Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M303567200 on August 28, 2003

J. Biol. Chem., Vol. 278, Issue 46, 45563-45569, November 14, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/46/45563    most recent
M303567200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Scheidt, H. A.
Right arrow Articles by Huster, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Scheidt, H. A.
Right arrow Articles by Huster, D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Potential of Fluorescent and Spin-labeled Steroid Analogs to Mimic Natural Cholesterol*

Holger A. Scheidt{ddagger}, Peter Müller§, Andreas Herrmann§, and Daniel Huster{ddagger}

From the {ddagger}Junior Research Group, Solid-state NMR Studies of Membrane-associated Proteins, Biotechnological-Biomedical Center/Institute of Medical Physics and Biophysics, University of Leipzig, Liebigstr. 27, D-04103 Leipzig, Germany and the §Humboldt-University Berlin, Institute of Biology/Biophysics, Invalidenstr. 42, D-10115 Berlin, Germany

Received for publication, April 7, 2003 , and in revised form, August 21, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Cholesterol analogs are often used to investigate lipid trafficking and membrane organization of native cholesterol. Here, the potential of various spin (doxyl moiety) and fluorescent (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group) labeled cholesterol analogs as well as of fluorescent cholestatrienol and the naturally occurring dehydroergosterol to mimic the unique properties of native cholesterol in lipid membranes was studied in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes by electron paramagnetic resonance, nuclear magnetic resonance, and fluorescence spectroscopy. As cholesterol, all analogs undergo fluctuating motions of large amplitude parallel to the bilayer normal. Native cholesterol keeps a strict orientation in the membrane with the long axis parallel to the bilayer normal. Depending on the chemical modification or the position of the label, cholesterol analogs may adopt an "up-side-down" orientation in the membrane or may even fluctuate between "upright" and up-side-down orientation by rotational motions about the short axis not typical for native cholesterol. Those analogs are not able to induce a comparable condensation of phospholipid membranes as known for native cholesterol revealed by 2H nuclear magnetic resonance. However, cholesterol-induced lipid condensation is one of the key properties of native cholesterol, and, therefore, a well suited parameter to assess the potential of steroid analogs to mimic cholesterol. The study points to extreme caution when studying cholesterol behavior by the respective analogs. Among seven analogs investigated, only a spin-labeled cholesterol with the doxyl group at the end of the acyl chain and the fluorophore cholestatrienol mimic cholesterol satisfactorily. Dehydroergosterol has a similar upright orientation as cholesterol and could be used at low concentration (about 1 mol %), at which its lower potential to enhance lipid packing density does not perturb membrane organization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Cholesterol constitutes a major component of mammalian cellular membranes. Its correct distribution among intracellular membranes and the plasma membrane is essential for the homeostasis of mammalian cells. Thus, intracellular trafficking plays a major role in the correct disposition of internalized cholesterol and in the regulation of cholesterol efflux (for a recent review, see Ref. 1). Furthermore, lateral and transbilayer organization of cholesterol in the plasma membrane determines membrane structure and dynamics. However, neither its intracellular pathways of trafficking nor its precise lateral organization, e.g. in cholesterol-enriched microdomains such as rafts (2-9), and transbilayer distribution between the two leaflets of biological membranes are well known.

To characterize intracellular trafficking and membrane organization of cholesterol, various fluorescent and spin-labeled cholesterol analogs have been employed bearing a probe group either at the polar end or at the short hydrocarbon chain of the molecule (10-15). Although spin-labeled analogs are useful for studying the bilayer organization and dynamics of cholesterol and its influence on membrane structure (15, 16), fluorescent analogs provide a tool to study intracellular trafficking of cholesterol by microscopy techniques (14, 17). However, bulky reporter groups may determine membrane properties of analogs. Therefore, steroid molecules of intrinsic fluorescence as dehydroergosterol (DHE)1 or cholestatrienol (CTL) have become popular cholesterol analogs (14, 17-24). DHE is a naturally occurring sterol comprising up to 20% of the total sterol in yeast. Its fluorescent-conjugated triene system leaves the 3-{beta}-hydroxyl group and the alkyl tail of the cholesterol backbone unperturbed. Nevertheless, DHE differs from cholesterol in having three additional double bonds and an extra methyl group, whereas CTL has only two additional double bonds compared with native cholesterol. Indeed, modifications of the cholesterol backbone and/or the presence of a reporter group may cause differences in the physico-chemical properties and thus in the behavior of analogs as compared with natural cholesterol (1, 23, 25, 26).

In previous studies some of these cholesterol analogs have also been studied by monolayer and permeability experiments. In these studies, the correlation between sterol structure and the influence of sterols on permeability and ordering effects in lipid membranes was investigated (27-30, 58). To set a basis for selecting appropriate spin-labeled and fluorescent analogs of cholesterol, we have studied the behavior of a variety of those analogs (Fig. 1) in lipid membranes in comparison to native cholesterol. By using NMR, fluorescence, and EPR spectroscopy we investigated in particular the bilayer orientation and dynamics of analogs and their influence on membrane structure and lateral lipid organization. In particular, sterol-induced phospholipid condensation measured by 2H NMR order parameters provides a common basis for the comparison of these analogs. Based on these data we can provide clear recommendations for the capacity of the various analogs to mimic natural cholesterol.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 1.
Chemical structure of the cholesterol analogs used in this study: native cholesterol (A), SL-cholestane (B), SL-cholesterol (C), SL-androstane (D), 22-NBD-cholesterol (E), 25-NBD-cholesterol (F), CTL (G), and DHE (H). On molecules A, G, and H, the methyl group at carbon position C-18, which is used for 1H NOESY measurements is marked with an asterisk.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d31), and 25-{N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino}-27-norcholesterol (25-NBD-cholesterol) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3{beta}-ol (22-NBD-cholesterol) and 1-palmitoyl-2-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]-sn-glycero-3-phosphocholine (C6-NBD-PC) were from Molecular Probes (Leiden, The Netherlands). Ergosta-5,7,9(11), 22-tetraen-3{beta}-ol (DHE), 3-doxyl-17{beta}-hydroxy-5{alpha}-cholestane (doxyl-androstane), and 3{beta}-doxyl-5{alpha}-cholestane (doxyl-cholestane) were purchased from Sigma. 25-doxyl-cholesterol and cholesta-5,7,9,(11)-trien-3{beta}-ol (CTL) were synthesized as described in Refs. 16 and 31, respectively. Purity of CTL and DHE was checked using MALDI-TOF mass spectrometry. All cholesterol analogs are shown in Fig. 1. The spin-labeled PC analog 1-palmitoyl-2-(4-doxylpentanoyl)-sn-glycero-3-phosphocholine (SL-PC) was synthesized as described in Ref. 32.

