The NADPH-protochlorophyllide oxidoreductase is the major protein constituent of prolamellar bodies in wheat (Triticum aestivum L.)

A fraction of highly purified prolamellar bodies was isolated from etioplasts of wheat (Triticum aestivum L. cv. Starke II, Weibull), as previously described by Ryberg and Sundqvist (1982, Physiol. Plant., 56, 125–132). Studies on the protein composition revealed that only one major polypeptide of an apparent molecular weight of 36000 is present in the fraction of prolamellar bodies. This polypeptide was identified as the NADPH-protochlorophyllide oxidoreductase. The highest specific activity of the enzyme in etiolated leaf tissue was confirmed to be in the fraction of prolamellar bodies.Abbreviations PChlide protochlorophyllide - PLB prolamellar body - PT prothylakoid     

Development and ageing of etioplast structures in dark grown leaves ofAvena sativa (L.)

Summary Etioplasts of dark grown plants contain a large paracrystalline prolamellar body (PLB) and, attached to this there are prothylakoid membranes (PTs).PLB-tubules inAvena are composed mainly of two saponins and include only a low percentage of other lipids, protochlorophyll(ide) and proteins.Following the development of etioplasts in darkness from the very beginning until plants loose turgescence one can observe marked changes in ultrastructure. In the early stage of development predominantly PTs are seen in small etioplasts. Wide-type PLBs are small. After eight days there is a well developed stage with the well-known big and highly crystalline PLBs, which are connected to many long PT-membranes. After 13 days the PLBs are not significantly changed, while number and length of PTs are strongly reduced.These morphological observations are quantified by measurements of PLB-area and PT-length per plastid section. Saponin content as a marker for PLB-tubules and protochlorophyll(ide)-content as a marker for PT-membranes were measured. Both methods of determination show in good agreement a peak of development for PTs around day 6–7, and for PLBs around 9–10. Beginning senescence affects PT-membranes and PChl(ide) strongly, while saponins resp. PLBs persist better. These results are presented in view of thylakoid formation during greening, starting from the different etioplast stages.Abbreviations Chl(ide) chlorophyll(ide) - EM electron-microscope - PChl(ide) protochlorophyll(ide) - PLB prolamellar body - PT prothylakoid - TLC Thin layer chromatography A preliminary report has been presented at the V. Intern. Congress on Photosynthesis at Chalkidiki, Greece 1980.     

Formation of the Pigment Apparatus in Etiolated Barley Leaves under the Influence of Levulinic Acid

The effects of levulinic acid (LA) on the synthesis of pigments and the membrane system of etioplasts were studied in etiolated leaves of barley (Hordeum vulgare L.). Growing in the solution of LA during a six-day period, which started one day after the soaking of seeds, resulted in a retardation of leaf growth, more than a twofold decrease in the level of carotenoids and protochlorophyllide (Pd) in the leaf tissue, and suppression of the synthesis of long-wave form of Pd₆₅₅; these effects depended on the LA concentration. In etioplasts isolated from the seedlings treated with 50 M LA and containing predominantly a short-wave form of Pd with a peak of fluorescence at 632 nm (–196°C), there was a membrane fraction whose location in the sucrose density gradient was identical to that of prolamellar bodies (PLB) in the control plants. The content of Pd and carotenoids in this fraction calculated on a protein basis was 2.46 and 1.3 times lower than in control seedlings, while the relative content of Pd oxidoreductase (POR) essentially did not change. Thus, the suppression of Pd synthesis did not affect translocation of POR from the cytoplasm to the membranes of etioplasts. In the PLB membranes, there was no transfer of energy from the molecules of lipophilic fluorescent probe pyrene (excitation at 337, 278, and 296 nm) to Pd; however, under pigment deficiency, the production therein of pyrene excimer form at the expense of energy transfer from protein tryptophanyls (excitation at 278 and 296 nm) became more efficient, which indicated changes in protein–lipid interactions. The obtained results suggest that the short-wave form of Pd₆₃₂ accumulating in etioplasts under the suppressed synthesis of tetrapyrroles is not a free pigment.     

