Lipid composition and molecular organization in plasma membrane-enriched fractions from senescing cotyledons

Plasma membrane fractions isolated from cotyledons of Phaseolus vulgaris L. cv. Kinghorn at various stages of senescence showed no significant change in fatty acid saturation with advancing senescence. However, the steroliphospholipid ratio increased by about 400% as senescence intensified. The lipid phase transition temperature of the membranes, which was measured by wide-angle x-ray diffraction, also rose from a point well below the growing temperature for young tissue to about 50°C for membrane from extensively senescent 9-day-old tissue. This means that by day 4 of germination there was a mixture of liquid-crystalline and gel phase phospholipid in the membrane matrices. Crystallinity attributable to sterol-sterol interaction was also apparent in the diffraction patterns for senescent membranes. The co-existence of gel and liquid-crystalline phase phospholipid in the aging membranes as well as the crystalline sterol aggregates presumably render the storage cells of cotyledons leaky and may thus facilitate the translocation of hydrolyzed food reserves into the vascular network.     

Isolation and characterization of deteriosomes from rat liver

Deteriosomes, a new class of microvesicles, have been isolated from rat liver tissue. These microvesicles are similar to those isolated previously from plant tissue [Yao et al., Proc Natl Acad Sci USA 88:2269–2273, 1991] in that they are nonsedimentable and enriched in membrane catabolites, particularly products of phospholipid degradation. Liver deteriosomes range in size from 0.05 μm to 0.11 μm in radius. They are also much more permeable than microsomal membrane vesicles indicating that the deteriosome bilayer is perturbed. The data are consistent with the proposal that deteriosomes are formed from membranes by microvesiculation and that they represent an intermediate stage of membrane deterioration. Furthermore, liver deteriosomes were found to contain phospholipase A₂ activity. This suggests that they not only serve as a means of moving destabilizing macromolecular catabolites out of membranes into the cytosol but also possess enzymatic activity. The fact that the specific activity of phospholipase A₂ is higher in deteriosomes than in deteriosome-free cytosol suggests that some of the enzymatic activity traditionally assumed to be cytosolic may in fact be associated with deteriosomes.     

Induction of gel-phase lipid in plasma membrane of chick intestinal cells after coccidial infection

When chickens are infected with the coccidial parasite Eimeria necatrix, the plasma membrane of intestinal cells harbouring second-generation schizonts becomes refractory to mechanical shearing, hypotonic shock and ultrasonication. Plasma membrane from these infected cells was isolated to high purity as judged by enriched levels of ouabain-sensitive (Na⁺ + K⁺)-stimulated Mg²⁺-dependent ATPase activity and sialic acid content, the lack of detectable cytochrome oxidase and glucose-6-phosphatase activities and electron microscopic analysis of the final preparation. Wide-angle X-ray diffraction patterns recorded from the isolated membranes revealed that during the later stages of parasite maturation the host cell plasma membrane acquires increasing proportions of gel-phase lipid. By contrast, purified membrane from isolated parasites is in a liquid-crystalline state. The transition temperature of host cell plasmalemma at 100 h postinfection is 61°C, about 20°C above physiological temperature. By contrast, liposomes of plasma membranes from infected cells undergo a thermal transition at about 28°C. The accumulation of gel-phase lipid in the host cell plasma membrane is not attributable either to an increase in the constituent ratio of saturated to unsaturated fatty acids or to a significant change in the cholesterol to phospholipid ratio. During the late stages of infection, the cells become stainable with trypan blue which suggests that the acquisition of crystalline phase lipid disrupts the permeability of the host cell plasmalemma.     

Calcium- and calmodulin-regulated breakdown of phospholipid by microsomal membranes from bean cotyledons

Evidence for the involvement of Ca²⁺ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U-¹⁴C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U-¹⁴C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca²⁺, whereas lipolytic acyl hydrolase proved to be insensitive to Ca²⁺. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca²⁺. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC₅₀ values ranging from 10 to 15 micromolar. Thus the Ca²⁺-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca²⁺ on phospholipase D is independent of calmodulin. The role of Ca²⁺ as a second messenger in the initiation of membrane lipid degradation is discussed.     