For NMR measurements, mixtures of phospholipids and cholesterol analogs were prepared in chloroform. After evaporating the chloroform under a stream of nitrogen, the samples were redissolved in cyclohexane and lyophilized. Samples were hydrated to 40 wt % D2O for 1H NMR or deuterium depleted H2O for 2H NMR measurements and equilibrated by freeze-thaw cycles and gentle centrifugation. Samples were transferred into 4-mm high resolution MAS rotors for 1H MAS NMR experiments or filled into 5-mm glass vials for static 2H NMR experiments.

For fluorescence or EPR experiments, lipid mixtures containing the respective analog after evaporating the chloroform were hydrated in phosphate-buffered saline (150 mM NaCl, 5.8 mM Na2HPO4/NaH2PO4, pH 7.4), and large unilamellar vesicles (LUV) were prepared by the extrusion method (33).

1H MAS NMR spectra were acquired on a DRX600 NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten) using a 4-mm high resolution MAS probe at a spinning speed of 10 kHz. Typical {pi}/2 pulse lengths were 8.5 µs. T1 relaxation times were measured using the inversion recovery pulse sequence with 13 delays between 1 ms and 4 s and a relaxation delay of 4 s. In case of spin-labeled analogs, due to the unpaired electron of the doxyl group a fast paramagnetic relaxation mechanism is introduced and the total relaxation rate R1 = 1/T1 is the sum of paramagnetic (R1,p) and dipolar relaxation rate (R1,d). After measuring R1,d from a sample of pure POPC, R1,p can be easily determined for each molecular segment of POPC from the total relaxation rate R1. Two-dimensional 1H MAS NOESY experiments of the sterol/POPC sample were carried out at various mixing times. Cross-peak volumes were integrated and NOE build-up curves were fitted to the spin pair model yielding cross-relaxation rates ({sigma}ij) as described in Ref. 34.

2H NMR spectra were recorded on a Bruker DRX300 NMR spectrometer at a resonance frequency of 46.1 MHz for 2H using a solids probe with a 5-mm solenoid coil. The 2H NMR spectra were accumulated using the quadrupolar echo sequence and a relaxation delay of 500 ms. The two 4.8 µs {pi}/2 pulses were separated by a 100 µs delay. 2H NMR spectra were depaked and order parameters for each methylene group in the chain were determined as described in detail in Ref. 35. All NMR spectra were acquired at a temperature of 30 °C.

For the reduction measurements of fluorescent and spin-labeled steroid analogs, LUV (lipid concentration 5 mM) containing NBD-labeled (2.5 µM) or spin-labeled (100 µM) analogues were mixed with dithionite (25 or 50 mM) or ascorbic acid (17 mM), respectively. Subsequently, the time-dependent evolution of signal intensities of fluorescence at 4 °C or of EPR at 30 °C (as taken from the height of the midfield peak) were followed. The signal intensities were related to the respective intensities before adding the reducing substance. Measurements were performed at an Aminco-Bowman Series 2 fluorescence spectrometer (Urbana, IL) and at a Bruker ECS 106 EPR spectrometer, respectively.

POPC, POPC/DHE, and POPC/CTL samples were checked for lipid oxidation over a time course of 24 h after preparation using MALDITOF mass spectrometry (36). No significant oxidation products were detected.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Transverse Distribution of Cholesterol Analogs in Membranes—To evaluate the potential of various analogs to mimic natural cholesterol first their orientation and transverse distribution along the membrane normal was determined and compared with native cholesterol. It is known that cholesterol molecules assume an upright position in the membrane with the polar OH group facing the aqueous phase (37). Due to the high mobility in the liquid crystalline phase, a distribution of the steroid parallel to the membrane normal has been observed and attributed to out of plane motions for the sterol (38). In the current study, the width of this distribution was determined by 1H MAS NOESY. Intermolecular cross-relaxation rates between cholesterol and phospholipid segments provide a measure for the strength of the interaction between molecular segments (39). As shown recently, the effects of spin diffusion do not contribute to the cross-relaxation process in liquid-crystalline membranes (40).

A characteristic NMR signal of the cholesterol moiety is the methyl group at carbon position C-18 (see Fig. 1). Quantitative analysis of the cholesterol-lipid cross-peaks, therefore, provides a measure of the interaction strength of the C-18 methyl protons with lipid segments and thus of the transverse distribution profile of the sterol in the membrane leaflet. In Fig. 2A, the cross-relaxation rates of these protons with various phospholipid segments are plotted. There is an interaction of the C-18 methyl group only with the phospholipid chains, indicating a transverse distribution of the molecule. The width of the distribution indicates relatively large fluctuations of cholesterol parallel to the bilayer normal. Such a broad distribution is in good agreement with the amplitude of out of plane motions of cholesterol of ~10 Å measured by neutron diffraction (38).



