Toward understanding interfacial activation of secretory phospholipase A2 (PLA2): membrane surface properties and membrane-induced structural changes in the enzyme contribute synergistically to PLA2 activation

Phospholipase A2 (PLA2) hydrolyzes phospholipids to free fatty acids and lysolipids and thus initiates the biosynthesis of eicosanoids and platelet-activating factor, potent mediators of inflammation, allergy, apoptosis, and tumorigenesis. The relative contributions of the physical properties of membranes and the structural changes in PLA2 to the interfacial activation of PLA2, that is, a strong increase in the lipolytic activity upon binding to the surface of phospholipid membranes or micelles, are not well understood. The present results demonstrate that both binding of PLA2 to phospholipid bilayers and its activity are facilitated by membrane surface electrostatics. Higher PLA2 activity toward negatively charged membranes is shown to result from stronger membrane-enzyme electrostatic interactions rather than selective hydrolysis of the acidic lipid. Phospholipid hydrolysis by PLA2 is followed by preferential removal of the liberated lysolipid and accumulation of the fatty acid in the membrane that may predominantly modulate PLA2 activity by affecting membrane electrostatics and/or morphology. The previously described induction of a flexible helical structure in PLA2 during interfacial activation was more pronounced at higher negative charge densities of membranes. These findings identify a reciprocal relationship between the membrane surface properties, strength of membrane binding of PLA2, membrane-induced structural changes in PLA2, and the enzyme activation.     

Differential mode of attack on membrane phospholipids by an acidic phospholipase A(2) (RVVA-PLA(2)-I) from Daboia russelli venom

An acidic phospholipase A(2) (RVVA-PLA(2)-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA(2)-I was non-lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA(2) enzyme. RVVA-PLA(2)-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA(2)-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA(2)-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA(2)s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA(2)-I suggested the correlation between catalytic and membrane damaging activities of this PLA(2) enzyme. Our study advocates that the presence of a large number of PLA(2)-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA(2) enzyme.     

Maintenance of epithelial surface membrane lipid polarity: A role for differing phospholipid translocation rates

Summary Large differences in lipid composition of apical and basolateral membranes from epithelial cells exist. To determine the responsible mechanism(s), rat renal cortical brush border and basolateral membrane phospholipids were labeled using³²P and either [³H]-glycerol or [2-³H] acetate for incorporation and degradation studies, respectively. Brush border and basolateral membrane fractions were isolated simultaneously from the same cortical homogenate. Different phospholipid classes were degraded at variable rates with phosphatidylcholine having the fastest decay rate. Decay rates for individual phospholipid classes were, however, similar in both brush border and basolateral membrane fractions. In phospholipid incorporation studies again, large variations existed between individual phospholipid classes with phosphatidylcholine and phosphatidylinositol showing the most rapid rates of incorporation. Sphingomyelin and phosphatidylserine showed extremely slow incorporation rates and did not enter into the isotopic decay phase for 48 hr. In contrast to degradation studies, however, the same phospholipid class labeled the two surface membrane domains at highly variable rates. The difference in these rates, with the exception of phosphatidylinositol, were identical to the differences in phospholipid compositions between the two membranes. For example, phosphatidylcholine was incorporated into the basolateral membrane 2.5 × faster than into the brush border membrane and its relative composition was 2.5 × greater in the basolateral membrane. The opposite was true for sphingomyelin. These results indicate incorporation and not degradation rates of individual phospholipids play a major role in regulating the differing phospholipid composition of brush border and basolateral membranes.     

