Model membrane studies of spin-label probes Part I. Mixed monolayers of 12-nitroxide stearic acid and myristic acid

Pure and mixed monomolecular films of a cell membrane spin label probe, 12-nitroxide stearic acid have been studied where myristic acid was selected as the host lipid. The behavior of 12-nitroxide stearic acid at the air water interface is understood in terms of two molecular configurations: erect (with only the carboxyl group in the interface) and bent (with both the carboxyl group and the oxazolidine ring in the interface). In mixed films both of these conformations play a role at high surface pressures. At low probe concentrations, 12-nitroxide stearic acid is primarily in an erect conformation, while at high probe concentrations the reverse is true. This particular host lipid appears capable of erecting the probe molecule with only small concentrations of myristic acid. In a condensed host lipid, the probe is partially immiscible, and segregates to form a heterogeneous film from which it is readily collapsed. The probe is seen to perturb the molecular packing in this mixed system and the perturbation to be dependent on both the molecular shape and nature of the probe.     

Correction of abnormal lipid fluidity and composition of rat ileal microvillus membranes in chronic streptozotocin-induced diabetes by insulin therapy

Diabetes was induced in rats by administration of streptozotocin. After 90-120 days, one group of chronic diabetic animals was treated with insulin for chronic diabetic animals was treated with insulin for 10 days. The lipid fluidity and composition of microvillus membranes prepared from ileal enterocytes of control, diabetic, and insulin-treated diabetic animals were determined. Lipid fluidity, as assessed by steady-state fluorescence polarization techniques using the probes 1,6-diphenyl-1,3,5-hexatriene, DL-2-(9-anthroyl)stearic acid and DL-12-(9-anthroyl)stearic acid, was decreased in membranes of diabetic animals compared to membranes of control and insulin-treated diabetic membranes. The differences in fluidity resulted from an increased cholesterol content and cholesterol/phospholipid molar ratio in membranes of diabetic animals. The activities of sucrase and alkaline phosphatase were also found to be higher in membranes of diabetic animals. Insulin treatment, however, failed to significantly influence the enzymatic activities of these membranes. These studies, therefore, demonstrate that alterations in the lipid fluidity, lipid composition, and certain enzymatic activities exist in microvillus membranes of enterocytes prepared from chronic streptozotocin-induced diabetic rats. Administration of insulin for 10 days to these animals restored membrane fluidity and lipid composition but not enzymatic activities to control membrane levels.     

Studies on spin-labelled egg lecithin dispersions.

ESR spectra of egg lecithin dispersions labelled with 5-nitroxide stearic acid are recorded with a 50 G field sweep, and also with a new technique which "expands" the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The expansion technique improves the precision of the order parameters determined from the hyperfine splitting measurements, and may prove useful in future spin label membrane studies. Approximate order parameters are derived to describe the fluidity of fatty acid spin-labelled membranes in those cases where either the inner or outer hyperfine extrema are not well defined. The ability of these expressions to measure the fluidity of labelled egg lecithin disperions for the temperature range 14-42 degrees C is examined.     

Studies on spin-labelled egg lecithin dispersions

ESR spectra of egg lecithin dispersions labelled with 5-nitroxide stearic acid are recorded with a 50 G field sweep, and also with a new technique which “expands” the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The expansion technique improves the precision of the order parameters determined from the hyperfine splitting measurements, and may prove useful in future spin label membrane studies.Approximate order parameters are derived to describe the fluidity of fatty acid spin-labelled membranes in those cases where either the inner or outer hyperfine extrema are not well defined. The ability of these expressions to measure the fluidity of labelled egg lecithin dispersions for the temperature range 14–42° C is examined.     