Phase properties of senescing plant membranes. Role of the neutral lipids

Wide-angle X-ray diffraction studies have indicated that rough and smooth microsomal membranes from bean cotyledons acquire increasing proportions of gel phase lipid at physiological temperature as the tissue senesces. In addition, for both types of membrane the lipid phase transition temperature, defined as the highest temperature at which gel phase lipid can be detected, progressively rises with advancing senescence. Liposomes prepared from total lipid extracts of the membranes show a similar increase in transition temperature with age, indicating that separation of the polar lipids into distinct gel and liquid-crystalline domains is not attributable to peculiar protein-lipid interactions. Liposomes prepared from purified phospholipid fractions of the membranes show little change in transition temperature with age, indicating that the altered phase properties of the lipid do not reflect an increase in fatty acid saturation. However, the formation of gel phase lipid that occurs naturally during senescence can be stimulated by preparing liposomes from a mixture of the phospholipid fraction from young membrane and the neutral lipid fraction from old membrane. By adding the separated components of the neutral lipid fraction to purified phospholipid it was found that sterol esters and several unidentified lipids are able to raise the transition temperature of the polar lipids. Sterols have no effect on the phospholipid transition temperature. The data have been interpreted as indicating that several neutral lipids, which presumably increase in abundance with advancing senescence, induce a lateral phase separation of the polar lipids resulting in distinct gel and liquid-crystalline domains of lipid in the senescent membranes.     

Evidence for the accumulation of peroxidized lipids in membranes of senescing cotyledons

Fluorescent products of lipid peroxidation accumulate with age in microsomal membranes from senescing cotyledons of Phaseolus vulgaris. The temporal pattern of accumulation is closely correlated with a rise in the lipid phase transition temperature reflecting the formation of gel phase lipid. Increased levels of fluorescent peroxidation products are also detectable in total lipid extracts of senescent cotyledons. Lipoxygenase activity increases with advancing age by about 3-fold on a fresh weight basis and 4-fold on a dry weight basis indicating that the tissue acquires elevated levels of lipid hydroperoxides. As well, levels of glutathione and superoxide dismutase activity decline on a dry weight basis as the cotyledons age, rendering the tissue more susceptible to oxidative damage. Catalase activity rises initially and then declines during senescence, but peroxidase activity rises steeply. Thus, apart from this increase in peroxidase, which would scavenge H₂O₂ only if appropriate cosubstrates were available, the defense mechanisms for coping with activated oxygen species (O₂⁻, H₂O₂, OH) are less effective in the older tissue. The observations support the contention that formation of gel phase lipid in senescing membranes is attributable to lipid peroxidation and suggest that the reactions of lipid peroxidation are utilized by the cotyledons to mediate deteriorative changes accompanying the mobilization and transport of metabolites from the storage tissue to the developing embryo.     

In vitro simulation of cytoplasmic membrane senescence in cotyledons

The loss of microsomal NADH-cytochrome c reductase activity (EC 1.6.99.3) in cotyledons, known to accompany germination of Phaseolus vulgaris and thought to reflect the progress of cytoplasmic membrane senescence, can be simulated in an in vitro system in which isolated microsomes from 2-day-old tissue are treated with cytosol fractions (microsomal supernatants). Inactivation of the enzyme is comparatively low when the microsomes are treated for 4 hours with cytosol fractions from 1- and 2-day-old tissue, but increases to about 68% upon treatment with a corresponding fraction from 3-day-old cotyledons. This temporal pattern is consistent with the pronounced in situ decline in NADH-cytochrome c reductase detectable between the 2nd and 4th days of germination. Extensive in vitro inactivation was also effected by cytosol fractions prepared from older tissue, including that harvested after 9 days of germination by which time the cotyledons were beginning to abscise.     

Effects of in vitro treatment with ozone on the physical and chemical properties of membranes

Treatment of microsomal membranes from cotyledons of Phaseolus vulgaris with ozone raises the liquid-crystalline to gel lipid phase transition temperature and results in the formation of distinct domains of gel phase lipid in the membranes. Liposomes prepared from the total lipid extracts of ozone-treated membranes undergo phase separations just a few degrees below the transition temperature for intact membranes, indicating that the formation of gel phase lipids is largely attributable to ozone-induced alterations in the membrane lipids. Levels of unsaturated fatty acids as well as the sterol to phospholipid ratio are markedly reduced in the ozone-treated membranes, and the neutral lipid fraction from treated membranes shows, an increased propensity to induce the formation of gel phase phospholipid when incorporated into liposomes of egg phosphatidylcholine. Since gel phase phospholipid also forms in naturally senescing plant membranes and appears to be attributable to changes in the neutral lipid fraction, the effects of natural senescence and ozone on membranes have been compared.     