View larger version (67K):
[in this window]
[in a new window]
  		FIG. 2.
Cross-relaxation rates between the methyl group at the C-18 position of cholesterol (A), DHE (B), and CTL (C) with phospholipid signals and paramagnetic relaxation rates of POPC segments in the presence of 0.5 mol % SL-androstane (D), SL-cholestane (E), and SL-cholesterol (F). Cross-relaxation rates were determined from 1H MAS NOESY experiments at a sterol concentration of 20 mol %. Cross-relaxation between the methyl group C-18 and the chain termini could not be analyzed because of signal overlap with the methyl groups C-26/27. Paramagnetic relaxation rates were determined from T1 relaxation time measurements. All measurements were carried out at a water content of 40 wt % and a temperature of 30 °C.

 
For comparison, the distribution of DHE and CTL in POPC bilayers are also shown in Fig. 2, B and C. As for cholesterol, similar patterns of cross-relaxation rates only with the phospholipid chains are observed, suggesting that both DHE and CTL are embedded similarly in the POPC bilayer.

Next, we investigated the orientation and distribution of three doxyl-labeled cholesterol analogs in the membrane. Unpaired electrons as present in the nitroxide spin labels introduce a fast paramagnetic relaxation mechanism that dominates all other interactions. As with cross-relaxation rates, the paramagnetic relaxation rates can be interpreted as contact probabilities between the doxyl group and various lipid segments (41). In Fig. 2, D-F, paramagnetic relaxation rates for SL-androstane (D), SL-cholestane (E), and SL-cholesterol (F) are plotted. For all three SL analogs a broad distribution of the doxyl groups around their average position is found reflecting their high mobility in the membrane. Even contacts of the doxyl group with the phospholipid headgroups and the chain termini are observed, indicating also a high degree of molecular disorder in the phospholipid membrane.

The distribution of the doxyl group of SL-androstane (Fig. 2D) has a broad maximum in the lower region of the phospholipid chain, whereas the probability of the probe to be in the glycerol/headgroup region is smaller. This suggests that the molecule faces the membrane interior with the doxyl group and the aqueous phase with the OH group, which places the ring system of SL-androstane exactly opposite to natural cholesterol. From the width of the distribution a highly dynamic reorientation can be concluded, which may include even rotations of the molecule within one membrane leaflet (perpendicular to the membrane normal) as suggested in Ref. 16.

The doxyl group of SL-cholestane (Fig. 2E) is also broadly distributed with its maximum in the lipid/water interface (upper chain/glycerol/headgroup region) of the membrane. Thus, the orientation of SL-cholestane in the membrane resembles that of native cholesterol, but the width of the distribution suggests a more dynamic reorientation of the analog.

For SL-cholesterol (Fig. 2F) a much sharper distribution of the doxyl group is observed with a maximum at the chain termini of the phospholipids. This is very similar to the orientation of the native cholesterol molecule. The probability for an unusual up-side-down orientation is very small.

To corroborate these results, we carried out EPR reduction experiments measuring the accessibility of the water soluble ascorbate toward spin-labeled cholesterol analogs. For reference, the ascorbate-mediated reduction of the spin label moiety of the phospholipid analog SL-PC was assessed. The label moiety of this analog linked to the short fatty acid chain is localized at the polar head group region allowing a rapid reduction by ascorbate (42). Indeed, we found a rapid reduction of SL-PC localized in the external membrane leaflet (Fig. 3A). Because ascorbate does not partition into the hydrophobic phase, and the transbilayer movement of SL-PC is very slow (43), analogs in the inner leaflet are shielded from reduction, therefore, only 50% of the label is reduced. Consistently, the rapid reduction of their EPR signal intensity of SL-androstane and SL-cholestane after addition of ascorbate underlines the preferential orientation of the label moiety in the lipid/water interface observed by NMR. The complete loss of signal intensity observed can be explained either by a preferential localization of the analogs in the outer membrane leaflet or by their rapid transbilayer movement. However, there is no reason to assume that the analogs are enriched in the outer leaflet. Numerous studies have given indications for a rapid translocation of cholesterol across membranes on the time scale of seconds or minutes even at 4 °C (15, 44-46). In contrast to cholestane and androstane, we observed only a slight reduction of signal intensity of SL-cholesterol, which provides independent evidence for a localization of its doxyl group in the membrane interior and its cholesterol like orientation.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 3.
Reduction of signal intensity of spin-labeled (A) and fluorescent lipids (B) after addition of ascorbate and dithionite, respectively. A, POPC-LUV were labeled with SL-PC (), SL-androstane ({diamondsuit}), SL-cholestane ({blacktriangleleft}), or SL-cholesterol ({blacktriangleright}), and the decrease of EPR signal intensity was measured at 30 °C. Data represent the mean of two independent experiments. B, POPC-LUV were labeled with C6-NBD-PC (lines a and b) or 22-NBD-cholesterol (lines c and d), and the decrease of fluorescence intensity was measured at 30 °C(lines b and d) or at 4 °C (lines a and c).

 
Using a similar approach, i.e. by measuring the kinetics of dithionite-mediated quenching of NBD fluorescence at 30 °C, we determined the orientation of the NBD moiety of 22-NBD-cholesterol and 25-NBD-cholesterol in POPC-LUV (Fig. 3B). For reference, we also measured the reduction of C6-NBD-PC fluorescence by dithionite. As it was previously shown the short fatty acid residue of the analog bearing the NBD group loops back to the membrane surface (47, 48). Thereby the NBD group is exposed to the lipid/water interface and thus easily accessible to dithionite as demonstrated by the rapid reduction of C6-NBD-PC in the external membrane leaflet (Fig. 3B, line b). Because of the slow transbilayer movement of C6-NBD-PC and the negligible permeation of dithionite across the membrane, analogs in the inner leaflet are efficiently shielded from reduction.

