Detergent-resistant membrane fractions contribute to the total 1 H NMR-visible lipid signal in cells Lesley C. Wright 1 , Julianne T. Djordjevic 2 , Stephen D. Schibeci 2 , Uwe Himmelreich 5 , Nick Muljadi 3,4 , Peter Williamson 2 and Garry W. Lynch 3,4 1 Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology & Medical Research; 2 Institute of Immunology & Allergy Research; 3 Centre for Virus Research, Westmead Millennium Institute, the Cellular and Molecular Pathology Research Unit, Department of Oral Pathology and Oral Medicine, Westmead Hospital Dental School and 4 National Centre for HIV Virology Research, University of Sydney, Westmead Hospital, NSW, Australia; 5 Institute for Magnetic Resonance Research, Westmead Royal North Shore Hospital, St. Leonards, NSW, Australia Leukocytes and other cells show an enhanced intensity of mobile lipid in their 1 H NMR spectra under a variety of conditions. Such conditions include stimulation, which has recently been shown to involve detergent-resistant, plasma membrane domains (DRMs) often called lipid rafts. As there is much speculation surrounding the origin of cellular NMR- visible lipid, we analysed subcellular fractions, including DRMs, by NMR spectroscopy. We demonstrated that DRMs isolated by density gradient centrifugation from lymphoid (CEM-T4, stimulated Jurkat cells), and mono- cytoid (THP-1) cells produced NMR-visible, lipid signals. Large scale subfractionation of THP-1 cells determined that while cytoplasmic lipid droplets constituted much of the total NMR-visible lipid, the contribution of DRMs was signifi- cant. Qualitative and quantitative lipid analyses revealed that DRMs and lipid droplets differed in their lipid composition. DRMs were enriched in cholesterol and ganglioside GM1, and contained relatively unsaturated fatty acids compared with the lipid droplets. Both lipid droplets and DRMs con- tained neutral lipids (triacylgycerols, cholesterol ester, fatty acids in THP-1 cells) that could, in addition to phospho- lipids, contribute to the NMR-visible lipid. The lipid droplets also exhibited different protein profiles and contained 500- fold less protein than DRMs, confirming that DRMs and droplets were fractionated as separate entities. The NMR- visible lipid in DRMs is therefore unlikely to be a contami- nant from lipid droplets. We propose a micropartitioning of the NMR-visible mobile lipid of whole cells between intra- cellular lipid droplets, where most of this lipid resides, and detergent-resistant plasma membrane domains. Keywords: lipid; membrane; domain; NMR; Triton X-100. The origin of prominent 1 H NMR signals from lipids in spectra from many different cell types has been the subject of controversy for almost two decades. Currently, two sources for the 1 H NMR-visible lipid have been suggested; these are the mobile acyl chains of triacylglycerol and/or cholesterol ester) localized to either membranes, or to EM-visible intracellular lipid droplets [1]. Ferretti et al. [2] concluded that these NMR signals originate from both cytoplasmic lipid droplets and intramembrane amorphous lipid vesicles. Highly intense lipid resonances have been associated with activation or proliferation of lymphocytes, macrophages and neutrophils [3–5], as well as T cell lines, many cancer cells, and cancer tissue both ex vivo and in vivo [6]. Other cellular conditions linked with the appearance of NMR- visible lipid include the antiproliferative effects of tetra- phenylphosphonium chloride on a transformed breast cell line [7], unstimulated human neutrophils in the presence of high levels of free fatty acids [8], treatment of thymic lymphocytes with anti-CD3 antibody [4], and the induction of apoptosis or activation in Jurkat T-lymphoblasts [9]. The conclusion to be drawn from these and many other studies is that no single event is linked with the appearance of NMR-visible lipid. Recently, much evidence has accumulated for the pres- ence of neutral lipid-containing plasma membrane domains that are resistant to solubilization with nonionic detergents at low temperatures and have a low buoyant density when subjected to density gradient centrifugation. Such domains have been given the term DRMs (detergent-resistant membranes), DIGS (detergent-insoluble glycolipid-enriched domains), GEMS (glycolipid-enriched membrane domains), rafts, or caveolae when the protein, caveolin, is present [10]. In this article, we refer to these membrane domains as DRMs and rule out the use of the term caveolae as caveolins are not expressed in hematopoietic cells such as the ones used in this study [10]. In comparison to the rest of the plasma membrane which is in the liquid-crystalline (lc) Correspondence to L. C. Wright, Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology & Medical Research, Westmead Hospital, Westmead NSW 2145, Australia. Fax: + 61 2 98915317, Tel.: + 61 2 98457367, E-mail: lesleyw@icpmr.wsahs.nsw.gov.au Abbreviations: DRM, detergent-resistant membrane; DSM, detergent soluble membrane; CT-B, cholera toxin biotin; PABA, p-aminoben- zoic acid; PE, phycoerythrin; NaCl/P i (–), phosphate-buffered saline without calcium and magnesium; STR-HRP, streptavidin-conjugated horseradish peroxidase; PtdCho, phosphatidylcholine. (Received 17 December 2002, revised 03 March 2003, accepted 19 March 2003) Eur. J. Biochem. 