Effects of lipid phase transition and membrane surface charge on the interfacial activation of phospholipase A2

Phospholipase A2 (PLA2) enzymes act at the membrane-water interface to access their phospholipid substrate from the membrane. They are regulated by diverse factors, including the membrane charge, fluidity, mode of membrane binding (insertion, orientation), and allosteric conformational effects. Relative contributions of these factors to the complex kinetics of PLA2 activation are not well understood. Here we examine the effects of thermal phase transitions and the surface charge of phospholipid membranes on the activation of human pancreatic PLA2. The temperature dependence of the initial catalytic rate of PLA2 peaks around the lipid phase transition temperature (Tm) when Tm is not too far from physiological temperatures (30-40 degrees C), and the peak is higher in the presence of anionic membranes. High PLA2 activity can be induced by thermal perturbations of the membrane. Temperature-dependent fluorescence quenching experiments show that despite dramatic effects of the lipid phase transition on PLA2 activity, the membrane insertion depth of PLA2 increases only modestly above Tm. The data show that membrane structural disorder, and not the depth of membrane insertion, plays a major role in PLA2 activity.     

The adipocyte fatty acid-binding protein binds to membranes by electrostatic interactions

The adipocyte fatty acid-binding protein (AFABP) is believed to transfer unesterified fatty acids (FA) to phospholipid membranes via a collisional mechanism that involves ionic interactions between lysine residues on the protein surface and phospholipid headgroups. This hypothesis is derived largely from kinetic analysis of FA transfer from AFABP to membranes. In this study, we examined directly the binding of AFABP to large unilamellar vesicles (LUV) of differing phospholipid compositions. AFABP bound LUV containing either cardiolipin or phosphatidic acid, and the amount of protein bound depended upon the mol % anionic phospholipid. The K(a) for CL or PA in LUV containing 25 mol % of these anionic phospholipids was approximately 2 x 10(3) M(-1). No detectable binding occurred when AFABP was mixed with zwitterionic membranes, nor when acetylated AFABP in which surface lysines had been chemically neutralized was mixed with anionic membranes. The binding of AFABP to acidic membranes depended upon the ionic strength of the incubation buffer: >/=200 mM NaCl reduced protein-lipid complex formation in parallel with a decrease in the rate of FA transfer from AFABP to negatively charged membranes. It was further found that AFABP, but not acetylated AFABP, prevented cytochrome c, a well characterized peripheral membrane protein, from binding to membranes. These results directly demonstrate that AFABP binds to anionic phospholipid membranes and suggest that, although generally described as a cytosolic protein, AFABP may behave as a peripheral membrane protein to help target fatty acids to and/or from intracellular sites of utilization.     

Inhibition of phospholipase A(2) as a therapeutic target

The hydrolysis of cell membrane phospholipids by phospholipase A(2) (PLA(2)) leads to the production of numerous lipid mediators of diverse pathological conditions, mainly inflammatory diseases. These include lysophospholipids and their derivatives, and arachidonic acid and its derivatives (the eicosanoids). Both these groups of mediators are produced predominantly by the secretory PLA(2)s (sPLA(2)s) which hydrolyze the phospholipids of the cell surface membrane. Protection of cell membrane from these 'inflammatory enzymes' can therefore be used for the treatment of inflammatory processes. A prototype of cell-impermeable PLA(2) inhibitors, which protect the cell membrane from different sPLA(2)s without affecting vital phospholipid metabolism, is presented and discussed in the present review.     

Human group IVC phospholipase A2 (cPLA2gamma). Roles in the membrane remodeling and activation induced by oxidative stress

To create the unique properties of a certain cellular membrane, both the composition and the metabolism of membrane phospholipids are key factors. Phospholipase A(2) (PLA(2)), with hydrolytic enzyme activities at the sn-2 position in glycerophospholipids, plays critical roles in maintaining the phospholipid composition as well as producing bioactive lipid mediators. In this study we examined the contribution of a Ca(2+)-independent group IVC PLA(2) isozyme (cPLA(2)gamma), a paralogue of cytosolic PLA(2)alpha (cPLA(2)alpha), to phospholipid remodeling. The enzyme was localized in the endoplasmic reticulum and Golgi apparatus, as seen using green fluorescence fusion proteins. Electrospray ionization mass spectrometric analysis of membrane extracts revealed that overexpression of cPLA(2)gamma increased the proportion of polyunsaturated fatty acids in phosphatidylethanolamine, suggesting that the enzyme modulates the phospholipid composition. We also found that H(2)O(2) and other hydroperoxides induced arachidonic acid release in cPLA(2)gamma-transfected human embryonic kidney 293 cells, possibly through the tyrosine phosphorylation pathway. Thus, we propose that cPLA(2)gamma is constitutively expressed in the endoplasmic reticulum and plays important roles in remodeling and maintaining membrane phospholipids under various conditions, including oxidative stress.     