Lipid crystallization in senescent membranes from cotyledons

Lipid transition temperatures for rough and smooth microsomal membranes isolated from bean (Phaseolus vulgaris) cotyledon tissue at various stages of germination were determined by wide angle x-ray diffraction. The transition temperatures were established by recording diffraction patterns through a temperature series until a sharp x-ray reflection centered at a Bragg spacing of 4.15 Å and denoting the presence of crystalline lipid was discernible. For rough and smooth microsomes from 2-day-old tissue, the transitions occurred at 0 C and 3 C, respectively, indicating that at this early stage in the germination sequence the membrane lipid is entirely liquid-crystalline at physiological temperature. By the 4th day of germination, the transition temperatures had increased to 32 C for smooth microsomes and 35 C for rough microsomes, indicating that at 29 C, which was the growth temperature, portions of the membrane lipid were crystalline. During the later stages of germination, the transition temperature for smooth microsomes continued to rise through 44 C at day 7 to 56 C at day 9, by which time the cotyledons were extensively senescent and beginning to abscise. There was also a dramatic increase in the proportion of membrane lipid in the crystalline phase at 29 C. By contrast, the rough microsomes showed little change in transition temperature and only a slight increase in the proportion of crystalline lipid during this late period in germination. The data indicate that substantial amounts of the lipid is senescing membranes are crystalline even at physiological temperature. Moreover, there is a temporal correlation between the appearance of this crystallinity and loss of membrane function, suggesting that the two may be causally related.     

RNA and DNA interactions with zwitterionic and charged lipid membranes — A DSC and QCM-D study

The aim of the present study is to establish under which conditions tRNA associates with phospholipid bilayers, and to explore how this interaction influences the lipid bilayer. For this purpose we have studied the association of tRNA or DNA of different sizes and degrees of base pairing with a set of model membrane systems with varying charge densities, composed of zwitterionic phosphatidylcholines (PC) in mixtures with anionic phosphatidylserine (PS) or cationic dioctadecyl-dimethyl-ammoniumbromide (DODAB), and with fluid or solid acyl-chains (oleoyl, myristoyl and palmitoyl). To prove and quantify the attractive interaction between tRNA and model-lipid membrane we used quartz crystal microbalance with dissipation (QCM-D) monitoring to study the tRNA adsorption to deposit phospholipid bilayers from solutions containing monovalent (Na⁺) or divalent (Ca²⁺) cations. The influence of the adsorbed polynucleic acids on the lipid phase transitions and lipid segregation was studied by means of differential scanning calorimetry (DSC). The basic findings are: i) tRNA adsorbs to zwitterionic liquid-crystalline and gel-phase phospholipid bilayers. The interaction is weak and reversible, and cannot be explained only on the basis of electrostatic attraction. ii) The adsorbed amount of tRNA is higher for liquid-crystalline bilayers compared to gel-phase bilayers, while the presence of divalent cations show no significant effect on the tRNA adsorption. iii) The adsorption of tRNA can lead to segregation in the mixed 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC)-1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) and DMPC-DODAB bilayers, where tRNA is likely excluded from the anionic DMPS-rich domains in the first system, and associated with the cationic DODAB-rich domains in the second system. iv) The addition of shorter polynucleic acids influence the chain melting transition and induce segregation in a mixed DMPC-DMPS system, while larger polynucleic acids do not influence the melting transition in these system. The results in this study on tRNA-phospholipid interactions can have implications for understanding its biological function in, e.g., the cell nuclei, as well as in applications in biotechnology and medicine.     

