Fluorescence polarization study of lipids and membranes prepared from brain hemispheres of a hibernating mammal

The physical behavior of total lipids, microsomes and microsomal lipids prepared from brain hemispheres of European Hamsters ($\text{Cricetus cricetus}$) was approached by the measure of the fluorescence polarization of the probe 1,6-diphenyl 1,3,5-hexatriene. We compare in this study the results obtained for two critical periods for a hibernator: winter (torpid state) and summer (active state). An increase in fluidity was noticed in the winter lipid and membrane preparations. The difference was however of very low magnitude, suggesting that only the microenvironment of some proteins was involved, rather than the bulk membrane fluidity.     

Seasonal Changes in Microsomal Fraction Enriched with Na,K-ATPase from Kidneys of the Ground Squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis>

The Na,K-ATPase activity in microsomal fraction isolated from kidneys of winter hibernating ground squirrels was found to be 1.8–2.0-fold lower than that in active animals in summer. This is partially connected with a decrease in Na,K-ATPase protein content in these preparations (by 25%). Using antibodies to different isoforms of Na,K-ATPase α-subunit and analysis of enzyme inhibition by ouabain, it was found that the decrease in Na,K-ATPase activity during hibernation is not connected with change in isoenzyme composition. Seasonal changes of Na,K-ATPase a-subunit phosphory- lation level by endogenous protein kinases were not found. Proteins which could be potential regulators of Na,K-ATPase activity were not found among phosphorylated proteins of the microsomes. Analysis of the composition and properties of the lipid phase of microsomes showed that the total level of unsaturation of fatty acids and the lipid/protein ratio are not changed significantly during hibernation, whereas the cholesterol content in preparations from kidneys of hibernating ground squirrels is approximately twice higher than that in preparations from kidneys of active animals. However, using spin and fluorescent probes it was shown that this difference in cholesterol content does not affect the integral membrane micro-viscosity of microsomes. Using the cross-linking agent cupric phenanthroline, it was shown that Na,K-ATPase in mem- branes of microsomes from kidneys of hibernating ground squirrels is present in more aggregated state in comparison with membranes of microsomes from kidneys of active animals. We suggest that the decrease in Na,K-ATPase activity in kidneys of ground squirrels during hibernation is mainly connected with the aggregation of proteins in plasma membrane.     

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.     

Fatty acids and cholesterol in the liver cell nuclei of hibernating Yakutian ground squirrels

The content of neutral lipids in tissue homogenates and liver cell nuclei of hibernating Yakutian ground squirrels was studied. In homogenates, hibernation increases the content of fatty acids and reduces the content of glycerides and cholesterol. When studying the liver cell nuclei of torpid winter ground squirrels, we detected a twofold increase in the content of fatty acids, cholesterol, and monoglycerides as compared to the “summer” ground squirrels. In the active “winter” ground squirrels, as compared to the torpid winter ones, the content of cholesterol did not change, whereas the content of fatty acids, monoglycerides, and diglycerides decreased but remained higher than in the “summer” ground squirrels.     

Characteristics of sarcoplasmic reticulum membrane preparations isolated from skeletal muscles of active and hibernating ground squirrel Spermophilus undulatus

The total Ca-ATPase activity in the sarcoplasmic reticulum (SR) membrane fraction isolated from skeletal muscles of winter hibernating ground squirrel Spermophilus undulatus is 2.2-fold lower than in preparations obtained from summer active animals. This is connected in part with 10% decrease of the content of Ca-ATPase protein in SR membranes. However, the enzyme specific activity calculated with correction for its content in SR preparations is still 2-fold lower in hibernating animals. Analysis of the protein composition of SR membranes has shown that in addition to the decrease in Ca-ATPase content in hibernating animals, the amount of SR Ca-release channel (ryanodine receptor) is decreased 2-fold, content of Ca-binding proteins calsequestrin, sarcalumenin, and histidine-rich Ca-binding protein is decreased 3-4-fold, and the amount of proteins with molecular masses 55, 30, and 22 kD is significantly increased. Using the cross-linking agent cupric–phenanthroline, it was shown that in SR membranes of hibernating ground squirrels Ca-ATPase is present in a more aggregated state. The affinity of SR membranes to the hydrophilic fluorescent probe ANS is higher and the degree of excimerization of the hydrophobic probe pyrene is lower (especially for annular lipids) in preparations from hibernating than from summer active animals. The latter indicates an increase in the microviscosity of the lipid environment of Ca-ATPase during hibernation. We suggest that protein aggregation as well as the changes in protein composition and/or in properties of lipid bilayer SR membranes can result in the decrease of enzyme activity during hibernation.     

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.     