Regional differences in the lipid composition and fluidity of rat colonic brush-border membranes

The lipid composition and fluidity of brush-border membranes prepared from rat proximal and distal colonocytes were determined. Fluidity, as assessed by steady-state fluorescence polarization techniques using the fluorophores 1,6-diphenyl-1,3,5-hexatriene, DL-2(9-anthroyl)stearic acid and DL-12(9-anthroyl)stearic acid, was decreased in distal compared to proximal plasma membranes. This pattern was similar to that previously described for both antipodal plasma membranes in rat enterocytes of the small intestine. The decrease in fluidity of the distal as compared to the proximal membranes resulted from an increase in cholesterol content, cholesterol/phospholipid molar ratio and degree of saturation of the fatty acid residues in the distal membranes. The specific activities of total alkaline phosphatase and cysteine-sensitive alkaline phosphatase, enzymes previously shown to be functionally dependent on the physical state of the colonic brush-border membrane's lipid, were also significantly lower in distal as compared to proximal clonic plasma membranes. These studies, therefore, demonstrate that differences in the lipid fluidity, lipid composition and certain enzymatic activities exist in brush-border membranes prepared from rat proximal and distal colonocytes. The regional variation in rat colonic luminal membrane lipid fluidity and composition may, at least partially, be responsible for differences in these enzymatic activities as well as in sodium and water absorption along the length of this organ.     

Effects of temperature and cholesterol on human erythrocyte membranes

The effects of temperature and cholesterol on the membrane fluidity of human erythrocytes were studied using 5-nitroxide stearic acid (5NS), 12-nitroxide stearic acid (12NS), and 16-nitroxide stearic acid (16NS). Human erythrocytes and their lipid vesicles were treated in the range of 5--55 degrees C. In erythrocytes, ESR signals for 12NS and 16NS showed line broadening above 40 degrees C, whereas those for 5NS became sharper with increasing temperature as was the case with the signals of lipid vesicles for each label molecule. Lipid extraction from the heated sample caused no radical reduction. Only in 12NS-labeled erythrocytes did a weakly immobilized component and a strongly immobilized component appear. In the time course at 50 degrees C, the former decreased and the latter remained constant. From the ratio of both components, it was found that the interaction of the label molecules with the binding sites was determined by the physical state of the membrane. Furthermore, the dependence on temperature of the molecular motion of the labels in the cell membrane was irreversible above 40 degrees C. On addition of cholesterol to the membrane, the outer hyperfine splittings for 12NS and 16NS increased but that for 5NS decreased at C/P greater than 1, perhaps indicating a spread between the head groups of phospholipids by cholesterol.     

Rat proximal small intestinal Golgi membranes: lipid composition and fluidity

The present studies were conducted to examine and characterize the lipid composition and physical state of the membrane lipids of rat proximal small intestinal Golgi membranes. Golgi membranes were purified from isolated enterocytes; lipids were extracted from these membranes and analyzed by thin-layer and gas-liquid chromatography. The 'static' and 'dynamic' components of fluidity of Golgi membranes and their liposomes were assessed by steady-state fluorescence polarization techniques utilizing r infinity and S values of 1,6-diphenyl-1,3,5-hexatriene and r values of DL-2-(9-anthroyl)- and DL-12-(9-anthroyl)stearic acid, respectively. Additional studies were also performed on these membranes, using benzyl and methyl alcohol, to examine the relationship between alterations in lipid fluidity and glycosphingolipid glycosyltransferase activities. The results of these studies demonstrated that: (1) the principal phospholipids and neutral lipids of intestinal Golgi membranes, respectively, were phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, and unesterified cholesterol and fatty acids; (2) the major fatty acids of Golgi membranes were palmitic (16:0), stearic (18:0), linoleic (18:2), arachidonic (20:4) and oleic (18:1) acids; (3) fluorescence polarization studies using diphenylhexatriene detected a thermotropic transition at 24-26 degrees C in Golgi membranes and liposomes prepared from lipid extracts of these membranes; (4) benzyl alcohol (25 and 50 mM) but not methyl alcohol (50 mM) significantly increased the fluidity of these membranes; and (5) at these same concentrations, benzyl alcohol was also found to increase significantly the specific activity of UDP-galactosyllactosylceramide galactosyltransferase but not CMP-acetylneuraminic acid: lactosylceramide sialyltransferase. Methyl alcohol was not found to influence either enzyme's activity in these membranes.     

