| For medical practitioners and the general public - Cholesterol Journal Article Catalog. |
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| Cholesterol diet-induced hyperlipidemia influences gene expression pattern of rat hearts: a DNA microarray study Puskas, L. G., Z. B. Nagy, et al. (2004), FEBS Lett 562(1-3): 99-104. Abstract: To profile gene expression patterns involved in the direct myocardial effect of cholesterol-enriched diet-induced hyperlipidemia, we monitored global gene expression changes by DNA microarray analysis of 3200 genes in rat hearts. Twenty-six genes exhibited significant up-regulation and 25 showed down-regulation in hearts of rats fed a 2% cholesterol-enriched diet for 8 weeks as compared to age-matched controls. The expression changes of 12 selected genes were also assessed by real-time quantitative polymerase chain reaction. Genes with altered expression in the heart due to hyperlipidemia included procollagen type III, cofilin/destrin, tensin, transcription repressor p66, synaptic vesicle protein 2B, Hsp86, chaperonin subunit 5epsilon, metallothionein, glutathione S-transferase, protein kinase C inhibitor, ATP synthase subunit c, creatine kinase, chloride intracellular channel 4, NADH oxidoreductase and dehydrogenase, fibronectin receptor beta chain, CD81 antigen, farnesyltransferase, calreticulin, disintegrin, p120 catenin, Smad7, etc. Although some of these genes have been suspected to be related to cardiovascular diseases, none of the genes has been previously shown to be involved in the mechanism of the cardiac effect of hyperlipidemia. |
| Cholesterol distribution between high-density-lipoprotein subfractions HDL2 and HDL3 determined in serum by discontinuous gradient gel electrophoresis Atger, V., D. Malon, et al. (1991), Clin Chem 37(7): 1149-52. Abstract: We used discontinuous gradients of polyacrylamide gel to determine the high-density-lipoprotein (HDL) subfractions HDL2 and HDL3 of serum lipoproteins. Serum (40 microL) prestained with Sudan Black was electrophoresed in cylindrical tubes over successive layers of 3.5%, 6%, 13%, and 17.5% acrylamide gels in a Tris.glycine buffer (3-4 h, 300 V). Very-low- (VLDL) and low-density lipoprotein (LDL) were retained by the 3.5% and 6% gels. HDL2 was concentrated at the interface between the 13% and 17.5% gels, and HDL3 migrated into the 17.5% gel. The distribution between HDL2 and HDL3 was obtained by densitometric scanning. Application of the respective percentages to HDL cholesterol assayed after phosphotung-state-Mg2+ precipitation of VLDL and LDL gave calculated concentrations of HDL2 and HDL3 cholesterol. The calculated values for HDL2 cholesterol were in excellent agreement with those for HDL2 isolated by ultracentrifugation (r = 0.920 for n = 120 sera; differences nonsignificant by Student's paired t-test). Besides being highly discriminating, the method is rapid, easily performed, and economical. |
| Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog Mukherjee, S., X. Zha, et al. (1998), Biophys J 75(4): 1915-25. Abstract: Cholesterol is an important constituent of most mammalian cell membranes and its concentration in various cellular membranes is tightly regulated. Although there is much information about cholesterol distribution and trafficking in cells, it is primarily derived from indirect measurements, and the results obtained using different approaches are often conflicting. A cholesterol analog that faithfully mimics the properties of cholesterol and can be followed in living cells would thus be very useful. In this study, we report the fluorescence imaging of such an analog, dehydroergosterol (DHE), in living cells. DHE differs from cholesterol in having three additional double bonds and an extra methyl group. In model systems, DHE closely mimics the behavior of native cholesterol. Using triple-labeling studies, we show that DHE colocalizes extensively with endocytosed transferrin, an endocytic recycling compartment marker, and with a marker for the trans-Golgi network, Tac-TGN38. This distribution of DHE is qualitatively similar to that observed when cells are labeled with the fluorescent cholesterol-binding polyene antibiotic, filipin, although there are differences in apparent