270, 2091–2100 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03586.x phase, DRM structure is more ordered giving it character- istics of being in the liquid-ordered (lo) phase [10]. DRMs are rich in cholesterol and sphingolipids (including GM1 and GM3) and selectively retain a number of proteins including CD4, GPI-anchored proteins such as CD48 [11,12], and proteins associated with T cell receptor (TCR) signalling such as the src-family tyrosine kinase Lck, and LAT [10]. In addition, TCR engagement with antigen or by anti-CD3 antibody crosslinking leads to an increased partitioning of TCR subunits and their associated signalling molecules into DRMs implicating their central role as a focal point for T cell activation. Interestingly, DRM- associated proteins, such as caveolins and GPI-anchored proteins, have been identified in the surface monolayer of lipid droplets [13], indicating a possible link between lipid droplets and DRMs [14]. ThesizeofDRMshasbeensuggestedbysometo be > 100 nm [15], and by others to be around 26 nm [16]. The smaller domain size coincides with that suggested for NMR-visible microdomains [6]. In addition, parallels can be drawn between the diversity of functions associated with DRMs and the appearance of NMR-visible lipid in cells. These factors, as well as the presence in lipid droplets of known components of rafts and caveoli, such as cholesterol and caveolin, prompted us to investigate whether DRMs contain NMR-visible lipid. In this study we show that DRMs containing NMR-visible lipid can be isolated from several cell lines under conditions where mobile lipid is evident in the 1 H NMR spectra of the intact cells. Based on their distinctive protein and lipid composition, these detergent-insoluble domains differ from lipid droplets, and can be separated from them on the basis of different buoyant densities on sucrose gradients. Materials and methods Antibodies and reagents Phycoerythrin (PE)-conjugated anti-CD69/anti-CD25 and biotin-conjugated cholera toxin (CT-B) were purchased from Becton Dickinson (San Jose, CA, USA) and Sigma Aldrich (St. Louis, MO, USA), respectively. Monoclonal anti-Lck and horseradish peroxidase (HRP)-conjugated antimouse Ig/streptavidin (STR-HRP) were obtained from Santa Cruz and Amersham Pharmacia Biotech Inc., respectively. Antibodies against Hck, protein disulphide isomerase and tubulin were purchased from Transduction Laboratories, Stressgen and Sigma Aldrich, respectively. Monoclonal anti-CD48 was provided by T. Henniker (Westmead Hospital, Sydney, Australia) and polyclonal anti-CD4 (T4-5) was a gift from R. Sweet (Smith-Kline Beecham, King of Prussia, PA, USA). Cell lines The CEM-T4 human T lymphoblastoid cell line was obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, MD, USA). The human monocytic leukemic THP-1 and the Jurkat T-cell lines (J32.2) were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Cell culture and stimulation All cell lines were maintained in RPMI/10% fetal bovine serum. For Jurkat cell stimulation, cells were seeded into serum-free RPMI/0.1% BSA (10 6 ÆmL )1 ) and incubated for 24 h (viability > 80%). Harvested cells were then seeded into RPMI/10% fetal bovine serum and were either left unstimulated or were stimulated with PMA (30 ngÆmL )1 ) and ionomycin (300 ngÆmL )1 ) for 24 h. Preparation and fractionation of plasma membranes Jurkat and CEM-T4 cell membranes and cytosol, prepared from 1 and 5 · 10 8 cells, respectively, were solubilized with 1% Triton X-100 (v/v) and fractionated by 5–40% sucrose gradient centrifugation as described previously [17]. THP-1 cells were used for quantitative NMR and lipid analytical work. Membranes from THP-1 cells (1.56 · 10 9 )were separated from the cytosol by ultracentrifugation at 105 000 g for 60 min. The membrane-containing pellet was then solubilized with 1% Triton X-100 and fractionated by sucrose gradient centrifugation. Thirteen fractions of equal volume were collected from the top to the bottom of each gradient. Cholera toxin dot-blotting and SDS/PAGE immunoblotting Sucrose gradient fractions were examined for GM1 ganglio- side and protein content by cholera toxin dot-blotting and immunoblotting of SDS/PAGE-separated proteins, respect- ively. For Western blotting, proteins were recovered from cell extracts with Strataclean resin (Stratagene, La Jolla, CA, USA), eluted by boiling in Laemmli sample buffer, subjected to SDS/PAGE (10% or 4–16%) and electrotransferred to nitrocellulose membranes. Membranes were probed with primary antibody to Lck, Hck, CD48, protein disulphide isomerase, tubulin or CD4 followed by HRP-conjugated anti-rabbit/mouse Ig. For cholera toxin dot-blotting, 1 lLof each fraction was spotted directly onto nitrocellulose prior to incubation with CT-B followed by STR-HRP. Chemi- luminescent bands and spots were detected on X-ray film. Flow cytometry Cells (2 · 10 5 ) were incubated with anti-CD69 or anti- CD25 for 30 min on ice, washed with NaCl/P i and then fixed with 1% paraformaldehyde prior to FACScan ana- lysis (Becton Dickinson). Data were analysed using CELL QUEST software (Becton Dickinson). 1 H NMR analysis of whole cells, cell fractions and sucrose gradient fractions Sample Preparation. Cells (5 · 10 7 ) were washed three times in NaCl/P i (–) containing 0.1 mgÆmL )1 BSA, then washed and resuspended in 400 lLNaCl/P i (–) made up in 2 H 2 O. Homogenates, supernatants, floating lipid droplets and gradient fractions were dialyzed against NaCl/P i (–), and NaCl/P i (–) 2 H 2 O added to a final concentration of 10%. Membrane pellets were washed three times in NaCl/P i (–) and resuspended in NaCl/P i (–) 2 H 2 O. For quantitation of lipid 2092 L. C. Wright et al.