Quick-freeze differential scanning calorimetry and saturation transfer electron spin resonance: novel techniques for assessing phase transitions in biological membranes

Quick-freeze differential scanning calorimetry (QF-DSC) and saturation transfer-electron spin resonance (ST-ESR) spectroscopy were used to study lipid gel-phase transitions in mature green tomato fruit microsomal membranes. ST-ESR of 12-doxyl methyl stearate labelled membranes proved to be reproducible and provided increased sensitivity to temperature-induced structural changes, allowing the detection of several transitions in isolated membranes (6 degrees C, 21 degrees C, 28 degrees C). QF-DSC led to the assessment of lipid gel phase transitions in isolated microsomal membranes and microsomal membrane lipids by enhancing the transition. A phase transition enthalpy of 114 J/g and an onset temperature of 29.8 degrees C were obtained for whole membranes while with isolated lipids values of 370 J/g and 19.9 degrees C were found.     

Membrane homeostasis: thermotropic behaviour of microsomal membrane lipids isolated from livers of rats fed cholesterol-supplemented diets

Differential scanning colorimetry (DSC) has been applied to study the phase transition properties of isolated lipids from liver microsomal membranes of rats fed high cholesterol diets with or without high levels of either saturated (coconut oil) or unsaturated (sunflower seed oil) fat. DSC of aqueous buffer dispersions of liver microsomal lipids exhibited two independent, reversible phase transitions. The dietary cholesterol treatments had their major effect on the temperature at which the lower phase transition (T1) occurred. This transition occurred at a lower temperature when cholesterol was added to the diet, irrespective of the nature of the fatty acid supplement. However the magnitude of decrease was more when cholesterol was fed with sunflower seed oil. Inclusion of cholesterol into the rat diets also lowered the enthalpy values for the lower phase transition (T1). No appreciable effect on the temperature of the higher phase transition (T2) was observed, however the enthalpy values were slightly decreased by cholesterol feeding. These results suggest that certain domains of microsomal lipids, probably containing some relatively higher melting-point lipids, independently undergo solidus or gel formation and this transition (T2) is not greatly affected by dietary cholesterol. On the other hand, domains representing the bulk of the microsomal lipids undergo a phase change (T1) at temperatures which are dependent on cholesterol content and fatty acid profiles of the membrane, which are in turn, modified by dietary cholesterol intake.     

Lipid peroxidation increases the molecular order of microsomal membranes

The effect of enzymatic lipid peroxidation on the molecular order of microsomal membranes was evaluated by ESR spectroscopy using the spin probes 5-, 12-, and 16-doxyl-stearic acid. Rat liver microsomal membranes were peroxidized by the NADPH-dependent reaction in the presence of the chelate ADP-Fe3+. Peroxidation resulted in a preferential depletion of polyenoic fatty acids and an increase in the percentage composition of shorter fatty acyl chains. There was no change in the cholesterol/phospholipid ratio of the peroxidized microsomes. The molecular order of both control and peroxidized membranes decreased toward the central region of the bilayer, and the order parameter (S) of each probe was temperature dependent. Peroxidation of the microsomal membrane lipids resulted in an increase in the order parameter determined with the three stearic acid spin probes. Of the three probes, 12-doxylstearic acid was the most sensitive to the changes in membrane organization caused by peroxidation. These data indicate that ESR spectroscopy is a sensitive method of detecting changes in membrane order accompanying peroxidation of membrane lipids.     

Effects of the catalytic hydrogenation of microsomal lipids upon four enzyme activities involved in oleic acid desaturation

Catalytical hydrogenation of the unsaturated fatty acyl residues of microsomal lipids was realized for different times. Progress of the reaction was followed by calculating the progressive loss of double-bonds in 100 initial acyl residues (percentage of hydrogenation). The maximum loss observed was 45% after 60 min.The drop in polyunsaturated faty acid content was coupled with an increase in the amount of stearic acid and oleic acid.The order parameter of microsomal lipids, measured by ESR, increased parallely to the reduction of double bonds. Maximum hydrogenation of microsomal lipids strongly (200–250%) stimulated microsomal NADH-ferricyanide reductase activity. NADH-cytochrome c reductase, lysophosphatidylcholine-acyl-transferase and oleoyl-phosphatidylcholine desaturase were inhibited (40%, 100% and 100% respectively). These modifications of enzyme activities are discussed in conjunction with the changes observed in membrane fluidity, following hydrogenation of microsomal lipids     