Acyl chain and head group regulation of phospholipid catabolism in senescing carnation flowers

Microsomal membranes from the petals of senescing carnation (Dianthus caryophyllus L.) flowers contain phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol. These phospholipid classes decline essentially in parallel during natural senescence of the flower and when microsomal membranes isolated from young flowers are aged in vitro. However, measurements of changes in the endogenous molecular species composition of microsomal phospholipids during natural senescence of the flower petals and during in vitro aging of isolated membranes have indicated that the various molecular species of phospholipids have quite different susceptibilities to catabolism. Acyl chain composition and the nature of the head group are both determinants of their susceptibility to catabolism. As well, a comparison of the phospholipid catabolism data for naturally senesced membranes and for membranes aged in vitro suggests that the phospholipid composition of membranes is continuously altered during senescence by acyl chain desaturation and possibly retailoring so as to generate molecular species that are more prone to catabolism. The results collectively indicate that provision of particular molecular species of phospholipids with increased susceptibility to degradation contributes to enhanced phospholipid catabolism in the senescing carnation petal.     

Effect of a freeze-thaw cycle on properties of microsomal membranes from wheat

A freeze-thaw cycle to −12°C induced several physical and compositional changes in the microsomal membranes isolated from crown tissue of winter wheat (Triticum aestivum L. cv Frederick). Exposing 7-day-old, nonacclimated seedlings to a single freeze-thaw cycle prevented regrowth of the crown and resulted in increased membrane semipermeability. The phospholipid and protein content of microsomal membranes isolated from the crowns decreased by 70 and 50%, respectively. Microsomal membranes isolated after the lethal freeze-thaw stress, and liposomes prepared from total membrane lipids, exhibited greater microviscosity, measured by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The number of free thiol groups per milligram membrane protein, measured using the specific fluorescent probe, N-dansylaziridine, decreased after freezing. In contrast, acclimated wheat seedlings which showed increased freezing tolerance, as indicated by survival and ion leakage, suffered almost no effects from the freeze thaw treatment as determined by measurements of membrane microviscosity, phospholipid content, protein content, or danzylaziridine fluorescence. An examination of membranes isolated from frozen tissue showed that most of the changes occurred during the freezing and not during the thawing phase.     

Microsomal membrane structure and function subsequent to calcium activation of an endogenous phospholipase

An endogenous system in the membranes of rat liver endoplasmic reticulum is capable upon Ca²⁺ activation of considerable disruption of normal structure and function. Phosphatidylethanolamine (PE) and to a lesser extent phosphatidylcholine (PC) are degraded to hydrophilic products. This lipid loss is greater at an alkaline pH, preferentially utilizes millimolar Ca²⁺ rather than Mg²⁺ ions, and is inhibited by KCl. Diethyl ether has no effect on the rate of loss of PE or PC, and the Ca²⁺ ionophore A23187 does not lower the Ca²⁺ requirement. Phospholipids are most likely lost from the membranes in a two-step process. Lysophospholipids generated in the first, Ca²⁺-dependent step are removed by an endogenous lysophospholipase demonstrated by the hydrolysis of either added lyso PE or lysophospholipids generated from endogenous substrates by Naja naja phospholipase A₂. The depletion of microsomal membrane phospholipid is accompanied by a loss of glucose 6-phosphatase and of cytochrome P-450. The latter is not associated with any change in total heme content. Polyacrylamide gel electrophoresis showed no difference between the pattern or relative amounts of solubilized membrane proteins before or after depletion of membrane phospholipid. It is concluded that activation of an endogenous phospholipase by Ca²⁺ can result in significant depletion of PE and PC that is accompanied by considerable disruption of membrane function. The significance of this system with respect to the maintenance of cell integrity and its possible role in cell injury are discussed.     

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A² treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.     

Induction of gel-phase lipid in plasma membrane of chick intestinal cells after coccidial infection.

When chickens are infected with the coccidial parasite Eimeria necatrix, the plasma membrane of intestinal cells harbouring second-generation schizonts becomes refractory to mechanical shearing, hypotonic shock and ultrasonication. Plasma membrane from these infected cells was isolated to high purity as judged by enriched levels of ouabain-sensitive (Na+ + K+)-stimulated Mg2-dependent ATPase activity and sialic acid content, the lack of detectable cytochrome oxidase and glucose-6-phosphatase activities and electron microscopic analysis of the final preparation. Wide-angle X-ray diffraction patterns recorded from the isolated membranes revealed that during the later stages of parasite maturation the host cell plasma membrane acquires increasing proportions of gel-phase lipid. By contrast, purified membrane from isolated parasites is in a liquid-crystalline state. The transition temperature of host cell plasmalemma at 100 h postinfection is 61 degrees C, about 20 degrees C above physiological temperature. By contrast, liposomes of plasma membranes from infected cells undergo a thermal transition at about 28 degrees C. The accumulation of gel-phase lipid in the host cell plasma membrane is not attributable either to an increase in the constituent ratio of saturated to unsaturated fatty acids or to a significant change in the cholesterol to phospholipid ratio. During the late stages of infection, the cells become stainable with trypan blue which suggests that the acquisition of crystalline phase lipid disrupts the permeability of the host cell plasmalemma.     