(Eur. J. Biochem. 270) Ó FEBS 2003 signals, a known volume (usually 20 lL) of 10 m M p-aminobenzoic acid (PABA) was added to all fractions isolated from the cells. Integrals of phase and baseline corrected NMR spectra were determined using XWINNMR 2.6 (Bruker Biospin, Rheinstetten, Germany). The integrals were normalized to the PABA resonances at 6.83 p.p.m and 7.83 p.p.m. The CH 2 resonance at 1.3 p.p.m. was utilized to quantify the fatty acid residues. This resonance contains contributions mainly from the protons on CH 2 groups that are adjacent to other CH 2 groups, rather than those adjacent to double bonds or the carbonyl group of the acyl chains [7]. From the biochemical fatty acid analysis it was estimated that the average number of such CH 2 groups per fatty acid residue was 9.1. This was used for calculation of the fatty acid residue concentrations based on the NMR integrals. For most fractions, only trace amounts of valine, leucine, threonine and isoleucine contributed also to the 1.3 p.p.m. resonance. However, for the THP-1 supernatant fraction, a substantial contribution from these amino acid residues was noted, resulting in overestimation of the fatty acid concentration by up to twofold. Some contribution from amino acids was also noted in the membrane and DSM fractions from THP-1 cells. NMR spectroscopy. For both cells and gradient fractions, NMR spectra were acquired at 37 °C with the sample spinning at 20 Hz, using a Bruker Avance 360 MHz spectrometer; parameters for NMR spectroscopy of the cells were essentially as in [8]. 1D 1 HNMR spectra of gradient fractions were run using a selective excitation field gradient method of water suppression [18], a spectral width of 4000 Hz, 256 accumulations, and total acquisition time per transient of 3.14 s. 2D NMR spectroscopy of sucrose gradient fractions was carried out by acquiring 1 H, 1 H COSY NMR spectra in magnitude mode. Remaining water was suppressed by selective excitation. A total of 2000 data points were collected in the t 2 timedomainwithaspectral width of 10 p.p.m. The evolution time (t 1 ) was incremented to obtain 200 free induction decays, each with 32 scans for cells, and for the rafts 160 free induction decays, each with 256 scans. The total relaxation delay per scan was 1.6 s. Sine–bell window functions were applied in the t 1 dimension, and Gaussian–Lorentzian window functions were applied in the t 2 dimension according to [7]. Zero filling was used to expand the data matrix to 1K in the t 1 dimension. Lipid extraction and thin layer chromatography (TLC) Gradient fractions were extracted in chloroform and methanol, partitioned against water, and the lipid species in the organic phase separated by TLC as previously described [5,8]. The solvent system for neutral lipids was petroleum ether (BP 60–80 °C)/diethyl ether/acetic acid (90 : 15 : 1 v/v/v) and for polar lipids was CHCl 3 /meth- anol/water (65 : 25 : 4 v/v/v). The lipids were visualized with iodine vapour and also stained with sterol spray reagent and Coomassie Blue [8]. Their identities were confirmed by comparison with authentic standards. Lipid analyses Cholesterol and triacylgycerol estimations were performed using the Roche Modular Analytical System with CHOD- PAP methodology for cholesterol and GPO/PAP metho- dology for triacylgycerols (Roche). Fatty acid analyses were conducted on saponified lipid extracts converted to fatty acid methyl esters by acid methanolysis. These were separated on an HP Series II 5890 gas chromatograph with an Agilent Ultra 2 capillary column 19091B-102, using the method of MIDI Inc. (Delaware, USA). Results are expressed as percentages of the sum of the areas of all peaks identified. Results Examination of stimulated Jurkat cells by NMR spectroscopy Stimulation of Jurkat T cells with PMA/ionomycin was assessed using flow cytometry as shown in Fig. 1A. Augmentation of the fluorescence levels attributable to surface expression of the T-cell activation markers, IL-2 receptor alpha chain (CD25) and CD69, was observed following stimulation. Fig. 1. Examination of stimulated Jurkat T cells by flow cytometry and 1 H NMR spectroscopy. PMA and ionomycin-stimulated and non- stimulated Jurkat cells were incubated with anti-IgG 1 (isotype control), anti-CD25 or anti-CD69 (all PE–conjugated) and fluorescence was assessed by flow cytometry (A). The shaded peaks represent the stimulated phenotype in each case. 1D 1 H NMR spectra of sti- mulated and nonstimulated Jurkat cells are shown in B, confirming the presence of more NMR-visible CH 2 resonances at 1.3 p.p.m. in the stimulated cells. Sample preparation and 1 H NMR spectroscopy for this and subsequent figures were carried out as in the Materials and methods. Ó FEBS 2003 1 H NMR of cells/Triton-insoluble membrane fractions (Eur. J. Biochem. 