For 22-NBD-cholesterol we observed a rapid and complete decline of fluorescence intensity upon addition of dithionite (Fig. 3B, line d) even at 4 °C (Fig. 3B, line c). This indicates that (i) the NBD moiety is preferentially localized in the polar interface and (ii) the analog has a high transbilayer mobility. A similar rapid reduction by dithionite was observed for 25-NBD-cholesterol (data not shown).

These results show that the orientation and the motion parallel to the membrane normal vary significantly between cholesterol analogs. Out of the seven analogs investigated, four molecules (SL-cholestane, SL-cholesterol, CTL, and DHE) are embedded into membranes with a similar orientation as natural cholesterol. SL-androstane and the NBD-labeled cholesterol analogs exhibit an up-side-down orientation in the membrane and, therefore, are not well suited as analogs for cholesterol.

Lipid Condensation in POPC/Cholesterol Analog Membranes—To further explore the potential of fluorescent or spin-labeled cholesterol analogs to mimic native cholesterol we studied packing properties of phospholipid/cholesterol analog mixtures. It is well known that cholesterol condenses lipid bilayers by reducing the area per phospholipid molecule in the membrane and increasing chain order parameters (35, 49, 50). This effect has been attributed to favorable van der Waals interactions between the steroid ring system and saturated phospholipid acyl chains (51). Because lipid condensation is a very unique property of cholesterol and strongly attenuated for other sterols (52), it is a valuable parameter to assess whether or not an analog has the same properties as native cholesterol.

Individual order parameters along the lipid chains can be calculated from the quadrupolar splittings of 2H NMR spectra of chain perdeuterated lipid molecules. In Fig. 4, order parameters for the palmitoyl chain in POPC in the absence and in the presence of 10 and 20 mol % cholesterol are plotted (black symbols). It is evident that the lipid chain order increases with increasing cholesterol concentration. In general, POPC chain order parameters in the presence of 20 mol % of the respective spin-labeled (Fig. 4A) and fluorescent cholesterol analogs (Fig. 4B) indicate a lower lipid condensation than for native cholesterol. Only for SL-cholesterol and CTL, lipid condensation is rather similar to that of cholesterol. At 20 mol %, DHE induces a lipid chain ordering comparable with less than 10 mol % cholesterol, whereas other analogs show an effect on the order of or less than that of 5 mol % cholesterol.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 4.
Smoothed order parameter profiles of POPC-d31 membranes in the presence of 20 mol % spin-labeled (A) and fluorescent (B) cholesterol analogs. SL-androstane ({diamondsuit}), SL-cholestane ({blacktriangleleft}), SL-cholesterol ({blacktriangleright}), 22-NBD-cholesterol (•), 25-NBD-cholesterol ({blacktriangleup}), DHE ({blacktriangledown}), and CTL ({star}). For comparisons order parameter plots for POPC-d31 membranes in the absence (X) and the presence of 10 mol % (+) and 20 mol % (*) cholesterol are also shown.

 
The same conclusion on the lipid condensing effect can be derived from plotting the data as the average order parameter of POPC bilayers (Fig. 5). The parameter correlates linearly with the concentration of cholesterol in the studied range from 0 to 20 mol %. For all analogs, the average order parameter is lower than that of native cholesterol as measured at 20 mol %. Assuming a linear relation between average order parameter and molar content of native cholesterol, the cholesterol concentration that has the same lipid condensation effect as 20 mol % of the respective analog can be calculated. Results given in Table I confirm that SL-cholesterol and CTL have the largest potential of all analogs to condense POPC bilayers and mimic the behavior of cholesterol best. Obviously, NBD-labeled cholesterols are poor analogs.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 5.
Average chain order parameter of POPC-d31 at varying cholesterol concentrations ({blacksquare}). The line represents a linear regression. The plot also shows POPC-d31 average order parameter in the presence of 20 mol % SL-androstane ({diamondsuit}), SL-cholestane ({blacktriangleleft}), and SL-cholesterol ({blacktriangleright}), 22-NBD-cholesterol (•), 25-NBD-cholesterol ({blacktriangleup}), DHE ({blacktriangledown}), and CTL (*).

 


View this table:
[in this window]
[in a new window]
  		TABLE I
Average order parameters of POPC-d31/cholesterol analog membranes (80/20, mol/mol) and the cholesterol concentration that induces such an order parameter in POPC membranes

 
Lipid Condensation in POPC/Cholesterol/DHE Membranes—Although we found a reduced potential of DHE to condense lipid bilayers, in the light of the useful properties of this analog for studying lipid trafficking we addressed whether DHE perturbs lipid condensation at concentrations typically used in those trafficking studies. Because fluorescence spectroscopy is a very sensitive method, only small concentrations of the label are necessary (typically about 1 mol % of the total lipids or even less). Therefore, we investigated lipid packing properties in POPC/cholesterol/DHE membranes at those low DHE concentrations. In Fig. 6, the average order parameter of POPC membranes is shown as a function of DHE concentration at a constant total sterol concentration of 20 mol % (cholesterol plus DHE). Within the experimental error, small concentrations of DHE have no influence on the lipid packing properties.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 6.
Average order parameters of the palmitoyl acyl chain in POPC-d31/cholesterol/DHE membranes as a function of the DHE concentration. The total sterol concentration (i.e. DHE plus cholesterol) in these preparations was 20 mol %.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
In our study, we compared the physico-chemical properties of various fluorescent and spin-labeled cholesterol analogs in lipid membranes with those of natural cholesterol. As is clearly shown, analogs differ significantly in their capacity to reflect the molecular topology, dynamics, and lipid-packing properties of natural cholesterol. Deviations from the behavior of cholesterol depend on the label moiety and its position on the steroid backbone as well as on the structure of the steroid backbone. Based on our approach, selection of appropriate analogs to study (intra)cellular trafficking and membrane organization of cholesterol can be achieved.

