absolute adsorption rates measured in this study are approximately 17% lower than those previously reported because of the use of a 50% lower colipase concentration and an 80% higher subphase stirring rate. The rate data obtained with the different phospholipids are shown in Figure 6A-D. The relative adsorption behavior obtained with POPC was very similar to that obtained earlier with SOPC,14 and the POPC data are shown in each panel as open symbols to facilitate comparison among the data obtained with the other phospholipids. Note that the adsorption rates obtained with POPC are near zero until the mole fraction of 1,3-DO exceeds 0.3, a value significantly in excess of the complex composition of 0.22 obtained in this study (Table 1). Qualitatively, this shows that at the high surface pressures near collapse used, the complexes appear to inhibit the colipase adsorption rate. With BLPI, POPS, and POPE,
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Figure 6. [14C]Colipase binding to mixed monolayers of 1,3dioleoylglycerol and phospholipids. Lines were generated using fitting parameters from Table 2. Each panel shows data obtained for mixtures of 1,3-DO with POPC (0) or (A) BLPI, (B) POPS, (C) POPE, and (D) POPA (9). Table 2. Cluster Analysis of Colipase Adsorption to Phospholipid/1,3-Dioleoylglycerol Monolayers phospholipid
m
n
AEX, Å2
r
BLPI POPS POPE POPA POPC
1 1 1 2 1
7 7 7 10 6
72 76 67 52 83
0.9894 0.8765 0.9847 0.8367 0.8266
the same general trend is observed except that inhibition is relieved at compositions 0.1-0.2 lower than with POPC. In each case, however, the adsorption rate is near zero until the complex composition for that lipid system (arrow) has been exceeded. The data obtained with POPA are an exception to the trend noted with the other phospholipids. Specifically, at the complex composition of 0.20 mole fraction of 1,3-DO, the adsorption rate is half-maximal with an abrupt increase in binding rate observed between 0.05 and 0.10 mole fraction of 1,3-DO. To more quantitatively test the hypothesis that phospholipids exhibit an excluded area that inhibits colipase adsorption, we applied the simple statistical model developed earlier and described in Materials and Methods. As shown by the lines in Figure 6, the binding rate data could be reasonably described in each case. The model makes no assumptions about the existence of complexes. It simply assumes that each phospholipid exhibits an excluded area, AEX, that is determined as a fitting parameter. The other fitting parameter is the number of sites, m, of an area equal to the excluded area and scaled to the size of a bound colipase molecule that must be nonexcluded for adsorption to be initiated. The values of these parameters are shown for each of the phospholipids studied in Table 2. Also shown in the table are n, the number of sites equivalent to a bound colipase molecule, and the coefficient of correlation for each fitted line shown in Figure 6. The exact values of m and AEX should not be overinterpreted because of the simple nature of the analytical
Figure 7. Identity of phospholipid area in monolayer complexes with 1,3-dioleoylglycerol and phospholipid excluded areas for colipase adsorption. Phospholipid areas in complexes with 1,3DO at monolayer collapse are from Table 1, and phospholipid excluded areas are from Table 2 (9). Data from a previous study of SOPC mixed with several non-phospholipids (ref 14) is included for comparison (O). The solid line is a best fit assuming proportionality.
model used, the scatter in the binding measurements (Figure 6), and the necessity to vary m and n ()500/AEX) as integers. Within the limits of interpretation, however, it is clear that the minimal fraction of surface that must be free of complex for colipase binding to occur under initial rate conditions, m/n, is small. When the results are considered as the minimal area of a site needed to initiate colipase adsorption, mAEX, the values range from 67 to 104 Å2. This can be compared to the area occupied by a colipase molecule fully bound to a monolayer of such lipids, 500 Å2.19 Comparison of the values of excluded areas of the phospholipids (Table 2) with their collapse areas of the phospholipids in the absence of 1,3-DO (Table 1) shows that excluded areas are considerably larger, except for POPA. This implicates 1,3-DO as a part of the area preventing colipase adsorption, except with POPA. Indeed, with the exception of POPA, the values of AEX (Table 2) appear comparable to those of the complexes, expressed per molecule of phospholipid (Table 1). The relationship between the physically measured areas of the complexes at collapse and excluded areas determined from the statistical analysis of the rate data (Table 2) is shown in Figure 7. The data from this study (filled symbols) are combined with data from the earlier study of SOPC mixed with several non-phospholipids (open symbols). On the basis of the properties of POPA adsorption described above, the collapse area of pure POPA was used instead of its complex area, that is, it was assumed that XC ) 0 for POPA. Inspection of the data shows that the results from this study are comparable to those from the earlier work and that there is an approximately proportional relationship between the excluded areas (Table 2) and complex collapse areas (Table 1). Assuming proportionality, the slope of the line, calculated with all data shown in Figure 7, is 1.05. This nearly 1:1 correlation supports the idea that complexes between phospholipids and non-phospholipids are responsible for the observed inhibition of colipase adsorption.
