Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decrease membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC) ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipoproteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80-85% of abetalipoproteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity (eta) between 5 and 42 degrees C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively at 10 degrees C; 4.63 vs 4.04 poise at 37 degrees C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Influence of cholesterol content on red cell membrane viscoelasticity and fluidity.

The purpose of this investigation was to correlate the viscoelastic properties and lipid fluidity of the red blood cell membrane to its lipid composition. The viscoelastic properties of human red cells that had been enriched or depleted in cholesterol were determined by the micropipette technique. The lipid fluidity of the outer and inner leaflets of the erythrocyte membrane was concurrently assessed by steady state fluorescence depolarization. The elastic modulus and the viscosity moduli of the erythrocyte membrane showed no significant differences between the cholesterol-modified and the control cells. Cholesterol enrichment decreased the lipid fluidity of the outer membrane leaflet alone, and cholesterol depletion increased the fluidity mainly of the inner leaflet.     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decreased membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC)§ ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipo-proteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80–85% of abetalipo-proteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity ({ie116-1}) between 5 and 42°C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively, at 10°C; 4.63 vs 4.04 poise at 37°C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Homoeostatic control of membrane cholesterol and fatty acid metabolism in the rat liver.

Experiments were designed to assess the effect of cholesterol feeding, with or without high levels of either saturated (coconut oil) or unsaturated (sunflower-seed oil) fat on the fatty acid composition of hepatic microsomal membrane lipids, as well as on the activities of several membrane-bound enzymes of cholesterol synthesis and metabolism. Administration of 2% (w/w) cholesterol in the rat diet inhibited hydroxymethylglutaryl-CoA reductase activity, and this inhibition was much more pronounced when cholesterol was fed in combination with unsaturated rather than with saturated fat. Cholesterol 7 alpha-hydroxylase activity was increased by all the high-cholesterol diets and inhibited by both the high-fat diets. Cholesterol esterification, as assessed by acyl-CoA:cholesterol acyltransferase (ACAT) activity, was enhanced after unsaturated-fat feeding. Cholesterol supplement, without any added fat, failed to elicit any significant increase in ACAT activity, whereas consumption of cholesterol in combination with unsaturated fat led to the greatest increase in ACAT activity. After cholesterol feeding, C18:1 and C18:2 fatty acids in the microsomal phospholipids were increased, with concomitant decreases in C18:0, C20:4 and C22:6 fatty acids, leading to an overall decrease in membrane unsaturation, irrespective of the particular fat supplement. It can be concluded that the inhibition of cholesterol biosynthesis and the enhancement of cholesterol utilization, either by increased bile formation or by increased cholesterol esterification, after cholesterol feeding, may not be enough to prevent cholesterol accumulation in the microsomal membranes. Then, to compensate for the altered fluidity resulting from cholesterol enrichment, the unsaturation of membrane phospholipids is decreased, which would in turn have an effect on membrane lipid fluidity opposite to that of increased cholesterol.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37°C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20°C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20°C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freezethaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Modulation of erythrocyte membrane proteins by membrane cholesterol and lipid fluidity.

Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide. Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups. The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64%. The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 0.4 nmol/mg of protein, or 14%. Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands. Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4. The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins.     

Influence of cholesterol and prostaglandin E1 on the molecular organization of phospholipids in the erythrocyte membrane. A fluorescent polarization study with lipid-specific probes

Anthryl-labeled fluorescent probes closely mimicking phosphatidylcholine and sphingomyelin were applied to study the state of these phospholipids in the rabbit erythrocyte membrane. At normal cholesterol levels both probes exhibited higher fluorescence polarization values in the membranes than in phospholipid vesicles of similar lipid composition, indicating a decreased fluidity of the probe environment in erythrocyte ghosts. In ghosts prepared from normal erythrocytes no evidence of lateral separation of phosphatidylcholine and sphingomyelin was found. At higher cholesterol levels, however, these lipids appear to segregate. Probably the effect of cholesterol on the erythrocyte membrane lipids involves lipid-protein interactions. At physiological concentrations, prostaglandin E1 only weakly affects the state of phosphatidylcholine and sphingomyelin in erythrocyte membranes. Cholesterol enrichment amplifies the effect of prostaglandin E1. Although the prostaglandin E1-induced changes depended much upon whether the ghosts were enriched with cholesterol in vitro or in vivo, with both types of ghosts effects of prostaglandin E1 were seen at extremely low effector concentrations that may have presented a few molecules of prostaglandin per ghost. The structural and functional significance of these findings is discussed.     

Increased Na(+)-dependent D-glucose transport and altered lipid composition in renal cortical brush-border membrane vesicles from bile duct-ligated rats.