proportions of DHE and filipin that are localized at the plasma membrane. Another cholesterol derivative, 25-NBD-cholesterol, has a structure that is compromised by the presence of a bulky NBD group and does not distribute to the same organelles as DHE or filipin. In addition, we show in this manuscript that kinetic processes can be followed in living cells by monitoring recovery of DHE fluorescence in a photobleached region over time. Our observations provide evidence for the presence of a large intracellular cholesterol pool in the endocytic recycling compartment and the trans-Golgi network that might play important roles in the trafficking of lipids, lipid-anchored proteins, and transmembrane proteins that preferentially partition into cholesterol-enriched membrane domains. In addition, this intracellular cholesterol pool might be involved in the maintenance of cellular cholesterol homeostasis. |
| Cholesterol distribution in rat heart myocytes Shmeeda, H., D. Petkova, et al. (1995), Am J Physiol 268(2 Pt 2): H759-66. Abstract: Cholesterol oxidase was used to investigate the distribution of free cholesterol between plasma membrane and intracellular pools in cultured neonatal rat heart myocytes. Only 20% of the total unesterified cholesterol was converted to delta 4-cholestenone by cholesterol oxidase in intact cells. With increasing age in culture and concurrent hypertrophy, there was an increase in unesterified cellular cholesterol and plasma membrane cholesterol; their relative distribution remained unchanged. Electron micrographs of negatively stained samples of day 4 cytosol revealed the presence of vesicles 50-200 nm in diameter. Cholesterol monohydrate crystals were found in the cytosol of hypertrophic day 14 cells. Treatment of day 14 cells with small unilamellar vesicles of egg phosphatidylcholine reduced plasma membrane and intracellular cholesterol levels, resulting in the disappearance of the cholesterol monohydrate crystals and the formation of vesicles smaller than those observed in day 4 cultures. |
| Cholesterol distribution in rat liver and brain mitochondria as determined by stopped-flow kinetics with filipin Cremel, G., D. Filliol, et al. (1990), Arch Biochem Biophys 278(1): 142-7. Abstract: Recently, analysis of protein distribution in rat brain mitochondria suggested the existence of distinct cholesterol domains in the outer membrane (Dorbani et al., 1987, Arch. Biochem. Biophys. 252, 188-196) while such domains were not detected in rat liver mitochondria (Jancsik et al., 1988, Arch. Biochem. Biophys. 264, 295-301). We studied cholesterol distribution in both types of mitochondria by analyzing the kinetics of filipin-cholesterol complex formation, using the stopped-flow technique. In liver mitochondria, the kinetics are characterized by a biphasic curve which presumably corresponds to the two membranes. This was confirmed by the finding that pretreatment with digitonin abolished one of the kinetic components. Sonication of the mitochondria increased the rate of the filipin-cholesterol complex formation and also abolished one of the two components. In the case of brain mitochondria, several distinct cholesterol domains could be revealed: one of them was cholesterol-free and it was directly accessible to filipin. Two other domains were revealed by differences found in the rate of the cholesterol-filipin complex formation. It is noteworthy that only a part of the cholesterol is accessible to filipin. Sonication of mitochondria decreased the proportion of cholesterol molecules accessible to filipin. This suggests specific interactions of cholesterol with other mitochondrial components, which occur only in brain mitochondria. |