Vitamin A and microsomal membranes: the effect of retinol deficiency on lipid microviscosity and phospholipid turnover in rat liver microsomes

Studies with the use of the fluorescent probe pyrene revealed that vitamin A deficiency in maturing male rats results in the increased microviscosity of liver lipids. This effect seems to be due to changes in the lipid composition of microsomal membranes (increased cholesterol/phospholipid ratio and lowered polyunsaturated fatty acid content) as well as to the low level of retinol. Analysis of microsomal phospholipids labeled with [3H]palmitate and [14C]glycerol revealed that vitamin A deficiency accelerates the turnover of the glycerol skeleton but sharply decelerates that of fatty acid residues. It is concluded that the observed effect of retinol on the structural and functional properties of biological membranes is due to its ability to control the microviscosity and turnover of membrane lipids.     

Selective degradation with phospholipases D and C of radioactive isomeric spin-labelled lipids bound to guinea pig liver microsomal membranes.

Membrane-bound lipids of isolated guinea pig liver microsomal membranes were selectively enzymatically labelled with isomeric (5-, 12-, and 16-)doxyl stearic acid. After reisolation, the membranes were degraded with phospholipases D and C under conditions not requiring detergents or organic solvent activators. The degradation of membrane-bound lipids occurred according to the recognized specificity of phospholipases D and C. Temperature-induced changes of degraded membranes containing radioactive spin-labelled isomeric lipids were followed by the electron spin resonance and spectral changes correlated with the lipid composition of membranes. Discontinuities in plots of experimental spectral parameters versus temperature detected in the case of microsomal membranes before and after degradation with phospholipases D and C were attributed to lipid-protein and lipid-lipid interaction(s). On the basis of these and control experiments, discontinuity at around 10-12 degrees C was attributed to the microsomal membrane phosphatidylcholine intrinsic microsomal membrane protein interaction(s), while discontinuities detected at 19-21 degrees C approximately and at 20-30 degrees C approximately were attributed to the phase separation of Ca or Zn salts of membranous phosphatidic acid and to the similar phenomenon involving membrane-bound diglycerides respectively.     

Temperature-induced changes of spin-labelled radioactive lipids in isolated guinea pig liver microsomal membranes before and in mitochondrial membranes after their translocation.

Lipids of isolated guinea pig liver microsomal membranes were labelled biosynthetically with isomeric doxyl stearic acid and temperature-induced changes of these membranes were studied by electron spin resonance. A noticeable discontinuity was detected at 10--12 degree C with 12- or 16-doxyl stearic acid containing membrane lipids which was attributed to the spin-labelled lipid--microsomal membrane protein interactions since no such discontinuity was detected in liposomes prepared from total lipid extracts of microsomal membranes. When microsomal membranes containing radioactive isomeric spin-labelled lipids were incubated with unlabelled mitochondria, reisolated mitochondrial membranes contained translocated radioactive isomeric spin-labelled lipids. Temperature-induced changes in these membranes showed no discontinuity with either isomeric doxyl stearic acid derivative, establishing a difference in the environment of translocated lipids in the membrane donor compared with that in the membrane acceptor. Microsomal membranes recovered from translocation experiments showed the same behaviour as the original membranes and exhibited the same discontinuity at 10--12 degree C, establishing that the translocation incubation itself did not alter the spin-labelled lipid interaction within these membranes. Studies of the loss of paramagnetism of spin-labelled lipids in microsomal membranes before and in mitochondrial membranes after their translocation showed a significant difference and suggested that both the outer and the inner mitochondrial membranes might have been involved.     