Effects of pH on membrane fluidity of human erythrocytes

The effects of pH on the membrane fluidity of intact human erythrocytes, ghosts, and their lipid vesicles were studied by spin label techniques in the range of pH 3.0 to 9.1. Two fatty acid spin labels, 5-nitroxide stearic acid (5NS) and 12-nitroxide stearic acid (12NS), and a maleimide spin label were used for the labeling of the membrane lipids and proteins, respectively. The outer hyperfine splitting (T parallel) was measured as a parameter of membrane fluidity. In the case of 5NS, the T parallel values for intact erythrocytes and ghosts remained almost constant over the entire pH range at 22 degrees C but those for their lipid vesicles changed slightly, indicating the vertical displacement of the labels in lipid bilayers. On the other hand, the ESR spectra of 12NS incorporated into intact erythrocytes and ghosts, as compared with their lipid vesicles, showed marked pH dependence. By means of spin labeling of membrane proteins, the conformational changes of the proteins were observed in the pH range mentioned above. These results suggest a possible association between the strong pH dependence of the T parallel values and the conformation changes of membrane proteins. The pH dependence of the membrane fluidity was also investigated in cholesterol-enriched and -depleted erythrocytes. The effects of cholesterol demonstrated that the membrane fluidity was significantly mediated by cholesterol at low pH, but not at high pH.     

Glutaryl phosphatidylcholine effects on pH and composition dependent behavior of liposomes studied by spin-labeling method

The influence of glutaryl phosphatidylcholine on the molecular organization of phosphatidylcholine liposomes was studied by spin-labeling technique. The ESR signals given by the 5-nitroxide stearic acid label showed that the presence of glutaryl lecithin (i) significantly increased the negative charge density of the polar liposome surface with increasing proton concentration depending on the bulk solution pH, and (ii) apparently decreased the packing (order) of the hydrophobic region close to the surface, essentially in the presence of saturated phospholipids. The spectral information--S (order parameter) and alpha N (isotropic nitrogen coupling constant)--resulted in the location of the probe near or in the polar zone of the membrane or in the hydrophobic region, depending on the protonation/deprotonation of the fatty acid carboxyl group of the probe. The microviscosity of the inner region of the membrane monitored by the 12- and 16-probes was not significantly altered by glutaryl lecithin. On the other hand, glutaryl lecithin has a lesser effect on liposomes containing anionic polar head groups, such as dipalmitoyl phosphatidylglycerol or phosphatidylinositol, the anionic charge of which already had the same effect on protonation of the polar surface. The temperature dependence of dipalmitoyl phosphatidylcholine liposome dynamic behavior indicates that the glutaryl lecithin effect is completely different above and below the gel-to-liquid crystalline phase transition point.     

Correction by 1-25-dihydroxycholecalciferol of the abnormal fluidity and lipid composition of enterocyte brush border membranes in vitamin D-deprived rats

Weanling male Wistar rats were deprived of dietary and light sources of vitamin D for 11-18 weeks along with age-matched diet vitamin D-repleted controls to evaluate the role of lipid fluidity in the stimulatory effect of calcitriol on Ca transport. The "static" component of fluidity of proximal small intestine brush border membrane, as assessed by steady-state fluorescence techniques using the fluorophore 1,6-diphenyl-1,3,5-hexatriene, was similar between these two groups. In contrast, the "dynamic" component of fluidity, as assessed by DL-2-(9-anthroyl)-stearic acid and DL-12-(9-anthroyl)-stearic acid, was decreased in membranes of D-deprived animals. Lipid composition was analyzed to evaluate the potential mechanism mediating these fluidity changes. In vitamin D-deprived rats, linoleic (18:2) and arachidonic (20:4) acids of the phosphatidylcholine and phosphatidylethanolamine fractions of the membrane were decreased, whereas palmitic (16:0) and stearic (18:0) acids were increased in the phosphatidylethanolamine fraction of the membrane. These associated fatty acyl alterations could explain, at least in part, the differences in membrane fluidity between D-repleted and D-deprived rats. Membrane fluidity, lipid composition, and duodenal Ca transport were also analyzed 1, 2, and 5 h after the acute administration of 1-25-dihydroxycholecalciferol to D-deprived animals. In D-deprived rats, within 1-2 h, this hormone restored to levels of vitamin D-repleted controls the dynamic component of fluidity and concentrations of the same membrane phospholipid fatty acids. Since these changes temporally precede detectable increases in Ca absorption (demonstrable only during the 5th h), these data support the hypothesis that alterations in membrane fluidity and lipid composition may play an important role in the stimulation of intestinal calcium transport by calcitriol.     