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Discussion studies,13,14
From the data presented here and earlier the formation in monolayers of complexes between phospholipids and non-phospholipids, such as 1,3-DO, seems to be a general phenomenon. The idea of phospholipids forming complexes with sterols, such as cholesterol, is not new (e.g., ref 31). Recent studies in this area have put such complexation on a sounder thermodynamic footing32 and have established that it occurs in both mono- and bilayer membranes.33,34 The ratio of sterol to phospholipid varies with the chain composition of the phospholipid, its headgroup, and temperature, relative to Tm.33,35 What appears to distinguish the complexes we have observed from those formed with cholesterol is their physical state. Cholesterol complexes form a liquid-ordered phase, which is intermediate between liquid-expanded and condensed phases, whereas the films we have studied appear to remain in the liquid-expanded state. Moreover, they appear to be miscible with excess non-phospholipid and, possibly, with excess phospholipid. Thus, the complexes formed between phospholipids and non-steroidal lipids such as diacylglycerols are more likely relevant to the inner leaflet of the plasma membrane, a major site for the generation of diacylglycerols and other lipid second messengers,18 rather than in sterol/sphingolipid “rafts”.36 The absence of a defined stoichiometry for 1,3-DO complexation with the phospholipids shows that complex formation, like that observed with cholesterol,33 does not have a simple basis involving specific interactions, such as the formation of a specific charge or hydrogen bond network. As shown herein, complexes can be formed between 1,3-DO and both charged and zwitterionic phospholipids. Nor can complexation be based totally on steric accommodation or “condensation” between the acyl moieties of the lipids. This latter point is emphasized by the observation that POPE and POPA show very little evidence of condensation (Figure 5C), whereas BLPI and POPC do (Figure 5A,D). Likewise, the dipole moments, compressibilities, and other parameters given in Table 1 do not reveal any consistent values or relationships. Thus, although the phase diagrams (Figure 2) and earlier studies clearly show complex formation, the analysis presently possible does not reveal its molecular origin. The complexes described in this study form in the absence of proteins; that is, they arise from interactions among the lipid molecules and interfacial water. Moreover, as noted in the Introduction they have been observed in both monolayer and bilayer systems. It is, therefore, to be expected that when lipid second messengers such as diacylglycerol are generated in the inner leaflet of the plasma membrane or in the nuclear membrane of a cell, they will complex with surrounding phospholipids. This is potentially important because, as shown for cholesterol in complexes,37 complexation markedly alters the chemical activity of the complexed molecules. Thus, complexation (31) Huang, C.-H. Lipids 1977, 12, 348-356. (32) Anderson, T. G.; McConnell, H. M. J. Phys. Chem. B 2000, 104, 9918-9928. (33) Keller, S. L.; Radhakrishnan, A.; McConnell, H. M. J. Phys. Chem. B 2000, 104, 7522-7527. (34) Anderson, T. G.; McConnell, H. M. Biophys. J. 2001, 81, 27742785. (35) Radhakrishnan, A.; McConnell, H. M. J. Phys. Chem. B 2002, 106, 4755-4762. (36) Simons, K.; Ikonen, E. Science 2000, 290, 1721-1726.
of diacylglycerols will affect their availability for interaction with enzymes that bind them, like members of the protein kinase C family.5,6 Also affected could be their rate of transmembrane “flip-flop”,15 although the biological consequences of such translocation are not known. Although complexes between phosphatidylcholine and non-phospholipids were identified some time ago,13 their effect on biological phenomena in cells can, at present, be only indirectly presumed, as described in the preceding paragraph. In vitro, it has been clearly shown that complexes between SOPC and several non-phospholipid second messenger molecules inhibit the translocation of colipase to interfaces. In the present study, it has been demonstrated that such inhibition of colipase translocation from the aqueous phase to an interface is not unique to SOPC or to phosphatidylcholines but extends to all the major phospholipid classes found exposed to the cytoplasm of cells. It remains to be seen if colipase is somehow unique in having its adsorption rate regulated by the extent to which molecules such as diacylglycerols are complexed or free. This is unlikely because colipase does not specifically bind to non-phospholipids in interfaces, although it does show a preference for packing with such molecules in interfaces, relative to phosphatidylcholine.19 If the behavior of colipase is not unique, then the generation of molecules such as diacylglycerol in phospholipid membranes could play a significant role in regulating the initial translocation of peripheral proteins to membranes in response to signaling events. The global abundance of molecules such as diacylglycerols does not exceed a few mole percent, for example,38 far below the 0.14-0.25 mole fractions needed to exceed its abundance in the complex. However, it is likely that there are regions of confinement39 in which, following cell stimulation, the local concentration of diacylglycerols may exceed their abundance in complexes and, potentially, trigger the binding of peripheral proteins as a part of signaling cascades. In that regard, it has been noted that resting cells contain finite amounts of diacylglycerols and that there may be a threshold level for their biological effects to be induced.18 The one phospholipid tested for which complex formation was not associated with inhibition of colipase adsorption was phosphatidic acid. This lipid is not a major component of resting cells but, like diacylglycerols, is generated by cell stimulation. Phosphatidic acid is considered a lipid second messenger in its own right,16,17 being generated by the action of phospholipase D on phospholipids such as those studied herein as well as by phosphorylation of diacylglycerol. Particularly when it is generated from phospholipids such as phosphatidylserine, our results suggest that the formation of phosphatidic acid could have the effect of changing the state of diacylglycerols with which it may be complexed, thereby generating a secondary lipid signal. Acknowledgment. This work was supported by USPHS Grant HL 49180 and the Hormel Foundation. LA026172M (37) Radhakrishnan, A.; McConnell, H. M. Biochemistry 2000, 39, 8119-8124. (38) Preiss, J.; Loomis, C. R.; Bishop, W. R.; Stein, R.; Niedel, J. E.; Bell, R. M. J. Biol. Chem. 1986, 261, 8597-8600. (39) Dietrich, C.; Yang, B.; Fujiwara, T.; Kusumi, A.; Jacobson, K. Biophys. J. 2002, 82, 274-284.