Phospholipid metabolism in membranes of senescing bean cotyledons

Mechanisms underlying the depletion of phospholipid in senescingmembranes have been examined using microsomes isolated frombean cotyledons (Phaseolus vulgaris) at various stages of development.As the cotyledons age, microsomal phospholipid levels relativeto protein decrease by 93% indicating that phospholipids areselectively depleted from senescing membranes. This reflectsactive phospholipid catabolism, but can also be attributed toa reduction in phospholipid synthesis. Specifically, the activitiesof choline phospho-transferase and ethanolamine phosphotransferase,enzymes mediating the terminal step in the synthesis of phosphatidylcholineand phosphatidylethanolamine, respectively, decrease dramaticallyas the cotyledons senesce. Phosphatidylcholine and phosphatidylethanolaminecomprise over 70% of the total phospholipid in these membranes,and this pronounced decline in their synthesis with advancingsenescence will lead to phospholipid depletion. There is alsoa decrease with age in the activity of acyl-CoA synthetase,which generates acyl-CoA for use in phospholipid synthesis.Microsomal phospholipid deacylation-reacylation activity declinesas well as the cotyledons senesce, but this can be accountedfor in terms of decreased levels of phospholipid available forthe reaction. Thus the depletion of phospholipid in senescingmembranes can be attributed to active catabolism in the faceof declining synthesis. Key words: Phospholipid synthesis, senescence, microsomes, Phaseolus vulgaris     

Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes

Two-photon excitation microscopy shows coexisting regions of different generalized polarization (GP) in phospholipid vesicles, in red blood cells, in a renal tubular cell line, and in purified renal brushborder and basolateral membranes labeled with the fluorescent probe laurdan. The GP function measures the relative water content of the membrane. In the present study we discuss images obtained with polarized laser excitation, which selects different molecular orientations of the lipid bilayer corresponding to different spatial regions. The GP distribution in the gel-phase vesicles is relatively narrow, whereas the GP distribution in the liquid-crystalline phase vesicles (DOPC and DLPC) is broad. Analysis of images obtained with polarized excitation of the liquid-crystalline phase vesicles leads to the conclusion that coexisting regions of different GP must have dimensions smaller than the microscope resolution (approximately 200 nm radially and 600 nm axially). Vesicles of an equimolar mixture of DOPC and DPPC show coexisting rigid and fluid domains (high GP and low GP), but the rigid domains, which are preferentially excited by polarized light, have GP values lower than the pure gel-phase domains. Cholesterol strongly modifies the domain morphology. In the presence of 30 mol% cholesterol, the broad GP distribution of the DOPC/DPPC equimolar sample becomes narrower. The sample is still very heterogeneous, as demonstrated by the separations of GP disjoined regions, which are the result of photoselection of regions of different lipid orientation. In intact red blood cells, microscopic regions of different GP can be resolved, whereas in the renal cells GP domains have dimensions smaller than the microscope resolution. Preparations of renal apical brush border membranes and basolateral membranes show well-resolved GP domains, which may result from a different local orientation, or the domains may reflect a real heterogeneity of these membranes.     

Hydrolysis of membrane phospholipids by phospholipases of rat liver lysosomes

(1) The hydrolysis of ³²P- or myo-[2-³H]inositol-labelled rat liver microsomal phospholipids by rat liver lysosomal enzymes has been studied. (2) The relative rates of hydrolysis of phospholipids at pH4.5 are: sphingomyelin>phosphatidylethanolamine>phosphatidylcholine> phosphatidylinositol. (3) The predominant products of phosphatidylcholine and phosphatidylethanolamine hydrolysis are their corresponding lyso-compounds, indicating a slow rate of total deacylation. (4) Ca²⁺ inhibits the hydrolysis of all phospholipids, though only appreciably at high (>5mm) concentration. The hydrolysis of sphingomyelin is considerably less sensitive to Ca²⁺ than that of glycerophospholipids. (5) Analysis of the water-soluble products of phosphatidylinositol hydrolysis (by using myo-[³H]inositol-labelled microsomal fraction as a substrate) produced evidence that more than 95% of the product is phosphoinositol, which was derived by direct cleavage from phosphatidylinositol, rather than by hydrolysis of glycerophosphoinositol. (6) This production of phosphoinositol, allied with negligible lysophosphatidylinositol formation and a detectable accumulation of diacylglycerol, indicates that lysosomes hydrolyse membrane phosphatidylinositol almost exclusively in a phospholipase C-like manner. (7) Comparisons are drawn between the hydrolysis by lysosomal enzymes of membrane substrates and that of pure phospholipid substrates, and also the possible role of phosphatidylinositol-specific lysosomal phospholipase C in cellular phosphatidylinositol catabolism is discussed.     

Age-induced changes in cellular membranes of imbibed soybean seed axes

The physical and chemical properties of microsomal membranes and cellular antioxidant systems were investigated in imbibed soybean ( Glycine max L. Merr. cv. Maple Arrow) seeds following aging for 5 years at room temperature. The loss of germination capacity in aged seeds was associated with increased solute leakage during imbibition and with a loss of membrane phospholipid. Higher levels of free fatty acids were observed in the microsomal membranes from aged seeds. However, there was no change in fatty acid saturation. Wide angle X-ray diffraction studies indicated the presence of gel phase in addition to liquid-crystalline phase lipid domains in the membranes of aged seeds. Those from fresh seeds were exclusively liquid-crystalline. Fluorescence depolarization, using diphenylhexatriene, suggested that the microviscosity of the membrane bilayer was increased by aging. Aged seeds had a lower antioxidant potential in the lipid fraction, lower tocopherol content, and reduced ascorbate:dehydroxyascorbate ratio indicating that the aging process was associated with exposure to an oxidative stress.     

Effects of adriamycin on lipid polymorphism in cardiolipin-containing model and mitochondrial membranes

(1) The effects of the anti-tumor drug adriamycin on lipid polymorphism in cardiolipin-containing model membranes and in isolated inner mitochondrial membranes has been examined by ³¹P-NMR. (2) Adriamycin binding does not affect the macroscopic structure or local order in the phosphate region of cardiolipin liposomes. (3) In cardiolipin liposomes and in cardiolipin-phosphatidylcholine (1:1) liposomes, the drug inhibits the ability of Ca²⁺ to induce the hexagonal H_II phase. (4) Adriamycin interaction with both dioleoylphosphatidylethanolamine-cardiolipin (2:1) and dioleoylphosphatidylethanolamine-phosphatidylserine (1:1) liposomes results in structural phase separation into a liquid-crystalline hexagonal H_II phase for the phosphatidylethanolamine and a liquid-crystalline lamellar phase for the negatively charged phospholipid. (5) Combined high-resolution ³¹P-NMR, electron microscopy and light scattering studies reveal the prominent fusion capacity of adriamycin towards cardiolipin-phosphatidylcholine small unilamellar vesicles. (6) Addition of Ca²⁺ to total rat liver inner mitochondrial membrane lipids, dispersed in excess buffer, results in hexagonal H_II formation for part of the phospholipids. By contrast, the original bilayer structure is completely conserved when the above experiment is performed in the presence of adriamycin. (7) ³¹P-NMR spectra of isolated inner mitochondrial membranes are indicative of a bilayer organization for the majority of the phospholipids. Approximately 15% of the signal intensity originates from phospholipids which experience isotropic motion. Adriamycin addition almost completely eliminates the latter spectral component. In the absence of adriamycin, Ca²⁺ addition greatly increases the percentage of the phospholipids giving rise to an isotropic signal possibly indicating the formation of non-lamellar lipid structures. Adriamycin which specifically binds to cardiolipin (K. Nicolay et al. (1984) Biochim. Biophys. Acta 778, 359–371) completely blocks the Ca²⁺-induced structural reorganization of the lipids in this membrane.