270) 2093 Stimulated and nonstimulated Jurkat cells were examined for mobile lipid content using 1 H NMR. As shown in Fig. 1B, there was a marked increase in NMR-visible, mobile lipid on stimulation with PMA plus ionomycin. Protons from the methylene groups of the lipid acyl chains are at 1.3 p.p.m., and other resonances have previously been assigned to protons from methyl groups (0.9 p.p.m) and choline-containing residues (3.2 p.p.m) [4]. The CH 2 /CH 3 ratio increased from 1.44 to 3.02 after stimulation, with a concomitant increase in the CH 2 /choline peak height ratio from 0.44 to 2.82, due to a decrease in choline. These trends have also been observed previously [4,9]. Examination of plasma membrane fractions from stimulated Jurkat cells by NMR spectroscopy Next we examined the plasma membrane distribution of mobile lipids by fractionating plasma membranes on the basis of Triton X-100 solubility and buoyant density using sucrose gradient ultracentrifugation. The separation of lipid domains to the lighter, detergent-insoluble gradient frac- tions (DRMs, 3–6) was indicated indirectly by detecting the DRM-resident proteins Lck and CD48, and directly by detecting the glycosphingolipid GM1 with cholera toxin (Fig. 2A–C). The DRM protein markers were also detected in the high density fractions (9–10) which contain cytosolic material and Triton-soluble membrane (DSM) components. Lck and CD48 were, however, preferentially associated with DRMs, which contained less than 2% of the total protein found within the DSM fractions (determined by densito- metric scanning of Coomassie Blue-stained gels). When the DRM and DSM fractions were analysed for mobile lipid content by 1 H NMR, lipid CH 2 and CH 3 resonances at 1.3 and 0.9 p.p.m. were present in the DRM fractions of stimulated cells (mean CH 2 /CH 3 ratio of 1.9 ± 0.2 SEM, n ¼ 3), but lipid was below the level of detection, or present at a reduced level (mean CH 2 /CH 3 ratio of 1.4 ± 0.06 SEM, n ¼ 3), in similar fractions from the same number of nonstimulated cells (Fig. 2D). Lipid resonances were not detected in the DSM fractions from either cell type (not shown). Carbohydrate/polyol residues (CHOH, 3.4–4 p.p.m. region), but not choline, were visible in both the DRM and DSM fractions, but these were found to be artefacts from residual sucrose and/or dialysis cassette materials (see later). Identification of mobile lipid in other cell types and membrane fractions by 1 H NMR spectroscopy CEM-T4 cells and membranes. NMR spectra of CEM-T4 lymphoblasts displayed less mobile lipid than the stimulated Jurkat cells (CH 2 /CH 3 ratio of 2.78), but the choline resonance was much more prominent (Fig. 3A). No increase in mobile lipid was observed upon stimulation (data not shown). NMR-visible lipid and carbohydrate (but not choline) were again present in the DRM fractions (4–5) isolated from these cells (Fig. 3A), but this lipid was not observed in the DSM fractions (not shown). The distribution of CD4 protein is shown in Fig. 3B and demonstrates that a portion of CD4 is present in the DRM fractions containing the NMR-visible lipid. The DRM fractions contained about 2% of the total membrane protein content. THP-1 cells and membranes. NMR spectra of intact cells of the human monocytoid cell line THP-1, were clearly dominated by protons arising from lipid (Fig. 4A). The CH 2 /CH 3 peak height ratio was calculated to be 5.2, which was much higher than the equivalent signal observed in stimulated Jurkat cells. In contrast, the intensity of the choline resonance was very low, compared with the unstimulated Jurkat or CEM-T4 cells. The THP-1 cells did not increase their NMR-visible lipid when stimulated (data not shown). As with CEM-T4 DRM fractions, THP-1 DRM fractions 4–6, which contain less than 2% of the total membrane pro- tein, were found to localize specifically the DRM-associating proteins CD4 and Hck, without contamination by either of the abundant endoplasmic reticulum (protein disulphide isomerase) [19] or cytoskeletal (tubulin) representative protein markers (Fig. 4B). All proteins colocalized with the DSM fractions 10–12. Notably, the spectra arising from these DRM fractions were similar to the whole cell spectra in that they were again dominated by lipid (CH 2 /CH 3 ratio of 5.0; Fig. 5A). A small peak from choline-containing compounds was visible at 3.2 p.p.m. As with the Jurkat and CEM T-4 gradients, carbohydrate/polylol resonances were distinguished in both the DRM and DSM fractions (Fig. 5A,C). These resonan- ces, and one at 5.4 p.p.m., were also found in spectra from dialysed blank sucrose gradient fractions (Fig. 5B) which were prepared under the same conditions as the membrane- containing gradients, and were therefore not cell-derived. In comparison, virtually no NMR-visible lipid was detected in the DSM fractions (Fig. 5C), which did, however, contain most of the extracted membrane proteins. Blank gradient fractions isolated from the same part of the gradient produced spectra that were identical to that in Fig. 5B (results not shown). An NMR spectrum of Triton X-100 is shown in Fig. 5D. Resonances from Triton were not found in DRM, DRM control or DSM fractions (Fig. 5A–C), or in DSM control fractions (not shown). Two-dimensional 1 H, 1 H correlation spectroscopy (COSY) confirmed that the resonance at 1.3 p.p.m. was indeed derived mainly from lipid (Fig. 6). The crosspeaks labelled A–G¢ in Fig. 6 indicate spin-spin coupling between protons on adjacent carbon atoms. Crosspeaks A–F have previously been assigned to resonances from acyl chain protons found in triacylglycerol and/or cholesterol esters [20,21]. These could also arise from phospholipids. G¢ is derived from the glycerol backbone of triacylglycerol [20]. The intensities of crosspeaks C and D (– CH 2 -CH 2 - CH¼CH- and ¼CH-CH 2 -CH¼CH-, respectively) indicate that relatively large amounts of unsaturated fatty acid residues are present. The absence of resonances from lactate, threonine, valine, leucine and isoleucine was confirmed by COSY and spin-echo experiments (TE ¼ 135 ms, data not shown). Distinguishing the NMR-visible lipids of DRMs and cytosolic lipid droplets Most of the cytosolic lipid droplets floated as a visible, milky layer on the top of the supernatant formed when the plasma membrane fraction was sedimented from the cellular homogenate. The remainder of the lipid droplets were 2094 L. C. Wright et al.