Effect of lecithin on liver microsomal lipid peroxidation]

The effect of exogeneous (egg) lecithin on peroxidation of microsomal lipids was studied with the view of elucidating the role of various components of lipid substrate in the overall oxidation rate of the lipids. The following processes were studied a) NADPH-dependent microsomal lipid peroxidation in the presence of lecithin; b) ascorbate-dependent microsomal lipid peroxidation in the presence of lecithin; c) oxidation of lipid mixture, isolated from the microsomes, and that of lecithin in the presence of the Fe2+ + ascorbate system; 4) oxidation of lecithin induced by the Fe2+ + ascorbate system. It was found that in the presence of exogeneous lecithin the oxidation of microsomal lipids in inhibited, which is probably due to the peculiarities of lecithin oxidation. It was shown that the specific rate of lecithin oxidation is decreased with an increase in lecithin concentration. Possible mechanisms of lecithin effect on microsomal lipid peroxidation are discussed.     

Selective enzymatic radioactive and spin labelling of phospholipids in biological membranes: application to study of temperature-induced changes of microsomal phosphatidylinositols and mitochondrial polyglycerophosphatides.

A new method for the covalent radioactive and spin labelling of lipids within isolated biological membranes has been described in detail and applied to studies of temperature-induced changes of microsomal and mitochondrial membranes. The method is based on the enzymatic use of radioactive substrates carrying covalently bound doxyl derivatives of stearic acid in the biosynthesis of phospholipids in isolated membranes. Radioactive-and spin-labelled lipids bound to the microsomal and mitochondrial membranes were then used as internal probes in monitoring temperature-induced changes of these membranes. Since the analysis of isolated radioactive-and spin-labelled lipids revealed the exact composition of membrane-bound labelled lipids, specific temperature-induced changes were correlated with specific lipids of examined membranes. Phosphatidylinositol of microsomal membranes and polyglycerophosphatides (phosphatidyl-glycerol and cardiolipin) of mitochondrial and inner mitochondrial membranes were found to be involved in the apparent formation of lipid clusters at around 20-30 degrees C. Cardiolipin was found to be involved in the fluidization of inner mitochondrial membranes. These findings are discussed in view of the present state of knowledge of the organization of lipids in biological membranes.     

Glucocorticoids increase the fluidity of the fetal-rat liver microsomal membrane in the perinatal period.

Dexamethasone, a synthetic glucocorticoid, was administered to pregnant rats during the last week of pregnancy in order to examine its effects on the fluidity of the developing fetal-rat liver microsomal membrane. This early prenatal exposure to dexamethasone, which preceded the natural appearance of fetal corticosteroids, markedly accelerated the normal perinatal course of fluidization of this membrane. The lipid apparent microviscosity, which was determined by measurement of fluorescence polarization, decreased in 21-days-old treated fetuses to values that were indistinguishable from those of untreated newborn rats. This dexamethasone-mediated acceleration of membrane fluidization was associated with an increase in the index of unsaturation of the fatty acyl moiety of microsomal lipids. Dexamethasone caused a significant increase in the microsomal content of polyunsaturated fatty acids (arachidonic and linoleic acid), which was accompanied by a decrease in content of monoenoic fatty acids (oleic and palmitoleic acid). This early exposure in utero to dexamethasone precociously induced the changes in fatty acid composition of fetal-rat liver microsomal lipids that normally occur between the last day of pregnancy and the first day of extra-uterine life. These results suggest that endogenous glucocorticoids play a major role in the perinatal fluidization of the rat liver microsomal membrane.     

Effect of dietary cholesterol on microsomal membrane composition, dynamics and kinetic properties of UDPglucuronyl transferase