The polypeptide composition of highly purified prolamellar bodies and prothylakoids from wheat (Triticum aestivum) as revealed by silver staining

The inner membranes from wheat ( Triticum aestivum L. cv. Walde, Weibull) etioplasts were separated by density centrifugation. The etioplasts were broken by osmotic shock and the inner membranes were split by the sheering forces when pressed through a syringe needle. Membrane fractions representative of prolamellar bodies and prothylakoids, respectively, were achieved by separation on a 20–50% continuous sucrose density gradient followed by different purification procedures. The membrane contents of the isolated fractions were characterized by low temperature fluorescence spectra, sodium dodecyl sulphate polyacrylamide gel electrophoresis and electron micrographs. The prolamellar body and the prothylakoid fractions had a fluorescence emission ratio 657/633 nm of 18 and 0.9, respectively. The main part of the total amount of PChlide was found in the prolamellar body fraction. The electrophoretograms stained with Coomassie Blue showed the presence of mainly two polypeptides. The NADPH-protochlorophyllide oxidoreductase was the dominating polypeptide in the prolamellar body fraction, and the α and β subunits of the coupling factor 1 of chloroplast ATP synthase the dominating polypeptides in the prothylakoid fraction. Silver staining revealed at least 4 additional prominent bands with molecular weights of 86, 66, 34 and 28 kDa. The polypeptide composition of the prolamellar body is thus more complex than earlier judged after Coomassie Blue staining. The function of these polypeptides is unknown, but the knowledge of their presence is important in understanding the formation and function of the prolamellar body.     

Visualization and characterization of prolamellar bodies with atomic force microscopy

Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of atomic force microscopy (AFM), transmission electron microscopy (TEM) and fluorescence spectroscopy. With AFM, PLBs were seen as spherical structures about 1–2 μm in diameter, more elastic than mica and poly-l-lysine substrate. TEM analyses confirmed that PLBs of wheat leaf etioplasts also had an average diameter of appr. 1 μm. Illumination induced the photoreduction of photoactive protochlorophyllide (Pchlide), i.e. Pchlide bound to protochlorophyllide oxidoreductase, which was shown in fluorescence spectra. The photoreduction was followed by the disruption of PLB structures, which started with the enlargement of PLB spheres and then their fragmentation into small balls as seen with AFM. Light-induced vesicle formation and the outgrowth of lamellar (pro)thylakoid membranes on the PLB surface were also confirmed by TEM analyses, and resulted in the apparent enlargement of the PLB diameter. The blue-shift of the fluorescence emission maximum of chlorophyllide observed for PLBs at room temperature after Pchlide photoreduction was completed within 25 min. However, structural changes in PLBs were still observed after the completion of the blue-shift. The incubation of PLBs in darkness with HgCl₂ also resulted in PLB enlargement and a loosening of their structure. AFM provides a unique opportunity to observe PLBs at a physiological temperature without the necessity of fixation.     

Proteomic analysis of highly purified prolamellar bodies reveals their significance in chloroplast development

The prolamellar body (PLB) proteome of dark-grown wheat leaves was characterized. PLBs are formed not only in etioplasts but also in chloroplasts in young developing leaves during the night, yet their function is not fully understood. Highly purified PLBs were prepared from 7-day-old dark-grown leaves and identified by their spectral properties as revealed by low-temperature fluorescence spectroscopy. The PLB preparation had no contamination of extra-plastidal proteins, and only two envelope proteins were found. The PLB proteome was analysed by a combination of 1-D SDS-PAGE and nano-LC FTICR MS. The identification of chlorophyll synthase in the PLB fraction is the first time this enzyme protein was found in extracts of dark-grown plants. This finding is in agreement with its previous localization to PLBs using activity studies. NADPH:protochlorophyllide oxidoreductase A (PORA), which catalyses the reduction of protochlorophyllide to chlorophyllide, dominates the proteome of PLBs. Besides the identification of the PORA protein, the PORB protein was identified for the first time in dark-grown wheat. Altogether 64 unique proteins, representing pigment biosynthesis, photosynthetic light reaction, Calvin cycle proteins, chaperones and protein synthesis, were identified. The in number of proteins’ largest group was the one involved in photosynthetic light reactions. This fact strengthens the assumption that the PLB membranes are precursors to the thylakoids and used for the formation of the photosynthetic membranes during greening. The present work is important to enhance our understanding of the significance of PLBs in chloroplast development.     