| Cholesterol distribution in renal epithelial cells LLC-PK1 as determined by cholesterol oxidase: evidence that glutaraldehyde fixation masks plasma membrane cholesterol pools el Yandouzi, E. H., P. Zlatkine, et al. (1994), Biochemistry 33(8): 2329-34. Abstract: Treatment with cholesterol oxidases has shown that cholesterol is heterogeneously distributed in brush border membranes isolated from the apical domain of the renal and intestinal epithelial cells Bloj, B., & Zilversmit, D. B. (1982) J. Biol. Chem. 257, 7608-7614; El Yandouzi, E. H., & Le Grimellec, C. (1992) Biochemistry 31, 547-551. Cholesterol distribution between plasma membrane and intracellular membranes of the corresponding cells remains unexplored. The effects of Brevibacterium sp. cholesterol oxidase on the cholesterol content of LLC-PK1 cells, an epithelial cell line with multiple differentiated characteristics of the renal proximal tubule, were investigated. In confluent living cells grown as a monolayer on solid support, a small but significant fraction (13%) of the cholesterol was oxidized during the first hour of the oxidase treatment. Glutaraldehyde fixation prior to treatment resulted in a nearly complete (86.1 +/- 1.8) oxidation of the cellular cholesterol according to first-order kinetics. Filipin labeling and oxidation at 15 degrees C confirmed that cholesterol was essentially confined to the plasma membrane in LLC-PK1 cells. When adding the oxidase either on the apical or on the basolateral side of cells grown on permeant support and fixed with glutaraldehyde, a comparable monophasic oxidation of cholesterol was observed, despite the presence of efficient tight junctions. Adding the oxidase to both sides simultaneously did not increase the rate of oxidation. Finally, fixation of isolated renal brush border membranes with glutaraldehyde rendered undiscernible their cholesterol pools. We conclude that glutaraldehyde fixation, a commonly used process in the analysis of cholesterol distribution in cells, can mask the existence of cholesterol pools in plasma membranes. |
| Cholesterol distribution in the Golgi complex of DITNC1 astrocytes is differentially altered by fresh and aged amyloid beta-peptide-(1-42) Igbavboa, U., J. M. Pidcock, et al. (2003), J Biol Chem 278(19): 17150-7. Abstract: The Golgi complex plays an important role in cholesterol trafficking in cells, and amyloid beta-peptides (Abetas) alter cholesterol trafficking. The hypothesis was tested that fresh and aged Abeta-(1-42) would differentially modify Golgi cholesterol content in DINTC1 astrocytes and that the effects of Abeta-(1-42) would be associated with the region of the Golgi complex. Two different methods were used to determine the effects of Abeta-(1-42) on Golgi complex cholesterol. Confocal microscopy showed that fresh Abeta-(1-42) significantly increased cholesterol and that aged Abeta-(1-42) significantly reduced cholesterol content in the Golgi complex. Isolation of the Golgi complex into two fractions using density gradient centrifugation showed effects of aged Abeta-(1-42) similar to those observed with confocal microscopy but revealed the novel finding that fresh Abeta-(1-42) had opposite effects on the two Golgi fractions suggesting a specificity of Abeta-(1-42) perturbation of the Golgi complex. Phosphatidylcholine-phospholipase D activity, cell membrane cholesterol, and apolipoprotein E levels were associated with effects of fresh Abeta-(1-42) on cholesterol distribution but not with effects of aged Abeta-(1-42), arguing against a common mechanism. Extracellular Abeta-(1-42) targets the Golgi complex and disrupts cell cholesterol homeostasis, and this action of Abeta-(1-42) could alter cell functions requiring optimal levels of cholesterol. |
| Cholesterol does not affect the toxicity of amyloid beta fragment but mimics its effect on MTT formazan exocytosis in cultured rat hippocampal neurons Abe, K. and H. Saito (1999), Neurosci Res 35(3): 165-74. Abstract: It has recently been reported that methyl-beta-cyclodextrin-solubilized cholesterol protects PC12 cells from amyloid beta protein (Abeta) toxicity. To ask if this is the case in brain neurons, we investigated its effect in primary cultured rat hippocampal neurons. In basal culture conditions with no addition of Abeta, methyl-beta-cyclodextrin-solubilized cholesterol at concentrations of 30-100 microM was toxic to neurons, but at concentrations of 1-10 microM promoted neuronal survival. Methyl-beta-cyclodextrin-solubilized cholesterol at 1-10 microM was also effective in protecting neurons from toxicity of 20 microM Abeta. However, these effects were all mimicked by methyl-beta-cyclodextrin alone, but not by cholesterol solubilized by dimethylsulfoxide or ethanol. The effects of methyl-beta-cyclodextrin-solubilized