Erythrocyte membranes of patients with liver disease are characteristically enriched in cholesterol, a change known to impair several carrier-mediated membrane transport functions. In the present study we have assessed whether experimental liver disease can affect the membrane lipid composition and transport function of kidney epithelial cells. Small (about 5%) but significant (P less than 0.01) increases were found in the cholesterol-to-phospholipid molar ratio (C/PL) of rat renal cortical brush-border membrane (BBM) vesicles 3, 8, and 15 days after bile duct ligation which correlated closely with increased fluorescence polarization, i.e., decreased membrane fluidity (r = 0.75, P less than 0.001; n = 27). A lipoprotein-mediated pathogenesis was suggested by the close relationship between BBM C/PL and plasma C/PL (r = 0.69, P less than 0.001). The mean high-affinity Na(+)-coupled D-glucose uptake by BBM vesicles was higher 1, 3, 8, and 15 days after ligation than in non-operated rats, significantly so at 3 and 8 days (611 +/- 37 and 593 +/- 22 vs. 507 +/- 21 pmol/mg protein per 4 sec; P less than 0.05), and was positively correlated with BBM C/PL (r = 0.58, P less than 0.01) and fluorescence polarization (r = 0.41, P less than 0.05). Brief incubation of BBM vesicles from normal rats with cholesterol-rich phospholipid liposomes simultaneously increased BBM C/PL and Na(+)-dependent D-glucose uptake. Stimulation of BBM Na(+)-glucose cotransport in ligated rats was not due to delayed dissipation of the Na+ gradient or to a more rapid development of membrane potential. High-affinity Na(+)-dependent D-glucose uptake kinetics in 3-day bile duct-ligated rats showed a lower Kt, without an alteration in maximum velocity, Vmax, compared to sham-operated animals (0.298 +/- 0.015 vs. 0.382 +/- 0.029 mM; P less than 0.05), whilst the binding dissociation constant, Kd of high-affinity phlorizin binding sites was reduced by ligation (0.453 +/- 0.013 vs. 0.560 +/- 0.015 microM; P less than 0.001). We conclude that an early effect of bile duct ligation is to enrich renal cortical brush-border membranes in cholesterol, thereby decreasing membrane fluidity and stimulating Na(+)-dependent D-glucose uptake by increasing the affinity of the carrier.     

Effect of cholesterol on the branched-chain amino acid transport system of Streptococcus cremoris.

The effect of cholesterol on the activity of the branched-chain amino acid transport system of Streptococcus cremoris was studied in membrane vesicles of S. cremoris fused with liposomes made of egg yolk phosphatidylcholine, soybean phosphatidylethanolamine, and various amounts of cholesterol. Cholesterol reduced both counterflow and proton motive force-driven leucine transport. Kinetic analysis of proton motive force-driven leucine uptake revealed that the Vmax decreased with an increasing cholesterol/phospholipid ratio while the Kt remained unchanged. The leucine transport activity decreased with the membrane fluidity, as determined by steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene incorporated into the fused membranes, suggesting that the membrane fluidity controls the activity of the branched-chain amino acid carrier.     

Cholesterol oxidase susceptibility of the red cell membrane

We have used the highly variable and conditional susceptibility of cholesterol oxidase to probe molecular rearrangements in the human red cell membrane. Cholesterol in the intact erythrocyte normally is not a substrate for this enzyme. Susceptibility was induced however, by these pretreatments: mild enrichment in membrane cholesterol, exposure to greater than or equal to 0.03% (3 mM) glutaraldehyde and warming in dilute salt solutions (mu approx. 0.001). Cholesterol reactivity in dilute salt solutions emerged only following a lag of 30 min or more. The lag time was shortened by raising the temperature, by reducing the salt concentration or by treating with glutaraldehyde. The induced sensitivity to the enzyme was inhibited by restoring physiologic ionic strength or by introducing 0.1 mol lysophosphatidylcholine per mol cholesterol into the membrane. (In striking contrast, lysophosphatidylethanolamine and lysophosphatidylserine did not inhibit oxidation). The various effectors of cholesterol oxidase sensitivity strongly influenced the impact of the others, suggesting that each shifted cholesterol toward or away from an enzyme-sensitive disposition. None of these effects was observed in pure cholesterol or red cell membrane lipids dissolved in detergent, which were uniformly highly reactive with the enzyme. We conclude that the observed variation in cholesterol oxidase sensitivity reflects changes in the organization of the bilayer, perhaps a lateral redistribution of lipids which creates cholesterol-rich phases or domains in which cholesterol is more or less accessible to the enzyme. If so, the time-dependent increase in cholesterol susceptibility during warming at low ionic strength might be a novel indicator of the kinetics of phase changes in the bilayer of the red cell.     

Modulation of sarcoplasmic reticulum Ca2+-pump activity by membrane fluidity

Intramolecular excimerization of 1,3-di-1-pyrenylpropane [Py(3)Py] was used to assess the fluidity of sarcoplasmic reticulum membranes (SR); on the basis of the spectral data, the probe incorporates completely inside the membrane probably somewhere close to the polar head groups of phospholipid molecules, however not in the very hydrophobic core. The excimerization rate is very sensitive to lipid phase transitions, as revealed by thermal profiles of dimyristoyl-phosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) bilayers. Cholesterol abolishes pretransitions and broadens the thermal profiles of the main transitions which vanish completely at 50 mol % sterol. Excimer formation in liposomes of SR total lipid extracts does not show any sharp transitions, as in the case of DMPC and DPPC. However, the plots display discontinuities at about 20 degrees C which are broadened by cholesterol and not observed at 50 mol % sterol. Also cholesterol has been incorporated in native SR membranes by an exchange technique allowing progressive enrichment without changing the phospholipid/protein molar ratio. As in liposomes, discontinuities of excimer formation at 20 degrees C are broadened by cholesterol enrichment. The full activity of uncoupled Ca2+-ATPase is only affected by cholesterol above a molar ratio to phospholipid of 0.4. However, a significant decrease in activity (about 20%) is only noticed at a ratio of 0.6 (the highest technically achieved); at this ratio, about 28 lipid molecules per Ca2+-ATPase are expected to be relatively free from cholesterol interaction. The vesicle structure is still intact at this high ratio, as judged from the absence of basal activity (not Ca2+ stimulated).(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2-3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.     

Alteration of plasma and erythrocyte membrane lipid components in hyperthyroid rats

Cholesterol and phospholipid have been measured in plasma and erythrocyte membrane of hyperthyroid rats. It has been found that while the former is reduced by about 30% in plasma and increased by the same amount in erythrocyte membranes, on the contrary, the latter increases by 35% in both plasma and red cell membranes. It seems that when serum triiodothyronine increases, a major cholesterol transfer occurs from plasma to erythrocyte. In this way, by the concomitant phospholipid increase, it is possible to avoid an alteration of the cholesterol/phospholipid molar ratio in the red cells, thus preventing their abnormal function in hyperthyroid rats. The proposal is made that an additional reason for the plasma cholesterol decrease in hyperthyroid subjects can be attributable to a net transfer of this compound from plasma to erythrocyte.     

Toluene-induced alterations in rat synaptosomal membrane composition and function

Toluene is a widely used organic solvent that can produce acute central nervous system (CNS) effects. Since toluene reaches relatively high concentrations in the CNS and is extremely lipophilic, we investigated its effects on rat brain membrane composition and function. Toluene (1 g/kg, lh) did not alter total brain microsomal phospholipid (PL) or cholesterol (CL) content. However, synaptosomal PL was decreased (24%), while synaptosomal CL was unaltered. The PL/CL ratio, an indirect index of membrane fluidity, did not change, suggesting that toluene did not affect membrane fluidity. Fluorescence polarization studies employing 1,6-diphenyl-1,3,5-hexatriene (DPH) showed that toluene did not alter synaptosomal membrane fluidity after administration in vivo (1 g/kg) or in vitro (0.5 to 5.0 mM). Dose-response and time-course studies showed that toluene maximally decreased synaptosomal PL after 1 g/kg, 1 h. The dose-response and time-course studies also showed that the toluene-induced decreases in PL were a result of specific decreases in phosphatidylethanolamine (PE). Since PE was decreased, we assessed whether toluene altered synaptosomal membrane function by investigating phospholipid methylation, a reaction which uses PE as its initial substrate. Toluene decreased the incorporation of methyl groups into lipid when [³H]-methionine was used as the methyl donor, but did not affect methylation when [³H]-adenosylmethionine was the methyl donor. These data suggest that toluene-induced specific decreases in synaptosomal PE and inhibition of phospholipid methylation may alter normal synaptic function and play a critical role in the mechanism(s) of action of toluene's CNS effects.     

Dual effect of membrane cholesterol on simple and mediated transport processes in human erythrocytes

The influence of cholesterol on simple and facilitated transport processes across the membrane of intact human erythrocytes was studied after graded depletion or enrichment of membrane cholesterol by incubation of the cells in phospholipid or phospholipid/cholesterol suspensions.

1. 1. The carrier-mediated transfer of L-lactate and of L-arabinose proved to be enhanced by cholesterol. In the case of L-lactate, a decrease in K_m seems to be involved in this effect. In contrast, the self-exchange of SO₄²⁻, mediated by the inorganic anion-exchange system, and the simple diffusion of erythritol via the lipid phase of the membrane are inhibited by cholesterol.

2. 2. Reversibility of these two opposite effects of cholesterol was demonstrated by measurements on cells depleted again after cholesterol enrichment and enriched again after previous depletion.

3. 3. Certain phospholipids used for preparing the lipid dispersions that are required for cholesterol variation have effects on permeability of their own, due, for example, to traces of contaminants. A discrimination of such artifacts from the effects of cholesterol is only possible by demonstrating reversibility.

4. 4. The opposite effects of cholesterol on various facilitated transfer processes, which have a correlation in the opposite effects of other modifications of the membrane lipid phase (Deuticke, B., Grunze, M. and Haest, C.W.M. (1979) Alfred Benzon Symposium 14, Munksgaard, Copenhagen, in the press), are indicative of different types of lipid-protein interaction in the erythrocyte membrane.

Characterisation of myogenic cell membrane: II. Dynamic changes in membrane lipids during the differentiation of mouse C2 myoblast cells

Proliferating mouse C2 myoblast cells resist haemagglutinating virus of Japan, Sendai virus (HVJ) mediated cell fusion. However, differentiating C2 cells can be induced to fuse by HVJ, suggesting that the rigid membrane of C2 cells changes during the differentiation. To investigate this phenomenon, changes in membrane lipids which affect fluidity were examined. Membrane cholesterol gradually decreased with the differentiation of C2 cells. However, spontaneous fusion to form myotubes and artificial fusion induced by HVJ were both inhibited when the level of cholesterol was prevented from falling in the cell membrane. The membranes of differentiating C2 cells contained more unsaturated fatty acids than those of proliferating cells. Thus, when differentiating C2 cells were treated with stearate (a saturated fatty acid), they failed to form myotubes and were insensitive to HVJ-mediated fusion. Whereas, if proliferating C2 cells were given linolenate (an unsaturated fatty acid), they became capable of HVJ-induced fusion. These results indicate that differentiating C2 cells change their fusion sensitivity by decreasing cholesterol, probably at the same time as they increase the unsaturated fatty acid content of the cell membrane.     

Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2-3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.     

Evidence for a relationship between protein glycation and red blood cell membrane fluidity

This study examines the relationship between protein glycation and membrane fluidity in RBC membranes. Incubation of RBC membranes of healthy subjects with 25mM glucose or galactose at 37 degrees C induced a 38% (p less than 0.02) increase in protein glycation (using furosine determination by HPLC) and higher fluidity (p less than 0.05) in DPH polarization ratio). However, incubation of RBC membranes from diabetic subjects under the same conditions did not modify either membrane fluidity or protein glycation; protein glycation was above normal before incubation because of the high diabetic plasma glucose. There was no difference in the membrane fluidities of 21 healthy subjects and 32 diabetic subjects, despite a significantly elevated protein glycation in diabetics. Furthermore, there was no change with respect to age in either population. We conclude that other in vivo factors, such as membrane lipid changes (increase in CL/PL ratio) or formation of advanced Maillard products and peroxidation in the diabetic subjects, could be responsible for the difference between these in vitro results and the in vivo situation.     

Protein stability and the evolution of the cell membrane

Cholesterol has been shown to regulate the activity of several membrane proteins. Although this phenomenon represents an important factor in the regulation of ion homeostasis, insights are needed to fully understand the role of this lipid in cell function in order to better comprehend the effect of bilayer components upon membrane function. Since evolution has shaped the composition of the membrane bilayer, it becomes of interest to study these changes in parallel with the many functions of membranes such as ion transport. The present study employing a plasma membrane preparation obtained from calf ventricular muscle demonstrates that cholesterol partially inhibits the Ca(2+),Mg(2+)-ATPase as the catalytic function of the calcium pump, when incubation reaction temperatures are below 42 degrees C. In contrast, when incubation reaction temperatures are above 42 degrees C, cholesterol apparently promotes enzyme stabilization reflected in higher activity. Although the activation energy values for the enzyme are almost the same at ranges between 15 and 40 degrees C, the use of elevated temperatures promote higher enzyme inactivation rates in control than in cholesterol enriched membranes. Cholesterol apparently is promoting stabilization that in turn protects the enzyme against thermal inactivation. This protective effect is reflected in a decrease of inactivation rate values and energy released during enzyme catalysis. The modification of many membrane properties throughout million of years made it possible for new evolutionary driving forces to show themselves as new characteristics in eukaryotes such as the one discussed in this study, dealing with the presence of cholesterol in the cell membrane directly associated to the promotion of protein thermostability.     

Aging of the erythrocyte IV. Spin-label studies of membrane lipids,proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.