(Eur. J. Biochem. 270) Ó FEBS 2003 observed floating on top of fraction 1 of the sucrose gradient used for separation of DRMs from DSMs. The lipid droplets isolated from THP-1 cells displayed an NMR spectrum (Fig. 7A) which resembled that of the DRM fraction shown in Fig. 5A, except that the CH 2 /CH 3 ratio was only 2 (compared with 5 for DRMs) and a resonance at 3 p.p.m. was more prominent than the choline peak at 3.2 p.p.m. Comparison of NMR spectra from THP-1 cells (Fig. 4A) and their sedimented plasma membranes (Fig. 7B) shows a marked reduction in NMR-visible lipid in the membranes, indicating removal of cytoplasmic lipid droplets. The relative reduction in the intensity of the choline resonance at 3.2 p.p.m. confirmed previous obser- vations that most of the choline signal visible in whole cells derives from intracellular metabolites such as free choline, choline phosphate and glycerophosphocholine [4,7]. A large-scale fractionation of THP-1 cells was carried out to quantify the contribution of the lipid droplets and DRMs to the total cellular NMR-visible lipid. Relative concentra- tions of the NMR-visible lipid in all subcellular fractions were calculated by comparing the integrals of the lipid methylene resonances at 1.3 p.p.m. to those of a known concentration of the internal standard, PABA (Table 1). The only fraction not included was the hard pellet at the bottom of the gradients, which presumably contains aggregated proteins and cytoskeletal elements, as it is Triton-insoluble, but does not float on a density gradient. We calculated that approximately 12.4% of the total Fig. 2. Identification and examination of Jurkat DRMs by 1 HNMR spectroscopy. Proteins in sucrose gradient fractions from nonstimu- lated Jurkat cells were fractionated by SDS/PAGE, electrotransferred to nitrocellulose and immunoblotted with antibodies to DRM protein markerssuchasLck(A)orCD48(B)(1lgÆmL )1 ) followed by ECL detection as in the Materials and methods. Sucrose gradient fractions were also spotted onto nitrocellulose and probed with CT-B (2 lgÆmL )1 ) followed by ECL detection of GM1, a DRM lipid marker (C). The 1 H NMR spectra of dialysed DRMs from 10 8 stimulated and nonstimulated Jurkat cells are compared in D, indicating the presence of NMR-visible CH 2 resonances at 1.3 p.p.m. in the former, and low amounts of these resonances (sometimes below the limits of detection, as shown here) in the latter. Fig. 3. Examination of CEM-T4 cells and DRMs by 1 HNMR spectroscopy. The 1 H NMR spectra of CEM-T4 cells and dialysed DRM fractions obtained from 5 · 10 8 cells are shown in A. The presence of CH 2 resonances at 1.3 p.p.m. can be seen in both cells and DRMs. The sucrose gradient distribution of the DRM marker, CD4, is shown by immunoblotting with anti-CD4 (1 lgÆmL )1 )followedby ECL detection in B. Ó FEBS 2003 1 H NMR of cells/Triton-insoluble membrane fractions (Eur. J. Biochem. 270) 2095 cellular NMR-visible lipid signal was derived from the DRM fractions, about 2.1% from DSM fractions, and about 62.5% from lipid droplets. The latter figure was calculated as the sum of the signal from the supernatant (assumed to be contaminated with lipid droplets) plus that from the isolated cytoplasmic lipid droplet fraction. The rest of the signal was found in the membranes and low speed pellet (which includes unbroken cells and organelles). The contribution of the insoluble membrane pellet, including the cytoskeleton, was not calculated, as the pellet could not be resuspended uniformly. Lipid composition of DRMs and lipid droplets The THP-1 cells contained high levels of cholesterol and triacylglycerol. The DRM and lipid droplet fractions differed considerably in their content of these two neutral lipids (Table 1). As expected, the DRM fractions were enriched in total cholesterol relative to triacylglycerol (cholesterol/triacylglycerol ratio of 3.0) when compared with the plasma membrane fraction from which they were derived (ratio of 1.5), and the DSM fractions (ratio 1.0). The DRM fraction contained about 35% of the total cellular cholesterol and 23% of the total cellular triacylglycerol. Conversely, the lipid droplet fraction was enriched in triacylglycerol (cholesterol/triacylglycerol ratio 0.6), as was the lipid droplet fraction floating on the top of the sucrose gradient (ratio 0.5, not shown). The lipid droplet fraction contained 15.4% of the total cellular triacylglycerol, and this fraction plus the supernatant fraction (which presumably also contains lipid droplets) contained 40.3% of the cellular triacylgycerols. The major neutral and polar lipid components of DRM and DSM fractions from CEM-T4, THP-1 and Jurkat cells were examined qualitatively by TLC. The dominant neutral lipid in the Jurkat DRM fractions (from both stimulated and nonstimulated cells) was free cholesterol, with a small amount of triacylglycerol and free fatty acid. CEM-T4 DRM fractions contained mainly free cholesterol and cholesterol ester, whereas THP-1 DRMs, by