cholesterol on neuronal survival and Abeta toxicity are probably attributed to the action of methyl-beta-cyclodextrin, but not cholesterol. Alternatively, we found that methyl-beta-cyclodextrin-solubilized cholesterol at lower concentrations (> 10 nM) inhibited cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) by promoting the exocytosis of MTT formazan. This effect was shared by dimethylsulfoxide- or ethanol-solubilized cholesterol, but not by methyl-beta-cyclodextrin, supporting that it is attributed to the action of cholesterol. These results suggest that cholesterol does not protect neurons from Abeta toxicity, or rather inhibits cellular MTT reduction in a similar manner to Abeta. |
| Cholesterol does not induce segregation of liquid-ordered domains in bilayers modeling the inner leaflet of the plasma membrane Wang, T. Y. and J. R. Silvius (2001), Biophys J 81(5): 2762-73. Abstract: A fluorescence-quenching method has been used to assess the potential formation of segregated liquid-ordered domains in lipid bilayers combining cholesterol with mixtures of amino and choline phospholipids like those found in the cytoplasmic leaflet of the mammalian cell plasma membrane. When present in proportions >20-30 mol %, different saturated phospholipids show a strong proclivity to form segregated domains when combined with unsaturated phospholipids and cholesterol, in a manner that is only weakly affected by the nature of the phospholipid headgroups. By contrast, mixtures containing purely unsaturated phospholipids and cholesterol do not exhibit detectable segregation of domains, even in systems whose components differ in headgroup structure, mono- versus polyunsaturation and/or acyl chain heterogeneity. These results indicate that mixtures of phospholipids resembling those found in the inner leaflet of the plasma membrane do not spontaneously form segregated liquid-ordered domains. Instead, our findings suggest that factors extrinsic to the inner-monolayer lipids themselves (e.g., transbilayer penetration of long sphingolipid acyl chains) would be essential to confer a distinctive, more highly ordered organization to the cytoplasmic leaflet of "lipid raft" structures in animal cell membranes. |
| Cholesterol does not remove the gel-liquid crystalline phase transition of phosphatidylcholines containing two polyenoic acyl chains Kariel, N., E. Davidson, et al. (1991), Biochim Biophys Acta 1062(1): 70-6. Abstract: Homoacid (single-acid) phosphatidylcholines containing two linoleate (18:2), arachidonate (20:4), or docosahexaenoate (22:6) chains were dispersed in water to form multilamellar vesicles. The influence of cholesterol on the gel to liquid phase transitions was studied by differential scanning calorimetry (DSC). The pure dipolyenoic phosphatidylcholines exhibited very broad endothermic transitions (widths of 28 to 38 C degrees at scanning rates of 5 C degrees/min) in the temperature range of about -80 degrees C to -30 degrees C. The mixing of cholesterol into the phospholipids in proportions up to 50 mol% had little effect on the temperatures, widths or enthalpy changes associated with the phase transitions. The data suggest that cholesterol does not interfere with the packing of these lipids in the gel state, possibly because the packing is already irregular or because the solubility of cholesterol in these lipids is low, or both. |
| Cholesterol domains in biological membranes Schroeder, F., J. K. Woodford, et al. (1995), Mol Membr Biol 12(1): 113-9. Abstract: Membrane cholesterol is distributed asymmetrically both within the cell or within cellular membranes. Elaboration of intracellular cholesterol trafficking, targeting and intramembrane distribution has been spurred by both molecular and structural approaches. The expression of recombinant sterol carrier proteins in L-cell fibroblasts has been especially useful in demonstrating for the first time that such proteins actually elicit intracellular and intraplasma membrane redistribution of sterol. Additional advances in the use of native fluorescent sterols allowed resolution of transbilayer and lateral cholesterol domains in plasma membranes from cultured fibroblasts, brain synaptosomes and erythrocytes. In all three cell surface