Phospholipids of liver cell nuclei during hibernation of Yakutian ground squirrel

In hibernating Yakutian ground squirrels S. undulatus, the content of total phospholipids in the nuclei of liver increased by 40% compared to that in animals in summer. In torpid state, the amount of sphingomyelin increased almost 8 times; phosphatidylserine, 7 times; and cardiolipin, 4 times. In active “winter” ground squirrels, the amount of sphingomyelin, phosphatidylserine, and cardiolipin decreased compared to the hibernating individuals but remained high compared to the “summer” ones. The torpor state did not affect the amount of lysophosphatidylcholine and phosphatidylinositol.     

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

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

Role of lipids in the assembly of endoplasmic reticulum and dictyosomes in neuronal cells from the cerebral cortex of Yakutian ground squirrel (Citellus undulatus) during hibernation

Phospholipids and cholesterol were assayed in homogenates and microsomal fractions from the cerebral cortex of summer-active, winter-torpid, and winter-active Yakutian ground squirrels (Citellus undulatus). Ultrastructural analysis of both microsomal fraction and intact neurons was performed by serial ultramicrotomy. The levels of sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PEA) were decreased in homogenates from the cerebral cortex of winter ground squirrels compared with the summer-active animals, while the levels of phosphatidylcholine (PC) and cardiolipin (CL) were increased. The level of cholesterol was decreased in the cerebral cortex of winter-torpid animals compared with both winter-active and summer-active animals, and the level of total phospholipids was decreased in comparison to the summer-active animals. Three-dimensional reconstruction of serial membrane profiles displayed the microsomal fraction to be an interconnected system of cisterns and vesicles, which corresponds to endoplasmic reticulum and dictyosomes (Golgi stacks) of intact neurons. In winter the content of PC was increased in the microsomal fraction, while the contents of lysophosphatidylcholine (LPC), PS, phosphatidylinositol (PI), and SM were decreased. In winter-torpid animals compared with the winter-active ones the contents of total phospholipids, PEA, LPC, and cholesterol were decreased. As for the winter-active ground squirrels, their lipid contents did not differ from those in the summer-active animals, but LPC content was decreased. The changes in microsomal lipid contents in intact pyramidal neurons throughout the hibernation were accompanied by disassembly of dictyosomes and endoplasmic reticulum (ER), including the decomposition of polyribosomes to monosomes. The ultrastructural analysis of nucleoli, ER, and dictyosomes of both winter-active and torpid ground squirrels showed a direct correlation between the increasing contents of both cholesterol and total phospholipids (mainly PEA and LPC) in microsomes and the structural recovery of endoplasmic reticulum, Golgi stacks, and nucleoli in intact pyramidal neurons. A role of seasonal variations in lipid contents of brain cells in their adaptation to low temperature is discussed. We also propose an involvement of cholesterol in the activation of protein-synthesizing function of endoplasmic reticulum and Golgi stacks in intact neurons.     

A comparative fluorescence polarization study of cis-parinaroyl-phosphatidylcholine and diphenylhexatriene in membranes containing different amounts of cholesterol

The steady state fluorescence anisotropy (rs) of 1-acyl-2-cis parinaroyl phosphatidylcholine (PnPC) was compared with that of diphenylhexatriene (DPH) in a variety of model- and biological membrane systems. The fluorescence anisotropy of both probes responded similarly to temperature changes and variations in the acyl chain composition in phosphatidylcholine (PC) liposomes. The presence of proteins and cholesterol increased rs for both DPH and PnPC in the biological membranes as compared to the isolated polar membrane lipids. Comparison of DPH and PnPC in dipalmitoyl-PC-liposomes with and without 50 mol% cholesterol, showed at temperatures above the phase transition of pure dipalmitoyl-PC the presence of cholesterol increased the rs-value for DPH strongly, whereas the rs-value for PnPC was much less affected. In the cholesterol-rich erythrocyte membrane as well as in microsomes from Morris hepatoma 7787, which have an increased cholesterol content as compared to normal rat liver microsomes, the rs of DPH was higher than that of PnPC. No large differences between the rs-values of both probes were evident in the normal cholesterol-poor rat liver microsomes. These effects are discussed in terms of structural differences between the probes and variation of cholesterol content. Alterations in the fatty acid composition of PC present in human erythrocyte membranes were introduced with the aid of a PC-specific transfer protein. Fluorescence anisotropy values of both probes hardly changed upon enrichment of the red cell membrane with either dipalmitoyl PC or 1-palmitoyl-2-arachidonyl PC.     