The effect of cholesterol administration in vivo on the lipid composition, dynamic properties of the microsomal membrane of guinea pig livers and the kinetic properties of UDPglucuronyl transferase were studied. Cholesterol administration in the diet evoked an increase of microsomal cholesterol, but no significant changes in the fatty-acid composition of total lipids or of each phospholipid class. Instead, the phosphatidylethanolamine, phosphatidylcholine molar ratio of the membrane was markedly decreased from 0.57 to 0.38. This decline was not enough to counterbalance the overall 'ordering' effect of cholesterol and consequently, the fluorescence anisotropy of the membranes labeled with 1,6-diphenylhexatriene was increased. The lateral diffusion evaluated by measuring the pyrene excimer formation was decreased by the cholesterol incorporation. These physical changes were associated with changes in the kinetic properties of UDPglucuronyl transferase: Vmax increased, while the Km of the different steps of the reaction decreased in the modified microsomes. Furthermore, a shift of the non-michaelian kinetics to michaelian, equivalent to a decrease of a negative homotropic effect and apparent cooperativity of UDPglucuronic acid was observed since the Hill coefficient changed, approaching 1. A non-michaelian kinetics of this enzyme is an indication of boundary lipids in the gel phase and a shift to michaelian, a change of the surrounding lipids to a liquid-crystalline structure. In consequence, our results suggest that cholesterol incorporation in the microsomal membrane while producing a condensing effect of bulk lipids would produce an opposite effect on the UDPglucuronyl transferase boundary lipids.     

Effects of diffusible products of peroxidation of rat liver microsomal lipids

The effects on cellular structures of products of peroxidation of rat liver microsomal lipids were investigated. A system containing actively peroxidizing liver microsomal fraction was separated from a revealing or target system by a dialysis membrane. The target system, contained in the dialysis tube, consisted of either intact cells (erythrocytes) or subcellular fractions (liver microsomal fraction). When liver microsomal fractions were incubated with NADPH (or an NADPH-generating system), lipid peroxidation, as measured by the amount of malonaldehyde formed, occurred very rapidly. The malon-aldehyde concentration tended to equilibrate across the dialysis membrane. When the target system consisted of erythrocytes, haemolysis occurred abruptly after a lag phase. The lysis was greatly accelerated when erythrocytes from vitamin E-deficient rats were used, but no haemolysis was observed when erythrocytes from vitamin E-treated rats were used. When, in the same system, freshly prepared liver microsomal fractions were exposed to diffusible factors produced by lipid peroxidation, the glucose 6-phosphatase activity markedly decreased. A similar decrease in glucose 6-phosphatase activity, as well as a smaller but significant decrease in cytochrome P-450, was observed when the target microsomal fractions were exposed to diffusible factors derived from the peroxidation of liver microsomal lipids in a separate preincubation step. These and additional experiments indicated that the toxicological activity is relatively stable. Experiments in which the hepatic microsomal fractions destined for lipid peroxidation contained radioactively labelled arachidonic acid, previously incorporated into the membranes, showed that part of the radioactivity released from the microsomal fraction into the incubation medium entered the dialysis tube and was recovered bound to the constituents of the microsomal fractions of the target system. These results indicate that during the course of the peroxidation of liver microsomal lipids toxic products are formed that are able to induce pathological effects at distant loci.     

Binding of products originating from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomal membrane.

The binding of products derived from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomes was studied. To this end arachidonic acid labelled with tritium at the positions of the double bonds was given to rats and allowed to incorporate into the membrane lipids of the liver cell. When liver microsomes containing labelled arachidonic acid were incubated aerobically in the NADPH-dependent system, a marked production of malonic dialdehyde (MDA) occurred and, concomitantly, there was a consistent release of radioactivity from the microsomes into the incubation medium. The addition of EDTA to the incubation medium prevented, to a large extent, both the MDA formation and the release of radioactivity. Chromatographic studies showed that the bulk of the radioactivity released from the incubated microsomes is not MDA. In the incubated microsomes, the radioactivity decreased in total lipids, while it increased by about 15 times in the non-lipoidal residue. A similar increase in radioactivity was seen in microsomal protein, while no increase was observed in microsomal RNA (the radioactivity was negligible in both the incubated and the non-incubated samples). It seems therefore that products originating from lipoperoxidation of arachidonic acid covalently bind to the microsomal protein. In order to investigate whether alterations similar to those observed in the in vitro peroxidation of liver microsomes could be detected in the in vivo intoxication with carbon tetrachloride, rats given labelled arachidonic acid as above, were poisoned with CCl4. Sixty minutes after poisoning, the radioactivity present in the microsomal lipids was generally lower in the intoxicated rats than in the controls, while the labelling of the non-lipoidal residue and of the protein was higher in the CCl4-poisoned rats.     