membranes, cholesterol is enriched in the inner, cytofacial leaflet. Up to three different cholesterol domains have been identified in the lateral plane of the plasma membrane: a fast exchanging domain comprising less than 10% of cholesterol, a slowly exchanging domain comprising about 30% of cholesterol, and a very slowly or non-exchangeable sterol domain comprising 50-60% of plasma membrane cholesterol. Factors modulating plasma membrane cholesterol domains include polyunsaturated fatty acids, expression of intracellular sterol carrier proteins, drugs such as ethanol, and several membrane pathologies (systemic lupus erythematosus, sickle cell anaemia and aging). Disturbances in plasma membrane cholesterol domains alter transbilayer fluidity gradients in plasma membranes. Such changes are associated with decreased Ca(2+)-ATPase and Na+, K(+)-ATPase activity. Thus, the size, dynamics and distribution of cholesterol domains within membranes not only regulate cholesterol efflux/influx but also modulate plasma membrane protein functions and receptor-effector coupled systems. |
| Cholesterol domains regulate the actin cytoskeleton at the leading edge of moving cells Manes, S. and A. C. Martinez (2004), Trends Cell Biol 14(6): 275-8. |
| Cholesterol dynamics in membranes Yeagle, P. L., A. D. Albert, et al. (1990), Biophys J 57(3): 413-24. Abstract: Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of 13C4 labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of 13C4 labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior. |
| Cholesterol dynamics in membranes of raft composition: a molecular point of view from 2H and 31P solid-state NMR Aussenac, F., M. Tavares, et al. (2003), Biochemistry 42(6): 1383-90. Abstract: Lipidic membrane systems that have been reported to be composed of sphingomyelin (SM)-cholesterol (Chol) microdomains or "rafts" by Dietrich et al. palmitoyloleoyl-phosphatidylcholine(POPC)/SM/Chol, 1/1/1; Dietrich, C., Bagatolli, L. A., Volovyk, Z. N., Thompson, N. L., Levi, M., Jacobson, K., and Gratton, E. (2001) Biophys. J. 80, 1417-1428 and by Schroeder et al. SCRL: Liver-PC/Liver-phosphatidylethanolamine/SM/Cerebrosides/Chol, 1/1/1/1/2; Schroeder, R., London, E., and Brown, D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 12130-12134 were investigated under the form of fully hydrated liposomes by the noninvasive solid-state (31)P and (2)H NMR method. Liposomes of binary lipid composition POPC/Chol and SM/Chol were also studied as boundary/control systems. All systems are found to be in the liquid-ordered phase (Lo) at physiological temperatures. Use of deuterium-labeled cholesterol afforded finding both the position of the sterol motional axis and its molecular order parameter. The axis of anisotropic rotation of cholesterol is such that the molecule is, on average, quasiperpendicular to the membrane plane, in all of the four systems investigated. Cholesterol order parameters greater than 0.8 are observed, indicating that the sterol is in a very motionally restricted environment in the temperature range 0-60 degrees C. The binary mixtures present "boundary" situations with the lowest values for POPC/Chol and the highest for SM/Chol. The SCRL raft mixture has the same ordering as the SM/Chol, i.e., the highest order parameter values over the temperature range. It demonstrates that in the SCRL mixture cholesterol dynamics is as in the binary system SM/Chol, therefore, suggesting that it might be depleted from the rest of the membrane to form complexes as if it were alone with SM. On the other hand, the mixture POPC/SM/Chol exhibits an intermediate ordering situation between those of SM/Chol and POPC/Chol. This strongly suggests that cholesterol could be in fast exchange, at the NMR time scale (milli- to microseconds), between two or more membrane regions of different dynamics and questions the statement of "rigid domains" made of SM and cholesterol in the model "raft" system POPC/SM/Chol. |