Sterol-induced Lipid Condensation Varies between the Analogs—Phospholipid condensation is a very specific effect of cholesterol. In particular, favorable van der Waals interactions of cholesterol with saturated chains lead to a strong increase of chain order accompanied by an extension of the lipid acyl chains and a reduction in lipid cross-sectional area and packing density in the membrane (51). Membranes of unsaturated lipids show a reduced area condensation in the presence of cholesterol (35). Therefore, measurements of cholesterol analog-induced lipid condensation by 2H NMR provide a precise and useful basis for the comparison and selection of appropriate cholesterol analogs.

Compared with cholesterol, only attenuated condensation has been observed for other naturally occurring sterols such as plant, yeast, or fungi sterols as well as their precursors (52-54). This is confirmed by our study on cholesterol analogs. None of the analogs produced a similarly strong lipid condensation as native cholesterol (Fig. 5). In terms of lipid condensation, SL-cholesterol and CTL are the most useful analogs of native cholesterol. Even though the bulky doxyl group supposedly disturbs lipid packing, SL-cholesterol has a strong potential to condense lipids. Both the orientation and the transverse dynamics of the analog in the membrane resemble those of natural cholesterol (see below). Because the doxyl group is inserted very deeply into the membrane, it can occupy some free volume, whereas the steroid ring system stabilizes lipid chains by van der Waals interactions. However, the presence of the doxyl group prevents stronger lipid condensation due to the packing imperfections.

In contrast, lipid condensation by SL-androstane and SL-cholestane is very low. Cholesterol and SL-cholestane differ by one double bond and the headgroup. The upright membrane orientation of the analog molecule places the bulky doxyl group in the lipid water interface of the membrane thereby preventing an increase in packing density. A similar mechanism accounts for the failure of SL-androstane to increase the lipid packing density because the location of the bulky doxyl group in the middle/lower chain region prevents lipid condensation. Consequently, these molecules experience more motional freedom in the bilayer compared with natural cholesterol (see below).

Similar to SL-androstane and SL-cholestane, the preferential localization of the charged and bulky NBD group of 22- and 25-NBD-cholesterol in the lipid water interface of the membrane separates lipid molecules laterally like a spacer, which lowers lipid chain order (48) or allows quick reorientation of the analogs in the bilayer. Accordingly, only a negligible lipid area condensation is measured for both molecules because the structural prerequisites for favorable van der Waals interactions are not fulfilled anymore.

CTL is the fluorescent cholesterol analog that mimics native cholesterol the best. However, the presence of just two double bonds in the ring structure of the molecule led to attenuated phospholipid condensation (Table I). This is in slight contrast to a monolayer and EPR study in which CTL was reported to produce the same POPC condensation as native cholesterol (30). However, we note that the direct 2H NMR measurement of the POPC chain order parameter represents a more direct and more precise approach than monolayer measurements or order parameters of doxyl-labeled free fatty acids. It has been noted before that the presence of additional double bonds in the sterol ring structure severely alters sterol lipid interactions (25, 26). Apparently, these modifications influence the van der Waals interactions between lipid chains and the sterol. Additional modifications of DHE such as the methyl group and the additional double bond in the chain region lead to further alterations of the lipid sterol interactions. At the moment it is not clear what exactly causes the specificity for interactions between cholesterol and lipid chains. Nevertheless, for DHE molecular modeling shows that the double bond in the chain of DHE causes kinks of phospholipid chains that may be responsible for the imperfect packing with lipids.

However, by investigating packing properties of POPC/cholesterol membranes in the presence of low DHE concentrations (about 1 mol %), the cholesterol-induced lipid packing is not significantly affected by DHE. Therefore, at the low DHE concentrations that are typically used to study, e.g. cholesterol trafficking in cells (14, 17), negligible perturbations of (sub)cellular membranes should be expected.

Cholesterol Analogs Differ in Their Membrane Orientation—Not only the lipid condensation of cholesterol analogs differs, some analogs show an unusual orientation in the lipid membrane. Because of its partially polar nature, cholesterol is embedded in the membrane with the hydrophilic OH headgroup facing the aqueous phase and with the acyl chain pointing toward the bilayer center for favorable hydrophilic and hydrophobic interactions (37). This orientation could be dramatically affected by the chemical nature of the analog. Although CTL, DHE, SL-cholestane, and SL-cholesterol are orientated upright like cholesterol, SL-androstane, 22-NBD-cholesterol, and 25-NBD-cholesterol show an up-side-down orientation in the membrane.

The additional double bonds of CTL and tail modifications of DHE with respect to cholesterol do not affect the dominating role of the polar hydroxyl group in determining the orientation in the membrane. For SL-cholesterol, the doxyl group is a rather bulky moiety with a volume of 142 Å3 representing a significant modification of the sterol molecule. Nevertheless, replacing the two terminal methyl groups of cholesterol (each has a volume of 54 Å3) by the doxyl group in SL-cholesterol does not alter the upright orientation of the analog in the membrane because of the hydrophobic properties of the doxyl group. The upright orientation is also preserved upon replacing the OH headgroup by the doxyl moiety as in SL-cholestane. However, the molecule is localized somewhat deeper in the membrane reflecting the less polar properties of the doxyl group.