contrast, contained predominantly free cholesterol, triacylglycerol, Fig. 5. Examination of THP-1 gradient fractions by 1 HNMRspectro- scopy. Spectra of dialysed DRM fractions (4–6) isolated from 1.56 · 10 9 cells, and the corresponding dialysed fractions from a blank (control) sucrose gradient are shown in A and B, respectively. The spectrum of the DRMs is dominated by CH 2 resonances at 1.3 p.p.m., which are absent in the control, and present at a much reduced level in the dialysed DSM fractions shown in C. DSM control fractions resembled those in B (not shown). The absence of detergent contami- nation in A, B and C can be confirmed by comparison with the spectrum of Triton X-100 [1% (v/v) in NaCl/P i (–) 2 H 2 O], shown in D. PABA (20 lL of a 10-m M solution) was added as an internal standard in A, B and C. Fig. 4. Examination of THP-1 cells by 1 H NMR spectroscopy and the distribution of proteins following sucrose gradient centrifugation. The 1D 1 H NMR spectrum of intact THP-1 cells, dominated by CH 2 reso- nances at 1.3 p.p.m., is shown in A. After fractionation of THP-1 membranes, proteins in each sucrose gradient fraction were subjected to SDS/PAGE, electrotransferred to nitrocellulose and immunoblot- tedwithantibodiestoDRMmarkers,CD4andHck,andantibodiesto protein disulphide isomerase (PDI, endoplasmic reticulum) and tub- ulin (cytoskeleton), followed by ECL detection, shown in B. Lysates were also loaded as controls. 2096 L. C. Wright et al.(Eur. J. Biochem. 270) Ó FEBS 2003 and small amounts of cholesterol ester, ether-linked triacyl- glycerol and free fatty acids. Phosphatidylcholine (PtdCho), sphingomyelin and other phospholipids were present in the polar lipid component of all DRMs. DSM fractions from CEM-T4 cells contained mainly free cholesterol and PtdCho, and those from THP-1 cells contained mainly cholesterol ester, triacylglycerol and PtdCho. Small amounts of other phospholipids were detected also. Apart from GM1, the glycolipid content of DRM and DSM fractions was not determined. The fatty acid composition of the total lipids extracted from the various fractions of the THP-1 cells is shown in Table 2. Surprisingly, the DRM fractions were not enriched in saturated fatty acids, relative to the cell homogenate and the total membrane fraction, except for a very small increase in myristic acid (14:0). Rather there was a small increase in palmitoleic acid (16:1), 18:2 + 18:3, and arachidonic acid (20:4) at the expense of palmitic, stearic and oleic acids (16:0, 18:0, 18:1, respectively). The DSM fractions contained higher levels of 18:0, 18:2 + 18:3 and 18:1 at the expense of 16:0, 14:0 and 16:1, whereas there was a marked increase in the amount of 16:0 and a decrease in the amount of 16:1 and 17:1 in the lipid droplet fraction (Table 2). The fatty acid analyses of fractions taken from blank gradients revealed no contamination from Triton X-100. We next compared the levels of the sphingolipid, GM1, in THP-1 DRMs and lipid droplets by dot-blot analysis using biotinylated cholera toxin (Fig. 8A). After accounting for fraction volumes, the enrichment of GM1 in DRMs relative to lipid droplets was found to be greater than 250-fold. Fig. 6. The 2D COSY spectrum of the THP-1 DRM fraction. The 1D spectrum is shown on top. Crosspeaks identified previously as arising from protons associated with lipid [20] are indicated by A–G¢,indi- cating that the DRM spectrum is dominated by resonances arising from lipid. Lys, lysine; CHOH, polyol and/or carbohydrate residues. Fig. 7. 1 H NMR spectra of THP-1 cytoplasmic lipid droplets and plasma membranes. Lipid droplets (A) were isolated by high speed centrifugation (105 000 g) of cellular, organelle-depleted homogenates and represented the milky layer at the top of the supernatant. This layer was dialysed against NaCl/P i (–) and NaCl/P i (–) 2 H 2 O was added to a final concentration of 10%. Intense CH 2 resonances were visible at 1.3 p.p.m. in the lipid droplets, but were obviously less prominent in the membrane-containing pellet (B) obtained after removal of the lipid droplets and supernatant and resuspended in NaCl/P i (–) 2 H 2 O. PABA (20 lLofa10-m M solution) was added as internal standard to both samples. Table 1. Cholesterol, triglyceride and NMR-visible lipid content of THP-1 cell fractions. Results are expressed as lmol of CH 2 equivalents (NMR-visible lipid) or lipid species (cholesterol and triacylglycerols) per 1.56 · 10 9 cells. The total cellular content is the sum of the top four fractions. Percentage compositions are shown in brackets. The method for calculation of the amount of NMR-visible lipid is described in the Materials and methods section. Fraction NMR-visible lipid Cholesterol Triacylgycerols Low speed pellet 2.47(13.7) 0.90(10.4) 0.42(9.7) Membranes 4.30(23.8) 3.41(39.4) 2.17(50.0) Supernatant 8.46(46.8) 3.93(45.5) 1.08(24.9) Lipid droplets 2.84(15.7) 0.40(4.6) 0.67(15.4) Total cellular content 18.07 8.64 4.34 DRM fractions 2.24(12.4) 3.00(34.7) 1.00(23.0) DSM fractions 0.38(2.1) 0.31(3.6) 0.31(7.1) Ó FEBS 2003 1 H NMR of cells/Triton-insoluble membrane fractions (Eur. J. Biochem. 