| Cholesterol dynamics in the foetal and neonatal brain as reflected by circulatory levels of 24S-hydroxycholesterol Lutjohann, D., I. Bjorkhem, et al. (2001), Acta Paediatr 90(6): 652-7. Abstract: Oxysterols, particularly those hydroxylated in the steroid side-chain, are formed from cholesterol by specific cytochrome P450 enzymes and may facilitate elimination of cholesterol from extrahepatic sources. In humans, the greatest portion of circulating 24S-hydroxycholesterol (24S-OH-Chol) is derived from the brain and the absolute concentration depends on age. In the present study, concentrations of 24S-OH-Chol and for comparison 27-OH-Chol were determined by a highly sensitive isotope dilution method using gas chromatography-mass spectrometry in serum samples from normal preterm and term neonates and those with Rhesus haemolytic disease, taken serially for diagnostic purposes. Serum concentrations of cholesterol, 24S-OH-Chol and 27-OH-Chol were similar in venous versus arterial cord blood of 6 term neonates. Serum concentrations of 24S-OH-Chol and 27-OH-Chol in 12 small for gestational age (SGA) preterm neonates were significantly lower than those in 12 appropriate for gestational age (AGA) preterm neonates (p < 0.001), and also lower than those in 12 SGA (0 < 0.001) and 12 AGA term neonates (p < 0.05). Serum cholesterol was significantly higher in preterm than in term neonates (p < 0.001). 24S-OH-Chol serially determined in 8 infants with Rhesus haemolytic disease increased 5-6-fold during the first 3 mo after birth (from 42 +/- 20 ng ml(-1) to 227 +/- 71 ng ml(-1)). 27-OH-Chol increased simultaneously from 30 +/- 14 ng ml(-1) to 100 +/- 39 ng ml(-1). Conclusion: Serum concentrations of 24S-OH-Chol increased 5-6-fold after birth. This could be an indication of normal cholesterol metabolism in the developing neonatal brain. |
| Cholesterol effect on the physical state of lipid multibilayers from the platelet plasma membrane by time-resolved fluorescence Velez, M., M. P. Lillo, et al. (1995), Biochim Biophys Acta 1235(2): 343-50. Abstract: There are indications that the plasma membrane lipid composition and, in particular, the cholesterol/phospholipid (C/PL) ratio, affects platelet function. As a first approximation to the molecular characterization of the effect of cholesterol on the order, fluidity and lateral heterogeneity of the platelet plasma membrane, the steady-state and time-resolved fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH) and trans-parinaric acid (tPnA) has been studied in multibilayer vesicles of phospholipids extracted from human platelet plasma membrane with different cholesterol/phospholipid molar ratios modified in vitro from 0.07 to 0.9. The DPH studies show that the increased presence of cholesterol has a stronger effect on the order than on the fluidity of the bilayer, as has been previously observed in other lipid membranes. On the other hand, from the analysis of the fluorescence kinetics of tPnA we conclude that a higher cholesterol content gives rise to an increase of the heterogeneity of the bilayer, due to a larger fraction of solid-like lipid domains. These domains contain a cholesterol concentration much higher than the macroscopic average value. |
| Cholesterol effect on thermostability of the (Ca2+, Mg2+)-ATPase from cardiac muscle sarcolemma Mas-Oliva, J. and J. Santiago-Garcia (1990), Biochem Int 21(2): 233-41. Abstract: When the cholesterol concentration in the sarcolemmal system is raised, the (Ca2+,Mg2+)-ATPase activity acquires an important degree of thermostability; phenomena that is completely lost if the experiment is carried out with cholesterol depleted sarcolemma. In this system, a gradual depletion of sarcolemmal cholesterol, renders the ATPase remarkably sensitive to temperature. At different concentrations of ATP, it is found that cholesterol affects the Vmax of the (Ca2+,Mg2+)-ATPase but not its Km. These results support our earlier suggestion of a direct effect of cholesterol upon the enzyme, and opens a possible mode of action of cholesterol on the enzyme. It is suggested that the inverse relationship between catalysis and thermostability is due to differences in the flexibility of the enzyme directly related to hydrophobicity changes caused by cholesterol. |