For SL-androstane, the effect of structural differences with respect to native cholesterol are more severe. The OH head-group is replaced by the doxyl moiety and the lipid chain by a hydroxyl group. Thus, the polar center of the molecule is shifted to the opposite side of the steroid ring, which is the driving force for the up-side-down orientation in the membrane. For the NBD-cholesterol analogs, an up-side-down orientation in the membrane was also found. This is explained by the charged NBD group, which is more polar than the OH moiety and, therefore, has a high propensity toward the aqueous environment. Indeed, it has been shown that the NBD group has a strong preference for the lipid-water interface of the membrane. When attached to the acyl chain of a phospholipid analog, a looping back of this entire chain is caused such that the NBD moiety has access to the aqueous phase (47, 48). In contrast to our results, it has been reported that the fluorescent group of 22-NBD-cholesterol is actually more deeply buried within the bilayer (47). If this is correct, it seems likely that the NBD groups would be moving to the membrane surface with a sufficient frequency for rapid dithionite reduction. This would imply a high mobility of the NBD-cholesterol analogs that explains why these analogs only very weakly condense lipid bilayers.

The Motion in the Membrane Varies between the Analogs—Liquid crystalline membranes are composed of very flexible and highly dynamic lipid molecules (39, 56). Accordingly, cholesterol molecules are also subject to various types of motions (57). In particular, transverse motions of large amplitude have been observed for cholesterol by neutron scattering data (38). Therefore, the location of cholesterol segments parallel to the membrane normal is best described by broad distribution functions. The distribution of (i) doxyl groups covalently attached to the sterol backbone or (ii) the C-18 methyl group of fluorescent analogs was determined by (cross-)relaxation rate analysis. Because the cholesterol analogs are relatively rigid molecules these distribution profiles determined from a single point on the molecules provide a measure for the amplitude of the transverse cholesterol motions. Although the anisotropic motions of cholesterol are rather complex (22, 55, 57), from the different transverse motions the suitability of a given cholesterol analog can be assessed.

We found cholesterol as well as all analogs to undergo transverse motions of large amplitude in the membrane. For SL-cholesterol, CTL, and DHE, the probability for an up-side-down membrane topology is rather low. However, although SL-androstane and SL-cholestane show a preferred membrane orientation, they may also experience other topologies by rotation perpendicular to the membrane normal. The different probability for rotation might be related to the fact that SL-cholesterol, CTL, and DHE condense lipid bilayers, thereby increasing the lipid packing density, which in turn confines the molecules almost exclusively to the most favorable energetic (upright) orientation. In contrast, SL-androstane and SL-cholestane only negligibly order lipid chains, which gives these molecules more motional freedom to adopt different orientations. It has been suggested that SL-androstane may undergo rotational motions within one membrane leaflet (16).


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
Our approach of assessing physico-chemical properties of various steroid analogs in membranes provides a basis for selection of appropriate analogs to characterize membrane behavior and cellular trafficking of native cholesterol. SL-cholesterol and CTL prove to be very useful in studying orientation, motion, and protein-lipid as well as lipid-lipid interaction of cholesterol in membranes. With regard to native cholesterol, these analogs have a very similar membrane topology and dynamics, and compare relatively well in terms of lipid condensation. The commercially available DHE may also provide a helpful analog because its membrane orientation and dynamics are similar to that of cholesterol. However, because its capacity to condense lipid bilayers is much lower, it should replace only a small fraction of the natural cholesterol. In that case, the perturbation of membrane structure can be neglected. All other analogs should be used with caution. Our approach clearly shows that they do not qualify to specifically mimic the molecular behavior of native cholesterol. Regardless of structural similarities between the different steroids, cholesterol-lipid interactions appear to be very specific and unique. To find appropriate cholesterol analogs for structural and functional studies by EPR and fluorescence spectroscopy is challenging. It may even turn out that specific questions require other analogs/approaches, e.g. the use of isotopically labeled cholesterol molecules.


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft. The Junior Research Group is funded by the Saxon State Ministry of Higher Education, Research and Culture. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 49-341-97-15706; Fax: 49-341-97-15709; E-mail: husd{at}medizin.uni-leipzig.de.

1 The abbreviations used are: DHE, dehydroergosterol; CTL, cholestatrienol; PC, phosphatidylcholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPC-d31, 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; 25-NBD-cholesterol, 25-{N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino}-27-norcholesterol; 22-NBD-cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3{beta}-ol; C6-NBD-PC, 1-palmitoyl-2-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]-sn-glycero-3-phosphocholine; NOESY, nuclear Overhauser enhancement spectroscopy; NMR, nuclear magnetic resonance; EPR, electron paramagnetic resonance; LUV, large unilamellar vesicles; SL, spin label; MALDI-TOF, matrix-assisted laser desorption/ionization-time-of-flight; MAS, magic angle spinning. Back


   ACKNOWLEDGMENTS
 
We thank Sabine Schiller and Dr. Joachim Leistner (both Humboldt-University Berlin) for technical assistance and Rosemarie Sü{beta} for carrying out MALDI-TOF measurements. Valuable discussions with Dr. Jürgen Schiller are gratefully acknowledged.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