Induction of microsomal cytochromes in the rat liver after intravenous administration of emulsions of perfluoro-organic compounds

Intravenous injections of perfluoroorganic emulsions to rats in a dose of 3 ml/kg led to changes in the composition and activity of enzymes of the liver microsomal membrane monooxygenase system. At the peak of induction, i. e., on the 3rd post-injection day, the levels of microsomal cytochromes P-450 and b5 increased 2.8- and 1.9-fold, respectively, as compared to the control. Simultaneously, the rate of NADPH oxidation in the microsomes and the rate of hydroxylation of substrates I and II showed an increase. Conversely, the rate of NADPH-dependent peroxidation of microsomal lipids on the 2nd-4th post-injection days reached its minimal values. These injections stimulated the detoxicating function of rat liver as evidenced from the duration of the hexenal sleep of the animals. All the changes in the monooxygenase system parameters were temporary and reached the control level on the 10th-14th days after injection. It was demonstrated that the main component of the perfluoroorganic emulsions, perfluorodecalin, was responsible for the induction of the monooxygenase system enzymes.     

Enzymic lipid peroxidation in the microsomal fraction of rat brain

Abstract: An enzymic lipid peroxidation system has been demonstrated in the microsomal fraction of rat brain and the requirements and optimal conditions for assay determined. The involvement of NADPH-cytochrome c reductase was demonstrated in vesicles reconstituted with lipids extracted from the brain microsomal fraction. Further characterization of the system made use of substances shown to inhibit the liver microsomal system. α-Tocopherol was shown to be an effective inhibitor of lipid peroxidation in the brain microsomal system, whereas Na₂SO₃ had no effect, which is indicative that free radical transfer occurs only in the hydrophobic regions. Neither superoxide dismutase nor catalase inhibited lipid peroxidation. The implications of an NADPH-cytochrome c reductase-dependent lipid peroxidation system that is not linked to a drug hydroxylation system and appears to differ from the liver microsomal system in a number of other ways are discussed.     

Drug induced alterations in some rat hepatic microsomal components: a comparative study of four structurally different antimalarials

Alterations in microsomal drug metabolizing enzymes, microsomal lipids and some serum enzymes following pre-treatment of rats with therapeutic doses of four structurally different antimalarial compounds, chloroquine (CQ), quinine (Q), quinacrine (QK) and primaquine (PQ) have been investigated. CQ and Q significantly decreased the activities of aminopyrene N-demethylase, aniline hydroxylase and both microsomal and cytosolic glutathione S-transferases. Only aniline hydroxylase was markedly decreased by QK, while PQ did not have much effect on any of these enzymes. CQ, Q and QK significantly increased the cholesterol:phospholipid ratio while all four compounds decreased the phosphatidyl choline:sphingomyelin (PC/S) ratio. All the drugs increased the activities of the serum enzymes glutamate-oxaloacetate transaminase, glutamate-pyruvate transaminase and alkaline phosphatase. The possible relationships of these results to structural variations in the four drugs being investigated has been discussed.     

Effects of growth at different temperatures on the physical state of lipids in native microsomal membranes from Tetrahymena

Fluorescence measurements of the probe 1,6-diphenyl-1,3,5-hexatriene in native Tetrahymena pyriformis microsomal membranes revealed characteristic "break points" in curves of polarization vs. temperature. In the 5--35 degree C range, membranes from cells grown at 39 degrees C exhibited two break points, one at 11.6 +/- 0.6 degrees C and another at 23.1 +/- 1.6 degrees C. Membranes from 15 degrees C grown cells also showed two break points, one at 8.0 +/- 1.7 degrees C and another at 17.7 +/- 1.7 degrees C. Complementary measurements of turbidity (absorbance at 360 nm) vs. temperature revealed break points at approximately the same temperatures as observed with the fluorescent probe, thus strengthening the likelihood that the break points signify the onset or termination of lipid phase separations or some other significant structural alteration of lipids. In general, break points measured in the native membrane samples occurred at slightly lower temperatures than did break points in lipids extracted from comparable membranes. This suggests two possible types of protein--lipid interaction. First, there may be a selective withdrawal of relatively highly saturated phospholipid molecular species from the bulk lipid phase and into protein annulus regions. Alternatively, the configuration of the hydrophobic core of certain key membrane proteins may be such that nonspecific interactions with the lipids stabilize the liquid-crystalline phase.     