Introduction of a high-resolution cytochemical method for studying the distribution of phospholipids in biological tissues

A novel cytochemical method for the in situ, ultrastructural localization of phospholipids in biological tissues is reported. The method is based on the enzyme-gold approach (M. Bendayan: J. Histochem. Cytochem. 29, 531, 1981). Phospholipase A2 from bee venom was adsorbed on colloidal gold particles (PLA2-gold) and applied for the specific labeling of its substrate, sn3-glycerophospholipids. The binding and enzymic competence of the PLA2-gold complex were confirmed by in vitro, preembedding experiments with erythrocytes and a crude lung surfactant preparation. The substrate specificity of the probe was assessed by labeling Epon thin sections of pure phospholipids. To test the potential applications of the PLA2-gold complex, lung and pancreatic tissues were fixed with glutaraldehyde-osmium and embedded in Epon for transmission electron microscopy (TEM). They were also prepared for critical-point-drying fracture-label (CPD-FL) replicas and thin-section fracture-label (TS-FL) specimens. On TEM thin sections incubated with PLA2-gold, all cellular membranes were labeled. The labeling density over each membrane compartment, as quantitated in lung type II pneumocytes, was classified in order of magnitude as follows: a) nuclear membranes; b) outer mitochondrial membrane and rough endoplasmic reticulm (RER); and c) Golgi complex, mitochondrial cristae and plasma membranes. In lung alveoli, the phospholipid-rich surfactant material was intensely labeled. Labeling of lung thin sections from chlorphentermine-treated rats (phospholipidosis-inducing drug) further demonstrates the reliability of PLA2-gold to label phospholipids. CPD-FL replicas and TS-FL specimens further extended the TEM observations: nuclear membranes and RER were more intensely labeled than plasma membranes. In exocrine pancreatic cells, two distinct labeling patterns were found for secretory granule membranes: sparse and dense. The specificity and reliability of the labeling were confirmed through several control experiments. The studies performed thus demonstrate the great potential of the PLA2-gold technique as a new approach to the high-resolution study of phospholipid distribution and density among biological structures.     

Synthesis of cell-impermeable inhibitor of phospholipase A2

Phospholipase A2 (PLA2) in the cell surface membrane is considered a regulator of cellular secretion. The distinction between the role of the cell surface and the intracellular PLA2 is not clear, since it has not been possible to differentiate unequivocally the activity of the enzymes in the various organelles. The use of an extracellular inhibitor of PLA2 can greatly contribute to the understanding of cell surface PLA2 function. In this paper, the preparation of a cell-impermeable inhibitor of PLA2 is presented. This substance incorporates into lipid membranes and is capable of blocking the hydrolysis of membrane phospholipids by snake venom as well as by cell membrane PLA2.     

Medium viscosity regulates the activity of membrane-bound and soluble phospholipase A2

Medium viscosity is a regulator of very low density lipoprotein production by cultured hepatocytes; their secretion and synthesis are inversely proportional to the extracellular fluid viscosity. The possibility that the mechanism of this extracellular effect on cell function involves modulation of cell membrane component(s) was considered. Along with this assumption, we studied the effect of medium viscosity on the activity of phospholipase A2 (PLA2), an enzyme present in the cell surface membrane, and the activity has been correlated with cellular secretion. We have found that culture medium viscosity inhibits the activity of PLA2 in the plasma membrane of cultured liver cells, concomitantly with the inhibition of lysosomal enzyme and lipoprotein secretion. It was also found that the degradation of liposomal phosphatidylcholine by soluble snake venom PLA2 is inversely proportional to the solvent viscosity. The possibility that the effect of medium viscosity on the enzymatic reaction involves the modulation of dynamic properties of membrane phospholipids was then considered. This hypothesis was examined by monitoring the fluorescence depolarization of fluorophores incorporated into phospholipid vesicles. No significant effect of the solvent viscosity on the phospholipid bilayer was observed. It is proposed that the regulation of cellular secretion by extracellular fluid viscosity involves modulation of the cell membrane PLA2 activity.     

Structure and morphology changes during in vitro degradation of electrospun poly(glycolide-co-lactide) nanofiber membrane

Electrospun poly(glycolide-co-lactide) (PLA10GA90, LA/GA ratio 10/90) biodegradable nanofiber membranes possessed very high surface area to volume ratios and were completely noncrystalline with a relatively lowered glass transition temperature. These characteristics led to very different structure, morphology, and property changes during in vitro degradation, which were examined systematically. A shrinkage study showed that the electrospun crystallizable but amorphous PLA10GA90 membranes exhibited a very small shrinkage percentage when compared with the electrospun membranes of noncrystallizable poly(lactide-co-glycolide) (PLA75GA25, LA/GA 75/25) and poly(d,l-lactide). Although the weight loss of electrospun PLA10GA90 membranes exhibited a similar degradation behavior as cast thin films, detailed studies showed that the structure and morphology changes in electrospun membranes followed different pathways during the hydrolytic degradation. After 1 day of degradation in buffer solution at 37 degrees C, electrospun PLA10GA90 membranes exhibited a sudden increase in crystallinity and glass transition temperature, due to the fast thermally induced crystallization process. The continuous increase in crystallinity and apparent crystal size, as well as the decrease in long period and lamellae thickness, indicated that the thermally induced crystallization was followed by a chain cleavage induced crystallization process. The mass loss rate was accelerated after 6 days of degradation. The increase in glass transition temperature during this period further confirmed that the degradation of PLA10GA90 nanofibers was initiated from the amorphous region within the lamellar superstructures. A mechanism of structure and morphology changes during in vitro degradation of electrospun PLA10GA90 nanofibers is proposed.     

Structural and functional effects of tryptophans inserted into the membrane-binding and substrate-binding sites of human group IIA phospholipase A2

Phospholipase A(2) (PLA(2)) enzymes become activated by binding to biological membranes and hydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatory mediators. To understand the functional significance of amino acid residues at key positions, we have studied the effects of the substitution of Val(3) (membrane binding surface) and Phe(5) (substrate binding pocket) of human group IIA PLA(2) by tryptophan on the structure and function of the enzyme. Despite the close proximity of the sites of mutations, the V3W mutation results in substantial enhancement of the enzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structural analysis of all three proteins free in buffer and bound to membranes indicates that large differences in activities result from distinct conformational changes in PLA(2)s upon membrane binding. Although PLA(2) and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, the F5W mutant experiences partial distortion of the alpha-helical structure presumably resulting from the tendency of Trp(5) to insert into the membrane. Furthermore, whereas the PLA(2) and the V3W mutant bind to the membrane at similar and apparently productive-mode orientation, the F5W mutant binds to membranes with a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutation and the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp(3) at the membrane binding face of PLA(2) facilitates the proper membrane binding of the enzyme, Trp(5) in the internal substrate binding site causes partial unwinding of the N-terminal helix in order to interact with the membrane.     

Liver ischemia increases the molecular order of microsomal membranes by increasing the cholesterol-to-phospholipid ratio

An accelerated degradation of phospholipid is the likely basis of irreversible cell injury in ischemia, and the membranes of the endoplasmic reticulum of the liver are a convenient system with which to study the effect of such a disturbance on the structure and function of cellular membranes. In the present report, electron spin resonance spectroscopy has been used to evaluate changes in the molecular ordering of microsomal membrane phospholipids in the attempt to relate the loss of lipid to alterations in membrane structure. The order parameter, S, was calculated from spectra reflecting the anisotropic motion of 12-doxyl stearic acid incorporated into normal and 3-h ischemic microsomal membranes. Over the temperature range 4-40 degrees C, the molecular order (S) of ischemic membranes was increased by 8-10%. This increase was reproduced in the ordering of the phospholipids in liposomes prepared from total lipid extracts of the same membranes. In contrast, after removal of the neutral lipids, liposomes prepared from phospholipids of ischemic and control membranes had the same molecular order. There were no differences in the phospholipid species of control and ischemic membranes or in the fatty acid composition of the phospholipids. In the neutral lipid fraction of ischemic membranes, however, triglycerides and cholesterol were increased compared to control preparations. There were no free fatty acids. The total cholesterol content of the liver was unchanged after 3 h of ischemia. The cholesterol-to-phospholipid ratio of ischemic membranes, however, was increased by 22% from 0.258 to 0.315 as a consequence of the loss of phospholipid. Addition of cholesterol to the control total lipid extracts to give a cholesterol-to-phospholipid ratio the same as in ischemic membranes resulted in liposomes with order parameters similar to those of liposomes prepared from ischemic total lipids. It is concluded that the degradation of the phospholipids of the microsomal membrane results in a relative increase in the cholesterol-to-phospholipid ratio. This is accompanied, in turn, by an increased molecular order of the residual membrane phospholipids.     

The transbilayer distribution of phospholipids in disc membranes is a dynamic equilibrium evidence for rapid flip and flop movement.

We studied the transbilayer redistribution of phospholipids in bovine rod outer segment membranes on thoroughly washed, Ficoll-floated osmotically intact disc vesicles; freshly prepared membranes separated from the disc stack by osmotic shock; and intact disc stacks with a permeabilized plasma membrane (A-discs, B-discs C-discs, respectively). In all cases, spin-labelled phospholipid analogues (SL-PL) with choline, serine and ethanolamine head groups (PtdCho, PtdSer and PtdEtn, respectively) were taken up into the outer leaflet of the membranes by > 90% and within less than 30 s after SL-PL addition, as deduced from the disappearance of spin-label from the suspension medium and from the specific ESR spectrum of membrane-associated spin-label. Using BSA extraction, the amount of SL-PL in the outer leaflet of the bilayer was determined. It decreased with a mean half-time of < 5 min at 25 degrees C, indicating rapid redistribution of all spin-labelled phospholipids into the inner leaflet of the disc membranes. After 1 h, PtdCho and PtdEtn were distributed almost symmetrically, whereas PtdSer was 35 : 65% (in/out). Using subsequent incubation with BSA, the outward movement (flop) of the analogues was observed directly, demonstrating that inward and outward movements proceed in thermodynamic equilibrium. No effect of N-ethylmaleimide or ATP on the redistribution could be measured, which makes it unlikely that energy-consuming translocase or flippase processes are involved in the redistribution in the dark. We reason that the solubilization zone around the photoreceptor rhodopsin may be the locus of rapid redistribution of the highly unsaturated disc phospholipid.     

Effect of membrane sterol content on the susceptibility of phospholipids to phospholipase A2

The effects of membrane sterol level on the susceptibility of LM cell plasma membranes to exogenous phospholipases A2 has been investigated. Isolated plasma membranes, containing normal or decreased sterol content, were prepared from mutant LM cell sterol auxotrophs. beta-Bungarotoxin-catalyzed hydrolysis of both endogenous phospholipids and phospholipids introduced into the membranes with beef liver phospholipid exchange proteins was monitored. In both cases, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were degraded at similar rates in normal membranes, while PC hydrolysis was specifically accelerated in sterol-depleted membranes. Additional data suggest that this preferential hydrolysis of PC is not a consequence of the phospholipid head group specificity of the phospholipase, nor of a difference in the accessibility of PC versus PE to the enzyme. Analysis of the reaction products formed during treatment of isolated membranes with phospholipase A2 showed almost no accumulation of lysophospholipids. This was found to be due to highly active lysophospholipase(s), present in LM cell plasma membranes, acting on the lysophospholipids formed by phospholipase A2 action. A soluble phospholipase A2 was partially purified from LM cells and found to behave as beta-bungarotoxin with regard to membrane sterol content. These results demonstrate that the nature of phospholipid hydrolysis, catalyzed by phospholipase A2, can be significantly affected by membrane lipid composition.     

Nonsedimentable microvesicles from senescing bean cotyledons contain gel phase-forming phospholipid degradation products

A mixture of liquid-crystalline and gel-phase lipid domains is detectable by wide angle x-ray diffraction in smooth microsomal membranes isolated from senescent 7-day-old cotyledons, whereas corresponding membranes from young 2-day-old cotyledons are exclusively liquid-crystalline. The gel-phase domains in the senescent membranes comprise phospholipid degradation products including diacylglycerols, free fatty acids, long-chain aldehydes, and long-chain hydrocarbons. The same complement of phospholipid degradation products is also present in nonsedimentable microvesicles isolated from senescent 7-day-old cotyledons by filtration of a 250,000g, 12-hour supernatant through a 300,000 dalton cut-off filter. The phospholipid degradation products in the microvesicles form gel-phase lipid domains when reconstituted into phospholipid liposomes. Nonsedimentable microvesicles of a similar size, which are again enriched in the same gel-phase-forming phospholipid degradation products, are also generated in vitro from smooth microsomal membranes isolated from 2-day-old cotyledons when Ca²⁺ is added to activate membrane-associated lipolytic enzymes. The Ca²⁺-treated membranes do not contain detectable gel-phase domains, suggesting that the phospholipid degradation products are completely removed by microvesiculation. The observations collectively indicate that these nonsedimentable microvesicles serve as a vehicle for moving phospholipid degradation products out of membrane bilayers into the cytosol. As noted previously (Yao K, Paliyath G, Humphrey RW, Hallett FR, Thompson JE [1991] Proc Natl Acad Sci USA 88: 2269-2273), the term “deteriosome” connotes this putative function and would serve to distinguish these microvesicles from other cytoplasmic microvesicles unrelated to deterioration.     

Differential hydrolysis of erythrocyte and mitochondrial membrane phospholipids by two phospholipase A2 isoenzymes (NK-PLA2-I and NK-PLA2-II) from the venom of the Indian monocled cobra Naja kaouthia

We previously demonstrated that venom from the Indian monocled cobra Naja kaouthia is a rich source of phospholipase A2 enzymes, and we purified and characterized a major PLA2 isoenzyme (NK-PLA2-I) from N. kaouthia venom. In the present study, we report the purification and biochemical characterization of a second PLA2 isoenzyme (NK-PLA2-II) from the same venom. A comparison of the membrane phospholipid hydrolysis patterns by these two PLA2s has revealed that they cause significantly more damage to mitochondrial membranes (NK-PLA2-I > NK-PLA2-II) as compared to erythrocyte membranes due to more efficient binding of the enzymes to mitochondrial membranes. Fatty acid release patterns by these PLA2s from the membrane phospholipid PC-pools indicate that NK-PLA2-I does not discriminate between saturated and unsaturated fatty acids whereas NK-PLA2-II shows a preference for unsaturated fatty acids during the initial phase of attack. The current investigation provides new insight into the molecular arrangement of NK-PLA2-sensitive domains in erythrocyte and mitochondrial membranes and highlights the contribution of polar, but uncharged, amino acids such as serine and cysteine in NK-PLA2 induced membrane damage.     

Involvement of distinct populations of phosphatidylglycerol and phosphatidylcholine molecules in photosynthetic electron-flow activities

When spinach thylakoid membranes were treated with pancreatic phospholipase A₂, phospholipids were degraded and the uncoupled non-cyclic electron-flow activity (from H₂O to NADP⁺) was progressively inhibited. To discriminate between the relative contributions of the hydrolysis products (free fatty acids and lysophospholipids) and of the phospholipid depletion per se to inhibit the activity, we made use of the known property of bovine serum albumin to remove such hydrolysis products from membranes. Using careful washings and adequate lipid extraction procedures, we could ascertain that all hydrolysis products generated by phospholipase A₂ were effectively removed from the thylakoid membrane by bovine serum albumin treatment. When bovine serum albumin was added to thylakoid membranes after various incubation times with the phospholipase A₂, the electron-flow activity was rapidly, but not completely restored. However, when phospholipid hydrolysis exceeded a certain extent (70–85%), the activity was totally inhibited and its restoration by albumin was no longer possible. Addition of EGTA to the phospholipase A₂-treated membranes blocked both the enzyme action and the progress of electron-flow inhibition. Under these conditions, the amplitude of the albumin-induced restoration of electron-flow rate did not depend on the time span between EGTA block and albumin addition. We show that phospholipid depletion of thylakoid membranes is entirely responsible for the irreversible (albumin-insensitive) inhibition of the electron flow from H₂O to NADP⁺ by phospholipase A₂. Plotting the extent (%) of this inhibition vs. the extent (%) of phospholipid depletion allowed us to distinguish three populations of both phosphatidylglycerol and phosphatidylcholine. The first one, which was easily accessible to the enzyme, did not support greatly the electron-flow activity (around 40% of each phospholipid destroyed vs. only 10% or less inhibition). On the other hand, the electron-flow activity strongly depended on the second, less accessible population of phospholipids (around 40% of each phospholipid destroyed vs. 90% inhibition). Finally, the third population of phospholipids was not involved in the uncoupled non-cyclic electron flow activity.     

Transbilayer phospholipid asymmetry and its maintenance in the membrane of influenza virus.

Two phospholipid exchange proteins and two phospholipases C have been employed to determine the phospholipid composition of the outer surface of the membrane of influenza virus. These four protein probes have defined the same accessible and inaccessible pool for each viral phospholipid. Phospholipids which are exchangeable or hydrolyzable are located on the outer surface, whereas the inaccessible pool is located at the inner surface of the viral bilayer. The two pools are unequal in size, with ca. 30% of the total phospholipid accessible to the four proteins, and ca. 70% inaccessible. The membrane is thus highly asymmetric with regard to the amount of phospholipid on each side of the membrane. There is also a marked asymmetry of phospholipid composition. Phosphatidylcholine and phosphatidylinositol are enriched in the outer surface, and sphingomyelim is enriched in the inner surface, whereas phosphatidylethanolamine and phosphatidylserine are present in similar proportions in each surface. This distribution is qualitatively different from that previously reported for the human erythrocyte. The close agreement between results obtained with excahnge proteins and phospholipases C demonstrates that the hydrolytic action of these enzymes does not alter phospholipid asymmetry. The nonperturbing nature of the exchange proteins has permitted the rate of transmembrane movement of phospholipids (flip-flop) in the intact virion to be studied. This process could not be detected after 2 days at 37 degrees C. It was estimated that the half-time for flip-flop is indeterminately in excess of 30 days for sphingomyelin and 10 days for phosphatidylcholine at 37 degrees C. These extremely long times provide a simple explanation for the maintenance of transbilayer asymmetry in influenza virions and possibly, other membranes. Since the viral membrane is acquired by budding through the host cell plasma membrane, the transbilayer distribution of phospholipids observed in the virions presumably reflects a similar asymmetric distribution of phospholipids in the host cell surface membrane. Because animal cells in culture do not incorporate extracellular phospholipid, our results demonstrate that individual cells have the capacity to generate asymmetric membranes.