  1. Maxfield, F. R., and Wüstner, D. (2002) J. Clin. Invest. 110, 891-898[CrossRef][Medline] [Order article via Infotrieve]
  2. Simons, K., and Ikonen, E. (1997) Nature 387, 569-572[CrossRef][Medline] [Order article via Infotrieve]
  3. Brown, R. E. (1998) J. Cell Sci. 111, 1-9[Abstract]
  4. Dietrich, C., Bagatolli, L. A., Volovyk, Z. N., Thompson, N. L., Levi, M., Jacobson, K., and Gratton, E. (2001) Biophys. J. 80, 1417-1428[Medline] [Order article via Infotrieve]
  5. Brown, D. A., and London, E. (1998) J. Membr. Biol. 164, 103-114[CrossRef][Medline] [Order article via Infotrieve]
  6. London, E., and Brown, D. A. (2000) Biochim. Biophys. Acta 1508, 182-195[Medline] [Order article via Infotrieve]
  7. Anderson, R. G., and Jacobson, K. (2002) Science 296, 1821-1825[Abstract/Free Full Text]
  8. Xu, X., and London, E. (2000) Biochemistry 39, 843-849[CrossRef][Medline] [Order article via Infotrieve]
  9. Xu, X., Bittman, R., Duportail, G., Heissler, D., Vilcheze, C., and London, E. (2001) J. Biol. Chem. 276, 33540-33546[Abstract/Free Full Text]
  10. Boesze-Battaglia, K., Clayton, S. T., and Schimmel, R. J. (1996) Biochemistry 35, 6664-6673[CrossRef][Medline] [Order article via Infotrieve]
  11. Cheng, K. H., Virtanan, J., and Somerharju, P. (1999) Biophys. J. 77, 3108-3119[Medline] [Order article via Infotrieve]
  12. Hapala, I., Kavecansky, J., Butko, P., Scallen, T. J., Joiner, C. H., and Schroeder, F. (1994) Biochemistry 33, 7682-7690[CrossRef][Medline] [Order article via Infotrieve]
  13. Mukherjee, S., and Chattopadhyay, A. (1996) Biochemistry 35, 1311-1322[CrossRef][Medline] [Order article via Infotrieve]
  14. Mukherjee, S., Zha, X. H., Tabas, I., and Maxfield, F. R. (1998) Biophys. J. 75, 1915-1925[Medline] [Order article via Infotrieve]
  15. Müller, P., and Herrmann, A. (2002) Biophys. J. 82, 1418-1428[Medline] [Order article via Infotrieve]
  16. Morrot, G., Bureau, J. F., Roux, M., Maurin, L., Favre, E., and Devaux, P. F. (1987) Biochim. Biophys. Acta 897, 341-345[Medline] [Order article via Infotrieve]
  17. Wüstner, D., Herrmann, A., Hao, M., and Maxfield, F. R. (2002) J. Biol. Chem. 277, 30325-30336[Abstract/Free Full Text]
  18. John, K., Kubelt, J., Müller, P., Wüstner, D., and Herrmann, A. (2002) Biophys. J. 83, 1525-1534[Medline] [Order article via Infotrieve]
  19. Smutzer, G., Crawford, B. F., and Yeagle, P. L. (1986) Biochim. Biophys. Acta 862, 361-371[Medline] [Order article via Infotrieve]
  20. Butko, P., Hapala, I., Nemecz, G., and Schroeder, F. (1992) J. Biochem. Biophys. Methods 24, 15-37[CrossRef][Medline] [Order article via Infotrieve]
  21. Kavecansky, J., Joiner, C. H., and Schroeder, F. (1994) Biochemistry 33, 2880-2890[CrossRef][Medline] [Order article via Infotrieve]
  22. Yeagle, P. L., Albert, A. D., Boesze-Battaglia, K., Young, J., and Frye, J. (1990) Biophys. J. 57, 413-424[Medline] [Order article via Infotrieve]
  23. Ohvo-Rekila, H., Akerlund, B., and Slotte, J. P. (2000) Chem. Phys. Lipids 105, 167-178[CrossRef][Medline] [Order article via Infotrieve]
  24. Nemecz, G., Fontaine, R. N., and Schroeder, F. (1988) Biochim. Biophys. Acta 943, 511-521[Medline] [Order article via Infotrieve]
  25. Wharton, S. A., and Green, C. (1982) Biochim. Biophys. Acta 711, 398-402[Medline] [Order article via Infotrieve]
  26. Child, P., and Kuksis, A. (1983) J. Lipid Res. 24, 1196-1209[Abstract]
  27. Demel, R. A., Bruckdorfer, K. P., and van Deenan, L. L. M. (1972) Biochim. Biophys. Acta 255, 321-330[Medline] [Order article via Infotrieve]
  28. Rogers, J., Lee, A. G., and Wilton, D. C. (1979) Biochim. Biophys. Acta 552, 23-37[Medline] [Order article via Infotrieve]
  29. Butler, K. W., and Smith, I. C. P. (1977) Can. J. Biochem. 56, 117-122
  30. Hyslop, P. A., Morel, B., and Sauerheber, R. D. (1990) Biochemistry 29, 1025-1038[CrossRef][Medline] [Order article via Infotrieve]
  31. Fischer, R. T., Stephenson, F. A., Shafiee, A., and Schroeder, F. (1984) Chem. Phys. Lipids 36, 1-14[CrossRef][Medline] [Order article via Infotrieve]
  32. Fellmann, P., Zachowski, A., and Devaux, P. F. (1994) in Methods in Molecular Biology (Graham, J., and Higgins, J. A., eds) pp. 161-175, Humana Press, Totowa
  33. Hope, M. J., Bally, M. B., Webb, G., and Cullis, P. R. (1985) Biochim. Biophys. Acta 812, 55-65[CrossRef]
  34. Macura, S., and Ernst, R. R. (1980) Mol. Phys. 41, 95-117[CrossRef]
  35. Huster, D., Arnold, K., and Gawrisch, K. (1998) Biochemistry 37, 17299-17308[CrossRef][Medline] [Order article via Infotrieve]
  36. Schiller, J., Arnhold, J., Benard, S., Müller, M., Reichl, S., and Arnold, K. (1999) Anal. Biochem. 267, 46-56[CrossRef][Medline] [Order article via Infotrieve]