Effect of three structurally related antimalarial drugs on liver microsomal components and lipid peroxidation in rats

Changes in microsomal drug oxidizing enzymes, microsomal lipids, hepatic glutathione (GSH), glutathione S-trans-ferase (GST) and malondialdehyde (MDA) formation following administration of rats with therapeutic doses of three structurally related antimalarial drugs, amodiaquine (AQ), mefloquine (MQ) and halofantrine (HF) were investigated. There was a significant decrease in the activities of aniline hydroxylase, p-nitroanisole O-demethylase and pentoxyresorufin O-dealkylase in AQ, MQ and HF treated rats. AQ elicited the greatest effect with 50, 37 and 67% reductions in the activities of aniline hydroxylase, p-nitroanisole O-demethylase and pentoxyresorufin O-dealkylase, respectively. All the drugs prolonged hexobarbital-sleeping time to varying extents. The three drugs increased significantly the cholesterol per phospholipid ratio. AQ, MQ and HF decreased significantly the GSH level, GST activity and increased the formation of MDA. The results indicate that the alterations in hepatic microsomal components and lipid peroxidation caused by the antimalarials are related to the structural differences in the compounds.     

Cytosol protein-independent translocation of isomeric spin-labelled radioactive lipids from isolated guinea pig liver microsomal to mitochondrial membranes

Intermembranous translocation of membrane-bound radioactive lipids covalently labelled with 5-, 12, and 16-doxyl stearic acid was studied. Guinea pig liver microsomal membranes containing known amounts of isomeric spin-labelled radioactive phosphatidic acid, phosphatidylcholine, and diglycerides were incubated with unlabelled mitochondria; reisolated mitochondria contained around 28-31% of microsomal labelled lipids above the microsomal contamination. The effect of adding crude or 'pH 5.1' 105 000 X g cytosol supernatant on the amount and composition of translocated labelled lipids was studied. While the translocation of labelled phosphatidylcholine was slightly stimulated by the addition of these cytosol supernatants, no significant increase of the amount of translocated labelled phosphatidic acic and diglycerides was observed by this addition. In view of these results, a probable mechanism for the cytosol protein-independent translocation of lipids between biological membranes is proposed.     

Phosphatidylcholine mobility in liver microsomal membranes

Purified phosphatidylcholine exchange protein from bovine liver was used to exchange rat liver microsomal phosphatidylcholine for egg phosphatidylcholine. It was found that at 25 and 37°C rat liver microsomal phosphatidylcholine was completely and rapidly available for replacement by egg phosphatidylcholine. In contrast, phosphatidylcholine in vesicles prepared from total microsomal lipids could only be exchanged for about 60%. At 8 and 0°C complex exchange kinetics were observed for phosphatidylcholine in rat liver microsomes. The exchange process had neither effect on the permeability of the microsomal membrane to mannose 6-phosphate, nor on the permeability of the phosphatidylcholine vesicles to neodymium (III) cations.Purified phospholipase A₂ from Naja naja could hydrolyze some 55–60% of microsomal phosphatidylcholine at 0°C, but 70–80% at 37°C. Microsomal phosphatidylcholine, remaining after phospholipase treatment at 37°C, could be exchanged for egg phosphatidylcholine at 37°C, but at a slower rate than with intact microsomes. Microsomal phosphatidylcholine remaining after phospholipase treatment at 0 and 37°C had a lower content of arachidonic acid than the original phosphatidylcholine.These results are discussed with respect to the localization and transmembrane movement of phosphatidylcholine in liver microsomes.