Ontogeny of proximal colon basolateral membrane lipid composition and fluidity in the rabbit.

Basolateral membranes from rabbit proximal colon were prepared from isolated colonocytes throughout postnatal maturation, using a modification of published techniques. In suckling (14-20 day) and post-weaning/mature (35-49 day) animals, membranes were purified approx. 10-fold, based upon the enrichment of ouabain-sensitive, sodium-potassium dependent adenosine triphosphatase activity. Membrane lipid analyses demonstrated age-dependent increases in total cholesterol and the cholesterol/phospholipid molar ratio, as well as decreases in phosphatidylethanolamine content and the fatty acid unsaturation index. Fluidity of basolateral membranes and membrane liposomes, determined from fluorescence anisotropy measurements using the lipid probes 1,6-diphenyl-1,3,5-hexatriene and DL-12-(9-anthroyl)stearic acid, demonstrated significant, ontogenic decreases in fluidity; and, additional studies showed that fluidity changes occurred early in the weaning period (by day 24 postnatally). Arrhenius plots of liposome anisotropies suggested a bilayer lipid thermotropic transition temperature of 22 degrees C in sucklings 26 degrees C in mature rabbits. These findings demonstrate that ontogeny of colonic basolateral membranes is associated with significant modulations in lipid composition and fluidity.     

The penetration of local anesthetics into the red blood cell membrane as studied by fluorescence quenching.

The local anesthetics procaine and tetracaine were found to quench the fluorescence of the probes N-octadecyl naphthyl-2-amine 6-sulfonic acid and 12-(9-anthroyl)stearic acid in the presence of erythrocyte membranes. This quenching was shown to be due to the aromatic amine of the procaine and tetracaine molecules. Lidocaine, an active anesthetic that does not contain an aromatic amine in the same position as does procaine and tetracaine did not quench either of the fluorophores. The preferential quenching of the fluorescent probes by procaine and tetracaine indicated a greater accessibility of tetracaine than of procaine to the hydrocarbon region of the membrane and a greater accessibility of procaine than of tetracaine at the membrane's surface. The addition of calcium was found to reverse the quenching of 12-(9-anthroyl)stearic acid by tetracaine in the presence of red cell membranes.     

The relation between lipid mobility and the specific hormone binding of thyroid membranes.

1. The specific binding of thyroid-stimulating hormone to isolated human thyroid membranes was examined under a variety of conditions. 2. In phosphate-saline buffer (in the presence of 0.14 M-NaCl) on increasing the temperature the binding of the hormone is increased, the plots of bound/free hormone against temperature showing a distinct break around 30 degrees C. 3. Detailed analysis showed that the increased binding is associated with an increase in the number of binding sites. 4. The motional characteristics of three membrane-bound fluorescent probes, 2-(9-anthroyl)palmitic acid, 12-(9-anthryl)stearic acid and N-1-naphthyl-N-phenylamine, were also examined as a function of temperature by measuring both fluorescence polarizations and lifetimes. 5. The results indicated that the 'fluidity' of membrane lipids also increased with temperature. The temperature-dependence of this property also shows a change at about 30 degrees C. 6. Bivalent cations decreased both membrane fluidity and hormone binding. 7. Similar correlations were found between the binding of adrenocorticotrophic hormone and the fluidity of the plasma membranes obtained from adrenal-cortical cells, with the discontinuity occurring in this case at 23 degrees C. 8. The possibility of lipid mobility being important in controlling hormone-receptor function is discussed.     

Convenient biosynthetic preparation of isomeric spin-labelled radioactive phosphatidic acids

A convenient method for the enzymatic preparation of sn-3-[2-3H]phosphatidic acids carrying also 5-, 12-, or 16-nitroxide stearic acids, from sn-3-[2-3H]glycerophosphate and isolated guinea pig liver microsomes, is described in detail. The procedure allows a simultaneous preparation of three spin-labelled sn-3-[2-3H]phosphatidic acids of yields 3-3.5 mumol of each compound which is greater than 99% pure in respect to the radioactivity and which contains 25 mol% of spin-labelled fatty acids. These phosphatidic acids were approximately equally distributed between the primary and the secondary hydroxyl when 12- or 16-nitroxide stearic acids were used or predominantly (75%) associated with the secondary hydroxyl of sn-3-[2-3H]phosphatidic acid when 5-nitroxide stearic acid was present in the incubation mixture.     

Lipid fluidity and composition of the erythrocyte membrane from healthy dogs and labrador retrievers with hereditary muscular dystrophy

Erythrocyte membranes and their liposomes were prepared from clinically normal dogs and Labrador retrievers with hereditary muscular dystrophy. The static and dynamic components of fluidity of each membrane were then assessed by steady-state fluorescence polarization techniques using limiting hindered fluorescence anisotropy and order parameter values of 1,6-diphenyl-1,3,5-hexatriene (DPH) and fluorescence anisotropy values ofdl-2-(9-anthroyl)-stearic acid anddl-12-(9-anthroyl)-stearic acid, respectively. Membrane lipids were extracted and analyzed by thin-layer chromatography and gas chromatography. The results of these studies demonstrated that the lipid fluidity of erythrocyte membranes, and their liposomes, prepared from dystrophic dogs were found to possess significantly lower static and dynamic components of fluidity than control counterparts. Analysis of the composition of membranes from dystrophic dogs revealed a higher ratio of saturated fatty acyl chain/unsaturated chains (w/w) and lower double-bond index. Alterations in the fatty acid composition such as decrease in levels of linoleic (18:2) and arachidonic (20:4) acids and increase in palmitic (16:0) and stearic (18:0) acids were also observed in the membranes of dystrophic animals. These associated fatty acyl alterations could explain, at least in part, the differences in membrane fluidity between dystrophic and control dogs.     

Effect of calcium, insulin and growth hormone on membrane fluidity. A spin label study of rat adipocyte and human erythrocyte ghosts

ESR spectra were recorded from rat epididymal adipocyte ghosts labeled with the 5-nitroxide stearic acid spin probe, I(12,3). Polarity-corrected and approximate order parameters, that are sensitive to the flexibility of the incorporated label, were used to evaluate the membrane lipid fluidity. Addition of CaCl2 a 37 degrees C decreased the fluidity, as indicated by positive increases in the order parameters. The ordering effect of Ca2+ was concentration-dependent, reached saturation at approx. 3--4 mM, and was completely reversed by excess EGTA. Previous studies indicated that low- and high-affinity sites on adipocyte plasma membranes are able to bind 45Ca2+, and our results suggest that Ca2+-induced alterations in the lipid fluidity involve cation binding to low-affinity sites. The cellular movements of Ca2+ and, in particular, the binding of Ca2+ to the plasma membrane may play important roles in insulin's action on fat cell function. The possibility that insulin directly alters the membrane fluidity was tested by adding hormone to freshly-prepared I(12,3)-labeled adipocyte ghosts. Insulin, at concentrations (10(-6) M) that enhance glucose uptake into intact adipocytes, did not affect the fluidity of ghosts suspended in buffers with or without Ca2+. The fluidities of I(12,3)-labeled rat adipocyte ghosts or human erythrocyte ghosts were also unaffected by various forms of human growth hormone.     

Abnormal fluidity state in membranes of malignant hyperthermia pig skeletal muscle

The fluidity state was analyzed on sarcoplasmic reticulum membranes and phospholipid vesicles prepared from normal and malignant hyperthermia susceptible pig muscle. Electron spin resonance studies were performed to determine the fluidity state at the region near the polar headgroups and in the central core of the bilayer using 5-nitroxide (5-NS) and 16-nitroxide stearic acid (16-NS), respectively. With the 5-NS label, no differences were found between normal and malignant hyperthermia sarcoplasmic reticulum (MH SR) membranes whereas with the 16-NS label, a significant increase of the activation energy was shown with MH membranes. Lower values of fluorescence anisotropy observed with DPH-labeled MH membranes as compared with normal ones, confirmed the higher abnormal fluidity state of these membranes. The fluidizing effect of halothane, a triggering agent of malignant hyperthermia syndrome, was also studied in these membranes. We show that a relatively low concentration of the drug destabilized not only the diseased sarcoplasmic reticulum membranes but also the vesicles made of total phospholipids extracted from MH skeletal muscle. Together, these findings strongly suggest that an overall increase in membrane fluidity may be implied in the MH disease, improving the general membrane defect hypothesis for this syndrome.     

Role of Mg2+ in lipid-protein interaction in reconstituted procine heart mitochondrial H+-ATPase

During reconstitution of pig heart mitochondrial H+-ATPase in soybean phospholipid liposomes by the cholate dialysis method, Mg2+ greatly enhances 32Pi-ATP exchange activity, ATPase activity and the sensitivity to oligomycin of the reconstituted enzyme complex. The effect of Mg2+ on the fluidity of the reconstituted proteoliposomes was measured by means of a fluoursecent probe. 1-anilinonaphthalene ?e-8-sulfonate, and spin-label probes, 5-nitroxide stearate, 12-nitroxide stearate and 16-nitroxide stearate. A difference in fluidity seems to be localized near the polar faces of the lipid bilayers of the reconstituted proteolipsomes. Fluidity was less in the presence of Mg2+ than it is absence. The conformations of the Mg2+-containing proteoliposomes was higher. We postulate that Mg2+ may play a role in altering the fluidity of the proteoliposomes, which would favor the formation of a conformation of the reconstituted H+-ATPase with higher activity.     

Spin label studies of erythrocytes with abnormal lipid composition: comparison of red cells in a hereditary hemolytic syndrome and lecithin: Cholesterol acyltransferase deficiency

Erythrocytes from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency have been shown to exhibit an increase in membrane fluidity which is surprisingly small in view of the extensive alterations both in membrane lipicl composition (namely, an elevation in cholesterol and phosphatidylcholine contents as well as a decrease in phosphatidylethanolamine) and in the functional properties of these cells. In the hope of deriving some information concerning the interrelationship between the structural and functional abnormalities, we have used the spin probe 5-doxyl stearic acid to investigate the temperature-dependent fluidity properties of red cells from two patients with a hereditary hemolytic syndrome (HHS) whose red cells are also characterized by qualitatively similar alterations in phosphatidylcholine and phosphatidylethanolamine but, unlike those in LCAT deficiency, have relatively normal levels of membrane cholesterol. A small increase in membrane fluidity of HHS erythrocytes equivalent to that previously observed in LCAT deficiency was found, indicating that membrane cholesterol level does not exert an important modulatory influence on membrane fluidity in these cells. It is concluded that while the distinct patterns of structural and functional erythrocyte alterations in these two disorders cannot be explained on the basis of differences in bulk membrane fluidity, the marginally increased fluidity may underlie the abnormalities in osmotic fragility and membrane p-nitrophenylphosphatase activity which are shared in common by both types of modified red cells.     

A spin-label study of sciatic nerves from quaking, jimpy and trembler mice.

The spin labels, 5-nitroxide stearic acid and 16-nitroxide stearic acid were incorporated into whole sciatic nerves dissected from normal, quaking, jimpy and trembler mice. With 5-nitroxide stearic acid, we have studied the thermal variation of the maximal apparent coupling constant (T) between 0 degrees C and 50 degrees C. Within this range of temperatures, we obtained identical values of 2 T for nerves from normal and jimpy mice, whereas 2 T was smaller for nerves from quaking and trembler mice. With 16-nitroxide stearic acid, composite spectra were recorded, particularly in the high-field range. A line characteristic of myelin was clearly observed in the spectra of nerves from normal and jimpy mice; its intensity was somewhat less in nerves from quaking mice and much less in spectra from trembler mice. A shoulder in the principal highfield line of the spectrum is modified only with nerves from jimpy mice. The results agree well with those obtained by electron microscopy, which reveal normal myelination in nerves from jimpy mice, a slight modification of the myelin from those of quaking mice and a practically complete demyelination in peripheral nerves from trembler mice. However, the structure of the nerves of jimpy mice also seems to be modified at an, as yet, undetermined level.