Light-induced structural changes during incubation of isolated maize etioplasts

Summary Etioplasts were isolated from leaves of 9-day-old etiolated maize (Zea mays L.) seedlings and incubated in a relatively simple medium in light and in the dark. During the first 5 h no changes occurred in the fine structure of the isolated etioplasts in the dark. In light the size of the prolamellar bodies decreased and significantly more plastid sections without prolamellar bodies were counted. The total length of the thylakoids per plastid section increased, but there was no evidence for bi- and polythylakoid formation. It is concluded that light induces the structural transformation of the prolamellar body membranes into primary thylakoids also in isolated etioplasts.     

Effect of Periodic Heat Shock on the Inner Membrane System of Etioplasts

Etiolated barley (Hordeum vulgare L.) seedlings were treated with heat shock (HS). The heat treatment was conducted daily for 1 h at 40°C over 6 days and led to shortening of leaves and coleoptiles, an increase in the etioplast volume and prothylakoid length, and to a decrease in the size of paracrystalline prolamellar bodies (PLB). As a result of HS treatment, stimulation of carotenoid and protochlorophyllide (Pchlide) synthesis as well as an increase in the relative content of the Pchlide short-wavelength form (Pchlide₆₃₀) were observed in the leaf tissue of seven-day-old seedlings 12 h after the last HS treatment. HS had no effect on the overall amount of Pchlide-oxidoreductase (POR) in leaves and PLB membranes and did not suppress the Pchlide photoreduction in vivo. PLB membranes, isolated from the HS-treated seedlings, possessed a higher Pchlide and carotenoid content as calculated on total protein basis. These membranes showed more intense protein fluorescence than PLB from untreated plants, whereas hydrophobicity of the microenvironment of the fluorescent amino-acid residues remained unchanged. Studies using pyrene (lipophilic fluorescent probe emitted in Pchlide and carotenoid absorption bands) showed that HS increases the fluidity of membrane lipids in PLB membranes and that the pigments accumulated in these membranes are located in the region of lipid–protein contact site. The results are discussed in relation to the adaptive role of protein–protein and pigment–protein–lipid interactions in etioplast membranes under stress.     

Chlorophyll synthetase is latent in well preserved prolamellar bodies of etiolated wheat

Membrane fractions containing intact etioplasts, etioplast inner membranes, prolamellar bodies or prothylakoids from wheat ( Triticum aestivum L. cv. Walde) were assayed for chlorophyll synthetase activity. Calculated on a protein basis, the etioplast inner membrane fraction showed a higher activity than the intact etioplasts. The activity was higher in the prolamellar body fraction than in the prothylakoid fraction. However, when the fractions were incubated in isolation medium with 50% (w/w) sucrose and 0.3 m M NADPH, chlorophyll synthetase activity could not be detected in the prolamellar body fraction, while the prothylakoid fraction maintained a high activity. The spectral shift to a shorter wavelength of the newly formed endogenous chlorophyllide was very rapid in the prothylakoid fraction but slow in the prolamellar body fraction. The relation between the spectral shift of chlorophyllide and the esterification activity in the fractions is discussed. Even exogenous short-wavelength chlorophyllide could not be esterified in well preserved prolamellar bodies. This indicates that chlorophyll synthetase is present in an inactive state in the prolamellar body structure. A large-scale method for the synthesis of geranylgeranylpyrophosphate, one of the substrates of the chlorophyll synthetase reaction, is also presented.     

Ultrastructural changes in isolated plastids

Summary Etioplasts were isolated from maize leaves and the changes in their ultrastructure were followed in light and in darkness for several hours. It has been shown that the regular crystalline structures of prolamellar bodies, present after the isolation in darkness, disappear after 30 to 60 minutes of illumination, and long straight tubules appear within prolamellar bodies. Their appearance is influenced by the molarity of the isolation medium used, by light intensity, duration of illumination and by the temperature at which the isolates are kept. Long tubules appear, however, also in isolated etioplasts incubated for several hours in complete darkness.In isolates illuminated for 2–3 hours long tubules disappear again, and prolamellar bodies produced eventually consist of irregularly connected short tubules. In prolamellar bodies, regions with regular and very dense arrangement of tubules sometimes develop at this stage. The thylakoids (usually perforated) are now arranged concentrically in the plastids. True grana or poly-thylakoids can never be found in isolated etioplasts, not even when the etioplasts have been illuminated for 6 hours or more (up to 24).The present investigations have indicated that in isolated etioplasts in light, tubular elements, which build up the prolamellar bodies, cannot normally be transformed into thylakoids as is the case with intact tissue.The survival of isolated etioplasts is limited at present, and for this reason changes in their fine structure could be followed successfully for as long as 6 hours (in light at 15 °C), although a certain percentage of plastids survive up to 24 hours.     

Localization of Galactolipid Biosynthesis in Etioplasts Isolated from Dark-Grown Wheat (Triticum aestivum L.)

Etioplasts were isolated from leaves of dark-grown wheat (Triticum aestivum L. var Starke II). Galactolipid biosynthesis was assayed in an envelope-rich fraction and in the fraction containing the rest of the etioplast membranes by measuring incorporation of ¹⁴C from uridine-diphospho[¹⁴C]galactose into monogalactosyl diacylglycerol and digalactosyl diacylglycerol. More than half of the galactolipid biosynthetic capability was found in the fraction of inner etioplast membranes. This fraction was subfractioned into fractions enriched in prolamellar bodies and membrane vesicles (prothylakoids), respectively. All membrane fractions obtained from etioplasts were able to carry out galactolipid biosynthesis, although the activity was very low in prolamellar body-enriched fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed markedly different polypeptide patterns between the different fractions. It is concluded that the capability of galactolipid biosynthesis of etioplasts probably is not restricted to the envelope, but is also present in the inner membranes of this plastid.     

Impaired carotenogenesis can affect organization and functionality of etioplast membranes

The effects of impaired carotenogenesis on plastid membrane organization, functionality and stability were studied in etiolated barley plants grown at 20 and 30°C. The plants were treated with norflurazon or amitrole, two herbicides affecting phytoene desaturation and lycopene cyclization, respectively. At 20°C, the amitrole-treated etioplasts, which accumulated lycopene in their inner membranes, exhibited disorganized prolamellar bodies, containing a prevalent form of non-phototransformable protochlorophyllide (Pchlide). They also showed a certain difficulty in reducing the phototransformable pigment to chlorophyllide when exposed to light, and were unable to reform the active ternary complex [protochlorophyllide–oxidoreductase (POR)–Pchlide–NADPH] when placed back in darkness. No ultrastructural alterations were found in norflurazon-treated etioplasts, with carotenogenesis inhibited at the phytoene desaturation step. In these latter organelles, Pchlide, whose forms were comparable with those of the control etioplasts, was photoreduced quickly after illumination and the ternary complex was reformed during a subsequent dark period. Thus, the impaired carotenogenesis leading to the accumulation of lycopene showed greater interference with the etioplast membrane arrangement and functionality than did the earlier interruption of the biosynthetic pathway at the phytoene level. This might be due to the different interactions of the distinct carotenoid precursors with other membrane components. However, in etioplasts of norflurazon-treated plants, a rise in growth temperature caused a partial demolition of prolamellar bodies, showing a lowered thermostability of the carotenoid-deficient membranes. This latter effect strengthens the concept that a correct and complete carotenogenesis pathway, leading to the synthesis of polar carotenoids (i.e. xanthophylls), is required for the maintenance of stable plastid membranes.     

An ultrastructural study of maize leaf etioplasts throughout their entire life-cycle

Summary The entire life-cycle of maize leaf etioplasts has been followed. Prolamellar bodies with different types of tubular membrane arrangement can be found in the juvenile stages of the organelles, while in mature etioplasts nearly all the prolamellar bodies exhibit an hexagonal ring arrangement, which, by optical diffraction, appears to be the most regular and compact possible.The prothylakoid membranes also undergo changes during organelle differentiation, and their different organization and arrangement produce a clear dimorphism between the etioplasts of mesophyll and bundle sheath cells.In senescent etioplasts the prothylakoids are more affected, while the prolamellar bodies appear rather stable, also in situations where protochlorophyll(ide) content is very low. The formation of clusters of osmiophilic globules is coupled with the breakdown of the etioplast membranes.     

Association of the NADPH:protochlorophyllide oxidoreductase (POR) with isolated etioplast inner membranes from wheat

Membrane association of NADPH:protochlorophyllide oxidoreductase (POR, EC: 1.6.99.1) with isolated prolamellar bodies (PLBs) and prothylakoids (PTs) from wheat etioplasts was investigated. In vitro-expressed radiolabelled POR, with or without transit peptide, was used to characterize membrane association conditions. Proper association of POR with PLBs and PTs did not require the presequence, whereas NADPH and hydrolysable ATP were vital for the process. After treating the membranes with thermolysin, sodium hydroxide or carbonate, a firm attachment of the POR protein to the membrane was found. Although the PLBs and PTs differ significantly in their relative amount of POR in vivo, no major differences in POR association capacity could be observed between the two membrane systems when exogenous NADPH was added. Experiments run with only an endogenous NADPH source almost abolished association of POR with both PLBs and PTs. In addition, POR protein carrying a mutation in the putative nucleotide-binding site (ALA06) was unable to bind to the inner membranes in the presence of NADPH, which further demonstrates that the co-factor is essential for proper membrane association. POR protein carrying a mutation in the substrate-binding site (ALA24) showed less binding to the membranes as compared to the wild type. The results presented here introduce studies of a novel area of protein-membrane interaction, namely the association of proteins with a paracrystalline membrane structure, the PLB.     

Influence of Divalent Cations and Chelators on the Structure of Prolamellar Bodies of Avena sativa

The influence of the cations Ca²⁺ and Mg²⁺ on the structureof oat prolamellar bodies (PLB), set free from etioplasts andinside intact etioplasts, was examined. The results presentedgive evidence for a relation between PLB-structure, its transformationand the presence of divalent cations, especially Ca²⁺. Underin vitro conditions the structure of isolated PLBs and of PLBsin intact etioplasts is stabilised by calcium. Deprivation ofdivalent cations by ethylenedi-aminetetraacetic acid (EDTA)or ethyleneglycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraaceticacid (EGTA) led to a breakdown of PLBs to structures very similarto those found in plastids after short time illumination ofetiolated leaves. Thus it seems reasonable, that Ca²⁺ is animportant factor in stabilising PLBs and in the regulation ofthe in vivo light triggered transformation of PLBs. (Received June 6, 1989; Accepted August 30, 1989)     

A Comparison between Prolamellar Bodies and Prothylakoid Membranes of Etioplasts of Dark-Grown Wheat Concerning Lipid and Polypeptide Composition

The aim of the present investigation was to find factors critical for the co-existence of prolamellar bodies and prothylakoids in etioplasts of wheat (Triticum aestivum L. cv Starke II). The lipid composition of the prolamellar body and prothylakoid fractions was qualitatively similar. However, the molar ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was higher in the prolamellar body fraction (1.6 ± 0.1), as was the lipid content on a protein basis. Protochlorophyllide was present in both fractions. The dominating protein of the prolamellar body fraction was protochlorophyllide oxidoreductase. This protein was present also in prothylakoid fractions. The other major protein of the prothylakoid fraction was the coupling factor 1, subunit of the chloroplast ATPase. From the lipid and protein data, we conclude that prolamellar bodies are formed when monogalactosyl diacylglycerol is present in larger amounts than can be stabilized into planar bilayer prothylakoid membranes by lamellar lipids or proteins.     

Effects of salt wash on the structure of the prolamellar body iembrane and the membrane binding of NADPH–protochlorophyllide oxidoreductase

NADPn–protochlorophyllide oxidoreductase (EC 1.6.99.1; PCR) is the major protein component of the prolamellar body (PLB) membrane of the etioplast. The interaction between the pigment–protein complex of PCR and the membrane lipids is of importance for the induction and maintenance of the regularly branched PLB structure. The isoelectric point of the PLB surface and the impact of salt treatment on the PLB structure, the PCR absorbance properties and the association of PCR to the membrane, have been studied in isolated fractions of PLBs from dark–grown wheat ( Triticum aestivum L. cv. Starke 11). We conclude that the PLB membrane has its isoelectric point at pH 4.5. which is similar to that of other plastid membranes. The PLB membrane and the pigment–protein complex of PCR are both affected by salt treatment. Concentrations below 50 mM MgCl₂, or 250 m M KCI tend to stabilize the regularly branched strueture. while higher concentrations of both mono– and divalent cations lead to disintegration of the membrane and shifts towards shorter wave–lengths of the in vivo absorbance spectra of protoehlorophyllide. PCR. the dominant PLB protein, however, seems to be intimately associated with the membrane lipids and is not washed off the membrane by repeated salt treatment.     

Etioplast and etio-chloroplast formation under natural conditions: the dark side of chlorophyll biosynthesis in angiosperms

Chloroplast development is usually regarded as proceeding from proplastids. However, direct or indirect conversion pathways have been described in the literature, the latter involving the etioplast or the etio-chloroplast stages. Etioplasts are characterized by the absence of chlorophylls (Chl-s) and the presence of a unique inner membrane network, the prolamellar body (PLB), whereas etio-chloroplasts contain Chl-s and small PLBs interconnected with chloroplast thylakoids. As etioplast development requires growth in darkness for several days, this stage is generally regarded as a nonnatural pathway of chloroplast development occurring only under laboratory conditions. In this article, we have reviewed the data in favor of the involvement of etioplasts and etio-chloroplasts as intermediary stage(s) in chloroplast formation under natural conditions, the molecular aspects of PLB formation and we propose a dynamic model for its regulation.     

Distribution of chloroplast coupling factor (CF1) particles on plastid membranes during development

Samples of internal membrane systems separated from lysates of intact plastids from dark grown Avena sativa L. (vars, Cooba and Mostyn) and Hordeum vulgare L. (vars, Himalaya and Deba Abed) given different periods of illumination before isolation were assayed for trypsin-activated Ca²⁺-dependent ATPase activities and also examined in the electron microscope after treatment in the manner described by Oleszko and Moudinanakis (1974) which assists the visualization of the chloroplast coupling factor (CF₁) particles. Concentrations of membrane-attached CF₁ particles were not observed on the membrane surfaces of the prolamellar bodies (PLBs) proper but only on the attached extruded lamellar membranes. Increasing lengths of illumination followed by plastid isolation and subsequent membrane separation had the effect of progressively increasing the mean distance between these individual lamellar-attached CF₁ particles. Measurements of trypsin-activated Ca²⁺-dependent ATPase activities during similar developmental regimes indicated that functions associated with CE₁ particles are relative constant and largely independent of the period of illumination if the values were expressed on a per plastid basis indicating that assembly of CF₁ particles may take place in either etioplasts, etiochloroplasts or mature chloroplasts.Abbreviations PLB prolamellar body - EDTA ethylene-diaminetetra-acetic acid - CF₁ chloroplast coupling factor particles - ATPase adenosine triphosphatase     

Molecular insights into the primary nucleation of polymorphic amyloid β dimers in DOPC lipid bilayer membrane

Alzheimer''s disease (AD) pathology is characterized by loss of memory cognitive and behavioral deterioration. One of the hallmarks of AD is amyloid β (Aβ) plaques in the brain that consists of Aβ oligomers and fibrils. It is accepted that oligomers, particularly dimers, are toxic species that are produced extracellularly and intracellularly in membranes. It is believed that the disruption of membranes by polymorphic Aβ oligomers is the key for the pathology of AD. This is a first study that investigate the effect of polymorphic “α‐helix/random coil” and “fibril‐like” Aβ dimers on 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) membrane. It has been found that the DOPC membrane promotes Aβ_1–42 “fibril‐like” dimers and impedes Aβ_1–42 “α‐helix/random coil” dimers. The N‐termini domains within Aβ_1–42 dimers play a role in Aβ aggregation in membrane milieus. In addition, the aromatic π–π interactions (involving residues F19 and F20 in Aβ_1–42) are the driving forces for the hydrophobic interactions that initiate the primary nucleation of polymorphic Aβ_1–42 dimers within DOPC membrane. Finally, the DOPC bilayer membrane thickness is locally decreased, and it is disrupted by an embedded distinct Aβ_1–42 dimer, due to relatively large contacts between Aβ_1–42 monomers and the DOPC membrane. This study reveals insights into the molecular mechanisms by which polymorphic early‐stage Aβ_1–42 dimers have distinct impacts on DOPC membrane.