270) 2097 Protein content of DRMs and lipid droplets The protein profiles of DRMs and lipid droplets were compared by SDS/PAGE (Fig. 8B). Prior to analysis, lipid droplets were subjected to sucrose density gradient centri- fugation to remove potentially contaminating cytoplasmic proteins, and the resulting milky layer at the top of fraction 1 was collected. We confirmed that these droplets also do not stain positive for GM1 (results not shown). Proteins were visualized by Coomassie Blue staining, and densitometric scanning revealed that the DRM fraction contained 500 times more protein than the lipid droplet fraction. This was calculated after taking into account that protein from 30% and 1.25% of the total lipid droplet and DRM fractions, respectively, was loaded onto the gel. Although there were protein bands of similar size in both fractions between 45 and 55 kDa, there were clearly bands unique to each fraction (see arrows). This finding, together with the relatively low level of the GM1 glycolipid in lipid droplets, and the differences in fatty acid and neutral lipid content, is further evidence that lipid droplets and DRMs are distinct subcellular fractions. Discussion Microscopically, detergent-resistant plasma membrane domains have proved difficult to detect [15]. However, we can now visualize by 1D and 2D 1 H NMR spectroscopy the mobile lipid component of such domains, which we have found to have a protein and lipid composition characteristic of DRMs or rafts, as described by others. While the membrane fractionation procedure may have altered the physical state of the lipids from that in the whole cells, the resonances appearing in DRM spectra are probably derived from the lipid acyl chains of triacylgycerols and cholesterol esters, as well as small amounts of free fatty acids. Some contribution could also come from the acyl chains of PtdCho or sphingomyelin; however, neither the choline headgroups of PtdCho and sphingomyelin (except for a small peak in THP-1) nor the sphingosine chain of sphingomyelin were visible in the NMR spectra of DRMs. In bilayer environments, the choline headgroups are relat- ively immobile and therefore of low visibility in NMR spectra [7]. This suggests that headgroup mobility might also be restricted in DRMs from some cells. Although DRMs from three different cell types produced NMR spectra dominated by resonances from lipid acyl chains, we found differences in the composition of the Fig. 8. Analysis of THP-1 DRMs and lipid droplets for GM1 and total protein. (A) THP-1 DRMs were isolated from total membranes by sucrose gradient fractionation as described in Materials and methods. Lipid droplets, prepared as in the legend to Fig. 7, were subjected to sucrose density gradient fractionation to remove any cytoplasmic proteins and were collected from the top layer (fraction 1). Pooled DRMs (fractions 4–8) and lipid droplets (0.5 lL of each) were spotted onto a nitrocellulose membrane which was subsequently probed with CT-B (2 lgÆmL )1 ). GM1 was detected by ECL. (B) Proteins from 1.25% and 30% of the total DRM and lipid droplet fractions, respectively, were captured with Strataclean resin and separated by SDS/PAGE. Proteins were visualized by Coomassie Blue staining. Molecular mass markers are shown in the left lane, and bands unique to each fraction are indicated by arrows. Table 2. The fatty acid composition of THP-1 cell fractions. Results are expressed as percentages of the total area under all peaks measured. Low concentration components with <1% of the area have been omitted from the calculations. Fatty acid Homogenate Membranes DRM DSM Lipid droplets 14:0 6.08 5.68 7.19 3.29 6.88 16:0 32.28 29.39 28.23 25.59 44.76 16:1x7c 18.27 18.75 22.07 14.88 12.06 17:1 5.57 5.64 6.62 7.06 1.88 18:2 + 18:3 1.57 1.59 2.63 2.91 1.79 18:1x9c 17.66 17.85 15.01 20.83 18.44 18:1x7c 7.78 7.86 6.36 8.99 8.84 18:0 6.59 9.05 5.45 11.06 6.05 20:4 4.19 4.18 6.41 5.37 2.29 2098 L. C. Wright et al.(Eur. J. Biochem. 270) Ó FEBS 2003 neutral lipids, which could potentially contribute to these spectra. Notably, the DRMs extracted from the monocytoid cell line, THP-1, contained high levels of triacylgycerols, whereas those from both of the lymphoid cells, CEM-T4 and Jurkat, were comprised mainly of cholesterol ester and free cholesterol/triacylgycerols, respectively. This indicates that just as there are cell-specific differences in the proteins found in DRMs, with cell-specific functions, there is cell-specific variability in the neutral lipid content of DRMs. While no quantitation was performed on the DRM lipids from stimulated and nonstimulated Jurkat cells, it appeared that the same components were present, therefore either an increase in the amount of DRMs present, or in the amount of lipid in the DRMs from the stimulated cells may explain their more intense lipid spectra. The fact that DRMs were difficult to detect in both stimulated and nonstimulated Jurkat cells may be due to the qualitatitive observation that little neutral lipid was present in this cell line and its DRMs, and also the use of much smaller cell numbers used to prepare gradients (10 8 ), compared with the THP-1 cells (1.56 · 10 9 ). While some neutral lipids, as well as phospholipids, were present in DSM fractions, their high protein to lipid ratio may account for the observation of very little NMR-visible lipid. Although triacylgycerols and cholesterol esters have been observed in intact cells and pure plasma membrane fractions by 1 H NMR spectroscopy and chemical analysis [5,8,20,21], triacylgycerols, as described for the THP-1 membrane fractions, have not previously been identified as a compo- nent of DRMs. The authenticity of our DRM fractions was confirmed not only by the presence of typical DRM protein