| Cholesterol effects on nonelectrolyte membrane transport in human erythrocytes: NMR magnetization transfer studies Nouri-Sorkhabi, M. H., B. E. Chapman, et al. (1994), Magn Reson Med 32(4): 505-10. Abstract: The cholesterol content of human erythrocytes was altered by incubating them with sonicated dispersions of cholesterol/phosphatidylcholine at 37 degrees C. 31P NMR saturation transfer experiments were used to measure the rate constant for efflux of dimethyl methylphosphonate (DMMP) from the cells, and thereby gain an estimate of the permeability coefficient. It was shown that up to 39% depletion of membrane cholesterol (cholesterol/phospholipid molar ratio of 0.46) increased the efflux rate constant and permeability coefficient of DMMP 1.55- and 1.86-fold, respectively. Enrichment of the membranes with cholesterol by 45% (cholesterol/phospholipid molar ratio of 1.57) on the other hand, decreased the efflux rate constant and permeability coefficient 1.63- and 1.79-fold, respectively. It was concluded that DMMP may be used as a probe molecule to study the functional consequences of changes in the lipid composition of erythrocytes in diseases that are associated with disorders of lipid metabolism. |
| Cholesterol effects on the phosphatidylcholine bilayer nonpolar region: a molecular simulation study Rog, T. and M. Pasenkiewicz-Gierula (2001), Biophys J 81(4): 2190-202. Abstract: A 15-ns molecular dynamics (MD) simulation of the fully hydrated dimyristoylphosphatidylcholine-cholesterol (DMPC-Chol) bilayer in the liquid-crystalline state was carried out to investigate the effect of Chol on the hydrocarbon chain region of the bilayer. The last 8-ns fragment of the generated trajectory was used for analyses. As a reference system, a pure DMPC bilayer (M. Pasenkiewicz-Gierula, Y. Takaoka, H. Miyagawa, K. Kitamura, and A. Kusumi, 1999, Biophys. J. 76:1228-1240) simulated for 14 ns was used. The study shows that a Chol-induced increase of the bulk molecular order parameter along both beta- and gamma-chain is mainly caused by a decrease of the average tilt of the chains, because the bulk average number of gauche rotamers/myristoyl chain is not significantly changed by Chol. Nevertheless, for DMPCs located near Chol molecules both the number of gauche rotamers/chain and the chain tilt are decreased. The magnitude of the Chol effect on the PC alkyl chains depends, in addition to the PC-Chol distance, on the side of the Chol molecule (alpha- or beta-face) that the chains are in contact with. This study provides some new insight into the properties of the coexistence region of the partial phase diagram for DMPC-Chol bilayers. |
| Cholesterol effects on the phosphatidylcholine bilayer polar region: a molecular simulation study Pasenkiewicz-Gierula, M., T. Rog, et al. (2000), Biophys J 78(3): 1376-89. Abstract: A molecular dynamics (MD) simulation of a fully hydrated, liquid-crystalline dimyristoylphosphatidylcholine (DMPC)-Chol bilayer membrane containing approximately 22 mol% Chol was carried out for 4.3 ns. The bilayer reached thermal equilibrium after 2.3 ns of MD simulation. A 2.0-ns trajectory generated during 2.3-4.3 ns of MD simulation was used for analyses to determine the effects of Chol on the membrane/water interfacial region. In this region, 70% of Chol molecules are linked to DMPC molecules via short-distance interactions, where the Chol hydroxyl group (OH-Chol) is 1) charge paired to methyl groups of the DMPC choline moiety (approximately 34%), via the hydroxyl oxygen atom (Och); 2) water bridged to carbonyl (approximately 19%) and nonester phosphate (approximately 14%) oxygen atoms, via both Och and the hydroxyl hydrogen atom (Hch); and 3) directly hydrogen (H) bonded to carbonyl (approximately 11%) and nonester phosphate (approximately 5%) oxygen atoms, via Hch (approximately 17% of DMPC-Chol links are multiple). DMPC's gamma-chain carbonyl oxygen atom is involved in 44% of water bridges and 51% of direct H bonds formed between DMPC and Chol. On average, a Chol molecule forms 0.9 links with DMPC molecules, while a DMPC molecule forms 2.2 and 0.3 links with DMPC and Chol molecules, respectively. OH-Chol makes hydrogen bonds with 1.1 water molecules, preferentially via Hch. The average number of water molecules H bonded to the DMPC headgroup is increased by 7% in the presence of Chol. These results indicate that inclusion of Chol decreases interlipid links and increases hydration in the polar region of the membrane. |