  37. Forslind, E., and Kjellander, R. (1975) J. Theor. Biol. 51, 97-109[CrossRef][Medline] [Order article via Infotrieve]
  38. Endress, E., Heller, H., Casalta, H., Brown, M. F., and Bayerl, T. M. (2002) Biochemistry 41, 13078-13086[CrossRef][Medline] [Order article via Infotrieve]
  39. Huster, D., Arnold, K., and Gawrisch, K. (1999) J. Phys. Chem. B. 103, 243-251[CrossRef]
  40. Huster, D., and Gawrisch, K. (1999) J. Am. Chem. Soc. 121, 1992-1993[CrossRef]
  41. Vogel, A., Scheidt, H. A., and Huster, D. (2003) Biophys. J. 85, 1691-1701[Medline] [Order article via Infotrieve]
  42. Seigneuret, M., Zachowski, A., Herrmann, A., and Devaux, P. F. (1984) Biochemistry 23, 4271-4275[CrossRef][Medline] [Order article via Infotrieve]
  43. Buton, N., Hervé, P., Kubelt, J., Tannert, A., Burger, K. N. J., Fellmann, P., Müller, P., Herrmann, A., and Devaux, P. F. (2002) Biochemistry 41, 13106-13115[CrossRef][Medline] [Order article via Infotrieve]
  44. Lange, Y., Dolde, J., and Steck, T. L. (1981) J. Biol. Chem. 256, 5321-5323[Abstract/Free Full Text]
  45. Schroeder, F., Nemecz, W. G., Wood, C., Joiner, C. H., Morrow, M. R., Ayraut-Jarrier, M., and Devaux, P. F. (1991) Biochim. Biophys. Acta 1066, 181-192
  46. Leventis, R., and Silvius, J. R. (2001) Biophys. J. 81, 2257-2267[Medline] [Order article via Infotrieve]
  47. Chattopadhyay, A., and London, E. (1987) Biochemistry 26, 39-45[CrossRef][Medline] [Order article via Infotrieve]
  48. Huster, D., Müller, P., Arnold, K., and Herrmann, A. (2001) Biophys. J. 80, 822-831[Medline] [Order article via Infotrieve]
  49. Oldfield, E., Meadows, M., Rice, D., and Jacobs, R. (1978) Biochemistry 17, 2727-2740[CrossRef][Medline] [Order article via Infotrieve]
  50. Ipsen, J. H., Mouritsen, O. G., and Bloom, M. (1990) Biophys. J. 57, 405-412[Medline] [Order article via Infotrieve]
  51. Davies, M. A., Schuster, H. F., Brauner, J. W., and Mendelsohn, R. (1990) Biochemistry 29, 4368-4373[CrossRef][Medline] [Order article via Infotrieve]
  52. Krajewski-Bertrand, M.-A., Milon, A., and Hartmann, M.-A. (1992) Chem. Phys. Lipids 63, 235-241[CrossRef]
  53. Yeagle, P. L. (1985) Biochim. Biophys. Acta 815, 33-36[Medline] [Order article via Infotrieve]
  54. Urbina, J. A., Pekerar, S., Le, H. B., Patterson, J., Montez, B., and Oldfield, E. (1995) Biochim. Biophys. Acta 1238, 163-176[Medline] [Order article via Infotrieve]
  55. Marsan, M. P., Muller, I., Ramos, C., Rodriguez, F., Dufourc, E. J., Czaplicki, J., and Milon, A. (1999) Biophys. J. 76, 351-359[Medline] [Order article via Infotrieve]
  56. White, S. H., and Wiener, M. C. (1996) in Biological Membranes: A Molecular Perspective from Computation and Experiment (Merz, K. M., and Roux, B., eds) pp. 127-144, Birkhäuser, Boston
  57. Bonmatin, J.-M., Smith, I. C., Jarrell, H. C., and Siminovitch, D. J. (2002) J. Am. Chem. Soc. 112, 1697-1704[CrossRef]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Biol. CellHome page
M. Mondal, B. Mesmin, S. Mukherjee, and F. R. Maxfield
Sterols Are Mainly in the Cytoplasmic Leaflet of the Plasma Membrane and the Endocytic Recycling Compartment in CHO Cells
Mol. Biol. Cell, January 1, 2009; 20(2): 581 - 588.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
K. Muller, P. Muller, G. Pincemy, A. Kurz, and C. Labbe
Characterization of Sperm Plasma Membrane Properties after Cholesterol Modification: Consequences for Cryopreservation of Rainbow Trout Spermatozoa
Biol Reprod, March 1, 2008; 78(3): 390 - 399.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. Du, Y. H. Pham, and A. J. Brown
Effects of 25-Hydroxycholesterol on Cholesterol Esterification and Sterol Regulatory Element-binding Protein Processing Are Dissociable: IMPLICATIONS FOR CHOLESTEROL MOVEMENT TO THE REGULATORY POOL IN THE ENDOPLASMIC RETICULUM
J. Biol. Chem., November 5, 2004; 279(45): 47010 - 47016.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
W. Stockinger, A. B. Castoreno, Y. Wang, J. C. Pagnon, and A. Nohturfft
Real-time analysis of endosomal lipid transport by live cell scintillation proximity assay
J. Lipid Res., November 1, 2004; 45(11): 2151 - 2158.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
T. A. Spencer, P. Wang, D. Li, J. S. Russel, D. H. Blank, J. Huuskonen, P. E. Fielding, and C. J. Fielding
Benzophenone-containing cholesterol surrogates: synthesis and biological evaluation
J. Lipid Res., August 1, 2004; 45(8): 1510 - 1518.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/46/45563    most recent
M303567200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Scheidt, H. A.
Right arrow Articles by Huster, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Scheidt, H. A.
Right arrow Articles by Huster, D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement