Phase transitions in liposomes formed from the polar lipids of mitochondria from chilling-sensitive plants

The thermal response of mitochondrial polar lipids from a variety of chilling-sensitive and chilling-insensitive plants was determined by differential scanning calorimetry. A phase transition was observed at 15°C for mitochondria from soybeam (Glycine max. cv Davis) hypocotyl, at 16°C for tomato (Lycopersicon esculentum cv Flora-Dade and cv Grosse Lisse) fruit, at 15°C for cucumber (Cucumus sativus L.) fruit, at 14°C for mung bean (Vigna radiata var Berken) hypocotyl, and at 15°C for sweet potato (Ipomea batatas L.) roots. The transition temperature was not significantly altered by the scan rate and was reversible. Changes in the temperature coefficient of motion for a spin label, intercalated with the polar lipids, occurred at a temperature slightly below that of the phase transition, indicating that the polar lipids phase separate below the transition. No phase transition was observed for mitochondrial polar lipids from barley (Hordeum vulgare) roots, wheat (Triticum aestivum L. cv Falcon) roots, and Jerusalem artichoke (Helianthus tuberosus L.) tubers. The results show that a phase change occurs in the membrane lipids of mitochondria a few degrees above the temperature below which chilling injury is evident in the sensitive species. Thus they are consistent with the hypothesis that sensitivity to chilling injury is related to a temperature-induced alteration in the structure of cell membranes.     

Anomaly of transport of dipicrylamine anions across the central barrier of planar dipalmitoylphosphatidylcholine bilayer membranes at the phase transition

The rate of translocation of the hydrophobic ion dipicrylamine across planar lipid membranes formed from dipalmitoyllecithin in n-decane was determined by voltage jump relaxation experiments. The activation energy of the rate constant shows a change from a positive to a negative value at about 42°C near the main phase transition temperature of this lipid. Below this temperature, the rate constant was found to increase with decreasing temperature. This anomalous behaviour extends over a temperature range of at least 10 K and may be formally interpreted as an enhanced mobility of dipicrylamine in the solid state of the membrane.     

Seasonal Changes in the Structure and Function of Mitochondrial Membranes of Artichoke Tubers: A Requisite for Surviving Low Temperatures during Dormancy

The temperature limits of the order-disorder transition, and the Arrhenius activation energy of succinate oxidase activity for mitochondria of Jerusalem artichoke (Helianthus tuberosus L.) tubers were determined from the initiation to the termination of dormancy. The temperature limits for the transition at the initiation of dormancy were 25 and 3 C. These changed to 9 and −5 C at mid-dormancy and returned to 25 and 2 C at the termination of dormancy. The Arrhenius activation energy measured in the temperature range above the transition was 35 kilojoules per mole at middormancy and decreased to 17 kilojoules per mole at the termination of dormancy when sprouting was evident. The coincidence of the changes in membrane structure and function with dormancy suggests that artichokes possess a mechanism for regulating membrane lipid structure so that cellular integrity of tuber tissue is maintained even when the tubers are exposed to low temperatures.     

The relationship between environmental temperature,cell growth and the fluidity and physical state of the membrane lipids in Bacillus stearothermophilus

A definite and characteristic relationship exists between growth temperature, fatty acid composition and the fluidity and physical state of the membrane lipids in wild type Bacillus stearothermophilus. As the environmental temperature is increased, the proportion of saturated fatty acids found in the membrane lipids is also markedly increased with a concomitant decrease in the proportion of unsaturated and branched chain fatty acids. The temperature range over which the gel to liquid-crystalline membrane lipid phase transition occurs is thereby shifted such that the upper boundary of this transition always lies near (and usually below) the temperature of growth. This organism thus possesses an effective and sensitive homeoviscous adaptation mechanism which maintains a relatively constant degree of membrane lipid fluidity over a wide range of environmental temperatures. A mutant of B. stearothermophilus which has lost the ability to increase the proportion of relatively high melting fatty acids in the membrane lipids, and thereby increase the phase transition temperature in response to increases in environmental temperature, is also unable to grow at higher temperatures. An effective homeoviscous regulatory mechanism thus appears to extend the growth temperature range of the wild type organism and may be an essential feature of adaptation to temperature extremes.Over most of their growth temperature ranges the membrane lipids of wild type and temperature-sensitive B. stearothermophilus cells exist entirely or nearly entirely in the liquid-crystalline state. Also, the temperature-sensitive mutant is capable of growth at temperatures well above those at which the membrane lipid gel to liquid-crystalline phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition upper boundary itself does not directly determine the maximum growth temperature of this organism. Similarly, the lower boundary does not determine the minimum growth temperature, since cell growth ceases at a temperature at which most of the membrane lipid still exists in a fluid state. These observations do not support the suggestion made in an earlier study, which utilized electron spin resonance spectroscopy to monitor membrane lipid lateral phase separations, that the minimum and maximum growth temperatures of this organism might be directly determined by the solid-fluid membrane lipid phase transition boundaries. Evidence is presented here that the electron spin resonance techniques used previously did not in fact detect the gel to liquid-crystalline phase transition of the bulk membrane lipids, which, however, can be reliably measured by differential thermal analysis.     

Membrane phase transitions and succinate oxidase activity in an extremely thermophilic bacterium

ESR and succinate oxidase activity were used to investigate the membrane phase-transitions of an extreme thermophile, Thermus T351, over an 80°C temperature range in whole cells, membrane particles, and extracted lipid suspension. Three phase transitions were observed using both techniques. These occurred at about 19°C, 39°C and 66°C. The transition at 19°C is unusual in that the Arrhenius plot for succinate oxidase is concave upwards, implying an increase in activation energy (E_a) with increased temperature.     

Temperature-induced Phase Changes in the Membranes of Glyoxysomes, Mitochondria, and Proplastids from Germinating Castor Bean Endosperm

Sucrose-gradient purified mitochondria, glyoxysomes, and proplastids from germinating castor bean (Ricinus communis L.) endosperm were examined by electron-spin resonance spectroscopy. A temperature-induced phase change was demonstrated in all of these organelles, their derivative membranes, and in micelles formed from the membrane phospholipids. The apparent transition temperature of the membrane lipids varied slightly between the samples, but in all cases, fell within the temperature range around 10 C where physiological and biochemical changes in the response to temperature for most chilling-sensitive plants occur.     

Effect of Temperature on Respiration of Mitochondria and Shoot Segments from Cold hardened and Nonhardened Wheat and Rye Seedlings

The effect of temperature on respiration of mitochondria and tissue segments from three wheat (Triticum aestivum L.) and one rye (Secale cereale L.) cultivar grown at 2 and 24 C has been examined. Discontinuities in Arrhenius plots of respiratory activity against temperature were observed for mitochondria and tissue segments from seedlings grown at both temperatures. The rates of respiration decreased abruptly below the transition temperatures, resulting in increased energy of activation values for respiration. Transition temperatures were observed from 6 to 10 C during tissue segment respiration, and from 10 to 14 C during respiration by isolated mitochondria. Respiratory control and efficiency of phosphorylation were not affected markedly by either reaction temperature or growth temperature of the seedlings. No correlation was observed between the cold hardiness of the cultivars and the temperature at which structural transitions occurred in the mitochondria. Dry matter content of the seedlings increased markedly during growth at 2 C, but no appreciable changes in the levels of mitochondrial protein were observed. The results support the view that changes other than fatty acid unsaturation are involved in the abrupt change in mitochondrial membrane properties at low temperature.     

Temperature dependence of delayed ehlorophyll fluorescence in intact leaves of higher plants. A rapid method for detecting the phase transition of thylakoid membrane lipids

The temperature dependence of the yield of in vivo prompt and delayed chiorophyll fluorescence was investigated in maize and barley leaves. In the chilling-sensitive maize, delayed fluorescence at steady-state level showed a maximum near the temperature at which thylakoid membrane lipids undergo a phase transition as revealed by differential scanning calorimetry measurements. In the chilling-resistant barley, no phase transition was detected above 0°C and the delayed light emission varied in a monotonic fashion. It was shown that measurements of delayed luminescence intensity in vivo can provide a rapid and sensitive method for detecting the phase change of membrane lipids in intact leaves of chilling-sensitive plant species such as tomato, cotton, cucumber, castor bean or avocado. In contrast, the use of steady-state prompt chlorophyll fluorescence as an indicator of membrane fluidity change was not successful.     

Seasonal changes in the structure and function of mitochondrial membranes of artichoke tubers: acyl Fatty Acid composition and the effect of growth conditions

Changes in the temperature response, fluidity, function and the acyl fatty acid composition, were determined for a mitochondria-rich membrane fraction from Jerusalem artichoke (Helianthus tuberosus L.) tubers during dormancy for a crop which matured in midsummer. The temperature of both the upper and lower limits of the membrane lipid transition decreased during dormancy from 26 C and 1 C to 4 C and −5 C, respectively. This was similar to the changes observed with crops maturing in late autumn. The order parameter of a spin label intercalated into the membrane lipids decreased from about 0.6 to 0.5 during dormancy and returned to the original value before sprouting, showing that membrane fluidity increased during dormancy. The activation energy of succinate oxidase of tuber mitochondria was generally high at middormancy when membrane lipids were more fluid and decreased as the membranes became more rigid at the end of dormancy. The fatty acid composition of the membrane lipids did not alter significantly during dormancy. The results indicate that neither decreasing day length nor low soil temperature during tuber maturation is essential for the initiation of the membrane changes necessary for tubers to avoid low temperature injury during dormancy. The increase in membrane fluidity during dormancy could not be accounted for by an increase in the proportion of unsaturated fatty acids in the membrane lipids.     

The influence of storage temperature on the transition, activation enthalpy, and activity of enzymes associated with inner mitochondrial membranes

The effects of storage at low temperature on the transition in enzyme function, Tf*, and the Arrhenius activation energy, Ea, were determined for several enzymes associated with the inner membrane of rat liver mitochondria. The enzymes studied were succinate:cytochrome c reductase, cytochrome c oxidase, beta-hydroxybutyrate dehydrogenase, and oligomycin-sensitive, Mg2+-activated ATPase. For freshly isolated mitochondria the Tf*, for succinate:cytochrome c reductase and cytochrome c oxidase, occurred at approximately 23 degrees C and was coincident with a transition in structure, Ts*, determined as the change in temperature coefficient of motion for a spin label intercalated with the membrane lipids. This suggest that the change in thermal response of the membrane-associated enzymes is related to a change in molecular ordering of the membrane lipids. When mitochondria were stored at -12 degrees C, the specific activities of succinate:cytochrome c reductase and cytochrome c oxidase decreased. Concomitant with these changes the Ea, above Tf*, increased. After 100 days storage at -12 degrees C, Ea above Tf* approached the value for Ea below Tf* such that the transition in thermal response could no longer be detected. In contrast, for mitochondria stored at -196 degrees C, although the specific activity declined over the 100 days storage, no changes in either Ea or Tf* were evident. The results indicate a need for caution in evaluating comparative studies of Tf and Ea, for membrane-associated enzymes, using mitochondria which have been frozen and stored.     

Thermal phase transitions in the polar lipids of plant membranes. Their induction by disaturated phospholipids and their possible relation to chilling injury

The phase behaviour of leaf polar lipids from three plants, varying in their sensitivity to chilling, was investigated by differential scanning calorimetry. For the lipids from mung bean (Vigna radiata L. var. Berken), a chilling-sensitive plant, a transition exotherm was detected beginning at $\text{10 ± 2°}\text{C}$. No exotherm was evident above 0°C with polar lipids from wheat (Triticum aestivum cv. Falcon) or pea (Pisum sativum cv. Massey Gem), plants which are insensitive to chilling. The enthalpy for the transition in the mung bean polar lipids indicated that only about 7% w/w of the lipid was in the gel phase at ?8°C. The thermal transition of the mung bean lipids was mimicked by wheat and pea polar lipids after the addition of 1 to 2% w/w of a relatively high melting-point lipid such as dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol or dimyristoylphosphatidylcholine. Analysis of the polar lipids from the three plants showed that a dipalmitoylphosphatidylglycerol was present in mung bean (1.7% w/w) and pea (0.3% w/w) but undetected in wheat, indicating that the transition exotherm temperature of 10°C in mung bean, 0°C in pea and about ?3°C in wheat correlates with the proportion of the high melting-point disaturated component in the polar lipids. The results indicate that the transition exotherm, observed at temperatures above 0°C in the membranes of chilling-sensitive plants, could be induced by small amounts of high melting-point lipids and involves only a small proportion of the membrane polar lipids.     

The effect of acclimation temperature on the activation energies of state III respiration and on the unsaturation of membrane lipids of goldfish mitochondria

The influence of the acclimation temperature on the thermotropic behaviour of mitochondrial respiration and on the degree of unsaturation of mitochondrial membrane lipids has been studied. The mitochondria were isolated from red muscle, white muscle and liver of goldfish acclimated to 5, 20 and 30°C. ADP-activated succinate oxidation was measured at different temperatures and resulted in non-linear Arrhenius-plots with breaks between 10 and 23°C. As for the break-temperatures, there was found a shift downwards in preparations of decreased acclimation temperatures. This could be caused by a changed composition of membrane lipids and a simultaneous shift of the membrane phase transition temperature. Therefore, the fatty acid composition of all membrane preparations was analyzed. However, no consistent change of the degree of unsaturation due to a changed acclimation temperature could be found.     

Lipid reorganization in biological membranes. A study by fourier transform infrared difference spectroscopy

The first application of infrared difference spectroscopy to the study of a natural biological membrane is described. Perdeuterated palmitic acid was incorporated biosynthetically into the lipids of the plasma membrane of Acholeplasma laidlawii and the temperature-induced structural rearrangement of the endogenous lipids monitored via their C²H vibrational modes. Changes in infrared parameters were studied between 0 and 50°C and contrasted with those occurring in the model membrane system of $\text{1,2-}\text{diperdeuteropalmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$. The phase transition of the biomembrane occurs over a 20°C range with the temperature of the maximum rate of change of absorbance coinciding with that of the sharp phase transition of the model membrane.     

The effect of altered membrane sterol composition on the temperature dependence of yeast mitochondrial ATPase

The sterol content of cells of $\text{Saccharomyces cerevisiae}$ was manipulated by growing the organism anaerobically in a medium containing excess supplements of unsaturated fatty acids and a range of supplements of ergosterol. Anaerobic mitochondrial precursor structures were isolated whose membrane lipids contain the same fatty acid composition but whose sterol content varies from 7 to 105 mg/g mitochondrial protein. Arrhenius plots of the mitochondrial ATPase activity of the different preparations show a discontinuity with Arrhenius activation energies of about +40 and +80 KJ/mole, respectively, above and below the transition temperature. However, the temperature of the transition is markedly dependent on sterol composition, and increases by up to 17° as the sterol content of the mitochondria is progressively decreased. These results support the concepts that membrane lipid composition influences the activity of membrane-bound enzymes, and that sterols promote the gel to liquid phase transition in biological membranes.     

Annular lipids determine the ATPase activity of a calcium transport protein complexed with dipalmitoyllecithin.

Pure complexes of dipalmitoyllecithin (DPL, 16:0) which Ca2+, Mg2+ dependent ATPase from sarcoplasmic reticulum are unusual in retaining significant ATPase activity down to about 30 degrees C, well below the transition temperature of the pure lipid at 41 degrees C. A minimum of about 35 lipid molecules per ATPase is required to maintain maximal ATPase activity, but the complexes are progressively and irreversibly inactivated at lower lipid to protein ratios. Complexes containing more than the minimum lipid requirement show very similar temperature profiles of activity about 30 degrees C over a wide range of lipid to protein ratios, up to 1500:1. Spin-label studies indicate that, at lipid to protein ratios of less than about 30 lipids per ATPase, no DPL phase transition can be detected, but at all higher ratios, a phase transition occurs at about 41 degrees C. In all of these complexes there are breaks in the Arrhenius plots of ATPase activity at 27--32 degrees C and at 37.5--38.5 degrees C. Experiments with perturbing agents, such as cholesterol and benzyl alcohol which have well-defined effects on the DPL phase transition, indicate that these breaks in the Arrhenius plots of ATPase activity cannot be attributed to a depressed and broadened phase transition in the lipids near the protein molecules. These results are interpreted as evidence for a phospholipid annulus of at least 30 lipid molecules with interact directly with the ATPase and cannot undergo a phase transition at 41 degrees C. This structural interaction of the ATPase with the annular DPL molecules has a predominant effect in determining the form of the temperature-activity profiles. However, the perturbation of the DPL phase transition does not extend significantly beyond the annulus since a phase transition which starts at 41 degrees C can be detected as soon as extraannular lipid is present in the complexes. We suggest that it may be a general feature of membrane structure that penetrant membrane proteins interact with their immediate lipid environment so as to cause only a minimal perturbation of the lipid bilayer.     

Temperature-induced transitions of porcine intestinal brush border membranes

Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33°C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35°C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35°C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35°C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33°C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.     

A lipid-phase separation model of low-temperature damage to biological membranes

An hypothesis is proposed to explain the damage caused to biological membranes exposed to low temperatures. The thesis rests on the general observation that the lipid components of most membranes are heterogeneous and undergo phase transitions from gel-phase lamellae to liquid-crystalline lamellae and some to a non-lamellar, hexagonal-II phase over a wide range of temperatures. As a consequence of these phase transitions the lateral distribution of the lipids characteristic of the growth temperature is disturbed and redistribution takes place on the basis of the temperature at which phase transitions occur. When membranes are cooled, first the non-lamellar forming lipids pass through a transition to a fluid lamellar phase and are miscible with bilayer-forming lipids into which they diffuse. On further cooling the high-melting-point lipids begin to crystallize and separate into a lamellar gel phase, in the process excluding the low-melting point lipids and intrinsic proteins. The lipids in these remaining regions form a gel phase at the lowest temperature. It is suggested that, because the non-lamellar lipids tend to undergo a liquid-crystalline to gel-phase transition at higher temperatures than lamellar-forming lipids, these will tend to phase separate into a gel phase domain rich in these lipids. Damage results when the membrane is reheated, whereupon the hexagonal-II-forming lipids give rise to non-lamellar structures. These probably take the form of inverted micelles sandwiched within the lipid bilayer and they completely destroy the permeability barrier properties of the membrane. The model is consistent with the phase behavior of membrane lipids and the action of cryoprotective agents in modifying lipid phase properties.     

The effect of acclimation temperature on the activation energies of state III respiration and on the unsaturation of membrane lipids of goldfish mitochondria.

The influence of the acclimation temperature on the thermotropic behaviour of mitochondrial respiration and on the degree of unsaturation of mitochondrial membrane lipids has been studied. The mitochondria were isolated from red muscle, white muscle and liver of goldfish acclimated to 5, 20 and 30 degrees C. ADP-activated succinate oxidation was measured at different temperatures and resulted in non-linear Arrhenius-plots with breaks between 10 and 23 degrees C. As for the break-temperatures, there was found a shift downwards in preparations of decreased acclimation temperatures. This could be caused by a changed composition of membrane lipids and a simultaneous shift of the membrane phase transition temperature. Therefore, the fatty acid composition of all membrane preparations was analyzed. However, no consistent change of the degree of unsaturation due to a changed acclimation temperature could be found.     

Two qualitatively different structuro-functional states of mitochondria

Low (120 mosM) tonicity of incubation media of mitochondria was found to be associated with anomalous phase transition at 19--26 degrees C. A rise in temperature caused a decrease in the pyrene excitation in border lipids of the mitochondrial membrane. Within this temperature range the quenching of intrinsic protein fluorescence by pyrene was sharply decreased. It may be inferred from these data that at 100mosM tonicity and temperatures below 19 degrees C, mitochondrial membrane proteins are in an aggregated state. At temperatures above phase transition protein deaggregation takes place. It was shown that a decrease in tonicity from 300 to 120 mosM at 15 degrees C or a rise in temperature from 15 degrees to 37 degrees C at 300 mosM tonicity increased the phosphorylation of the 52 kDa mitochondrial protein. It was assumed that swelling of mitochondria in hypotonic media simulates one of the steps of the hormone-induced signal transfer in mitochondria in vivo.