In vitro modification of cholesterol content of rat liver microsomes. Effects upon membrane 'fluidity' and activities of glucose-6-phosphatase and fatty acid desaturation systems

The cholesterol content of rat liver microsomal membranes was modified in vitro by incubating microsomes and cytosol with liposomes prepared by sonication of microsomal lipids and cholesterol. In this way, the cholesterol to phospholipid molar ratio was increased from 0.11-0.13 in untreated microsomes to a maximal of 0.8 in treated ones. Cholesterol incorporation in microsomes produced an increase in the diphenyl-hexatriene steady-state fluorescence anisotropy and a decrease in the efficiency of pyrene-excimer formation which indicated a decrease in the rotational and translational mobility, respectively, of these probes in the membranes lipid phase. Cholesterol incorporation in microsomes did not affect significantly the glucose-6-phosphatase activity in 0.1% Triton X-100 totally disrupted microsomes, but diminished the glucose-6-phosphatase activity of 'intact' microsomes. This indicates that possibly the glucose 6-phosphate translocation across the microsomal membrane is impeded by an increase in the membrane apparent 'microviscosity'. Cholesterol incorporation in microsomes decreased NADH-cytochrome c reductase without affecting NADH-ferricyanide reductase activity. The delta 9 desaturation reaction rate was enhanced by cholesterol incorporation at low but not at high palmitic acid substrate concentration. delta 5 and delta 6 desaturase reaction-rates were increased both at low and high fatty acid substrate concentrations. These results suggest that a mechanism involving fatty acid desaturase enzymes, might exist to self-regulate the microsomal membrane lipid phase 'fluidity' in the rat liver.     

COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG : II. Lipids

The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined. As a group, membranes of the smooth variety (i.e., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins. In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio. All membrane fractions were unusually high in their content of lysolecithin (up to ~20% of the total phospholipids) and of neutral lipids, especially fatty acids. The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media. Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant. E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant.     

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.     

Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae

Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.     

Blood Plasma Phospholipids and Cholesterol during Hibernation of the Long-Tailed Ground Squirrel

Seasonal changes in the levels of phospholipids, diglycerides, cholesterol, and total protein in the blood plasma were investigated during hibernation of the long-tailed ground squirrel Spermophilus undulatus. During the winter period, the levels of phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin phospholipids (per 1 mg of plasma protein) were increased in both torpid and active ground squirrels by 70–80, 50, 600–700, 70, and 150–200%, respectively; the level of phosphatidylserine did not change in comparison to the summer period. The plasma phospholipid composition differed between hibernating and active summer animals: in winter, the phosphatidylcholine mol % decreased by 20%, phosphatidylinositol and phosphatidylethanolamine increased by 3–4 times, and the phosphatidylserine mol % decreased by 50%, while sphingomyelin and lysophosphatidylcholine did not change in comparison to summer animals. In hibernating ground squirrels, the plasma cholesterol levels increased by two times, the diglyceride content diminished by 60%, and the level of protein (in milligrams per 1 mL plasma) increased by 20%. The simultaneous increase in the levels of cholesterol and total phospholipids, as well as the pronounced specific changes in the levels of individual phospholipids in the blood plasma of hibernating ground squirrels, indicate the involvement of plasma lipoprotein lipids in the molecular mechanisms of adaptation to natural hypobiosis in mammals and a possible role of these mechanisms in systemic reactions to damaging factors.     

Phosphorus nuclear magnetic resonance of Acholeplasma laidlawii cell membranes and derived liposomes.

1. The 129 MHz 31P-NMR spectrum of Acholeplasma laidlawii membranes is very similar to the spectrum of the derived liposomes and is a typical "solid state" spectrum in which the major contribution to the linewidth is made by the chemical shift anisotropy. From the value of the chemical shift anisotropy an order parameter of 0.15 is estimated for the lipid phosphates in both membranes. 2. The 31P-NMR spectrum of the A. laidlawii membrane is insensitive to pronase digestion of 4-60% of the membrane proteins and subsequent cytochrome C binding. These results indicate that either no strong lipid polar headgroup-protein interactions occur in the membrane or that the lipid-protein "complexes" in the membrane have a fast rotation (Tc shorter than 10(-6)S) along an axis perpendicular to the plane of the membrane. 3. Phospholipase A2 degrades all the phosphatidylglycerol in the membrane. The resulting membrane contains a phosphoglycolipid as the sole phosphorus-containing compound. The 31P-NMR spectrum of these membranes is identical to the spectrum of the native membranes suggesting a similar motion for the phosphate groups in both lipids. 4. Ca2+ binding to liposomes prepared from either the total polar lipids or the total phosphorus-containing lipids isolated from the A. laidlawii membrane does not affect the 21P-NMR spectrum. 5. The 31P-NMR spectrum of the membranes and derived liposomes, however, is sensitive to lipid phase transitions. When the membrane lipids are in the gel state a broadening of the 31P resonance occurs demonstrating that the polar head group motion in a biological membrane is more restricted below the lipid-phase transition temperature.     

Membrane lipids and morphology of brain cortex synaptosomes isolated from hibernating Yakutian ground squirrel

Synaptosomes were isolated from Yakutian ground squirrel brain cortex of summer and winter hibernating animals in active and torpor states. Synaptosomal membrane cholesterol and phospholipids were determined. The seasonal changes of synaptosomal lipid composition were found. Synaptosomes isolated from hibernating Yakutian ground squirrel brain cortex maintained the cholesterol sphingomyelin, phosphatidylethanolamine, lysophosphatidylcholine, cardiolipin, phosphatidylinositol and phosphatidylserine contents 2.5, 1.8, 2.6, 1.8, 1.6, and 1.3 times less, respectively, and the content of phosphatidylcholine twice as much as the one in summer season. The synaptosomal membrane lipid composition of summer animals was shown to be markedly different from that as hibernating ground squirrels and non-hibernating rodents. It is believed that phenotypic changes of synaptosomal membrane lipid composition in summer Yakutian ground squirrel are the important preparation step for hibernation. The phosphatidylethanolamine content was increased in torpor state compared with winter-active state and the molar ratio of cholesterol/phospholipids in synaptosomal membrane of winter torpor ground squirrels was lower than that in active winter and summer animals. These events were supposed to lead to increase of the synaptosomal membrane fluidity during torpor. Synaptosomes isolated from torpor animals have larger sizes and contain a greater number of synaptic vesicles on the synaptosomal profile area. The synaptosomal membrane lipid composition and synaptosome morphology were involved in phenotypic adaptation of Yakutian ground squirrel to hibernation.     

A decrease of lipid fluidity of the porcine intestinal brush-border membranes by treatment with malondialdehyde.

The effect of treatment of the porcine intestinal brush-border membranes with malondialdehyde (MDA) on their lipid fluidity was examined using a fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). When the membranes were treated with MDA, the fluorescence anisotropy of DPH-labeled membranes increased and the amount of DPH molecules incorporated into the membranes decreased from 3.25 to 2.23 nmol/mg protein. In addition, the response of the fluorescence anisotropy of DPH-labeled membranes to benzyl alcohol, a well-known fluidizer, was markedly suppressed by treatment of the membranes with MDA. These results suggest that treatment of the membranes with MDA causes a decrease of the membrane lipid fluidity. This interpretation was further supported by the increase observed in the fluorescence anisotropy of DPH-labeled liposomes prepared from the extracted lipids of MDA-treated membranes. The results of SDS-polyacrylamide gel electrophoresis suggested that the formation of high-molecular-weight aggregates of the membrane proteins is not involved in the increase of the fluorescence anisotropy of DPH-labeled membranes by treatment with MDA. On the basis of these results, changes in the physical properties of the intestinal brush-border membranes by treatment with MDA are discussed.     

Fluidity properties of isolated chloroplast thylakoid lipids

Chloroplast thylakoid lipids have been isolated free of photosynthetic pigments using a combination of high performance liquid and thin layer chromatography. The hydrophobic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) has been incorporated into aqueous dispersions of the isolated lipids in order to investigate dynamic and structural properties of the resulting bilayer membranes. Time dependent fluorescence anisotropy decays have been measured and analysed assuming the wobbling-in-cone model (Kinosita et al., Biophys J 20 (1977) 289–305). The DPH fluorescence lifetimes and the static and dynamic fluorescence anisotropy decay parameters for the probe in a total lipid mixture or in pure digalactosyldiacylglycerol (DGDG), changed in a predictable way with increasing temperature (10°–36°C). For a given temperature, it was found that the total lipid mixture was in general less ordered and showed greater dynamic motion as judged from DPH fluorescence anisotropy and compared with the pure DGDG system, although at 36°C differences in dynamic parameters were less evident. Overall the results obtained emphasize the highly fluid nature of thylakoid membrane lipids and give a basis for investigating how intrinsic proteins modify structural and dynamic properties of the in vivo membrane.     

Modulation of rat liver lipid metabolism by prolactin

The effect of chronic hyperprolactinemia on the delta6- and delta5-desaturation activity, total lipid and fatty acid composition, as well as fluorescence anisotropy, was studied in liver microsomes from anterior pituitary-grafted rats. We observed a depression in delta6-desaturation activity but no changes in the delta5-desaturation activity in the grafted animals. The microsomal fraction from the hyperprolactinemic rats contained significantly less amount of linoleic acid and a higher content of 20:4 n-6, 22:5 n-6 and 22:6 n-3 acids. Lipid rotational mobility was increased in microsomes as well as in liposomes obtained from the microsomes of transplanted animals. The fluidifying effect induced by PRL was located in the deepest zone of the membrane. The results obtained indicate that high levels of prolactin induce changes in polyunsaturated fatty acid distribution in liver microsomes, which regulates the lipid rotational mobility and hence membrane fluidity.