markers, but also by the presence of other DRM lipid components described in the literature, e.g. free cholesterol, sphingomyelin and gangliosides. There is precedence for intercalation of triacylgycerols and cholesterol esters, at least temporarily, into bilayers of some cell types. They form the cores of lipoproteins, which must be translocated through membranes for secretion [22]. Phospholipid bilay- ers can accommodate about 3% triacylgycerol and 5% sterol ester on a molar basis [23,24] before the neutral lipids phase-separate to form spherical domains sandwiched between the bilayer leaflets [25]. Such a mechanism has been invoked for the formation of lipid droplets in the endoplasmic reticulum [26] and in the secretion of milk [27]. Intracellular lipid droplets are surrounded by a mono- layer of amphipathic phospholipids, glycolipids and/or sterols that encircles the hydrophobic core of neutral lipids, such as triacylgycerols, diacylglycerols or sterol esters [13,14,25]. In THP-1 cells we have shown that lipid droplets have similar NMR spectra to DRMs (compare Figs. 5A and 7A), but contain different proportions of cholesterol and triacylgycerols, and almost no GM1. Lipid droplets were also enriched in saturated fatty acids, whereas DRM lipids were more unsaturated. We now propose that the mobile lipid visible in the NMR spectra of intact cells is derived from at least two pools – a large, intracellular, lipid droplet pool, and a smaller pool, specifically localized to detergent-insoluble, plasma membrane domains. The figure given for the contribution of acyl chains from DRM lipids (12.4%) to the THP-1 cell spectrum is a minimum percentage. Although no nonlipid contribution to the lipid signal at 1.3 p.p.m. could be detected in the low speed pellet, lipid droplets and DRM fractions, there was (as expected) a large contribution from nonlipid compo- nents in the supernatant fraction (see Materials and methods). The supernatant contains only 25% of the cellular triacylgycerol, but contributes 46.8% towards the mobile ÔlipidÕ signal (Table 1), therefore this latter figure would be a considerable overestimate, leading to an overestimation of the total cellular lipid signal. Because in THP-1 cells much of the lipid signal would derive from triacylgycerol, a better estimate may be obtained from the triacylgycerol content of the DRMs, namely 23% of the total, and for the supernatant plus lipid droplet fraction a figure of 40% might be more accurate (Table 1). Thus we would estimate the contribution of the DRMs to the cellular NMR-visible lipid in the range of 12–23%, with lipid droplet contribution at around 40%. Interestingly, the contribution of the total membrane fraction to the NMR- visible lipid is around 24%, and as the amount of triacylgycerol in this fraction is 50% of the total, almost all of the NMR-visible triacylgycerol in membranes must reside in the DRM fraction. It could be argued that the NMR signal detected in DRMs is merely contamination from cytoplasmic lipid droplets. This is unlikely, for the following reasons. Firstly, during method development, a lipid droplet preparation from THP-1 cells (rich in NMR-visible lipid) was incubated with membranes from CEM-T4 cells (poor in NMR-visible lipid). After washing, no increase in membrane lipid signal was observed, indicating that contamination by adherence of cytoplasmic lipid droplets to membrane components during preparation is unlikely (the small amount of droplets remaining are floated to the top of the sucrose gradients during fractionation). Secondly, the protein, lipid and fatty acid composition of droplets and DRMs are quite different, indicating that they are physically separate entities. If contamination with droplets did occur, it would have to be highly selective for the Triton-insoluble fraction of the membranes, because the Triton-soluble fraction (Fig. 5C) contains little NMR-visible lipid. Thirdly, the contamin- ation of the DRMs would also need to be very large, since the triacylgycerol content of DRMs is 23% of the total and the isolated lipid droplet fraction contains only 15.4% of the total. The relationship between the two lipid pools (DRMs and lipid droplets) is unclear at present. While the differences we have shown in lipid and protein content of lipid droplets and rafts from THP-1 cells suggest unrelated functions, both are believed to originate in the endoplasmic reticulum/Golgi apparatus, allowing the possibility of some physical inter- action between them. In addition, it has been suggested that free cholesterol may be channelled from triacylgycerol- containing lipid droplets in the endoplasmic reticulum to areas of tight contact between the droplet surface and the plasma membrane, where the free cholesterol could then be incorporated into the bilayer [14]. Both metabolic and spatial relationships between neutral lipids (triacylgycerols and sterol esters) and membrane phospholipids have been found, with growing evidence of direct physical continuities between lipid droplets and bilayer membranes [25,27]. This deserves further investigation, especially the possibility of translocation of lipid from droplets to DRM domains during stimulation of some cells (e.g. Jurkats), with subsequent effects on cell function. Ó FEBS 2003 1 H NMR of cells/Triton-insoluble membrane fractions (Eur. J. Biochem. 270) 2099 Acknowledgements We would like to thank Ms Leanne Hicks, Department of Infectious Diseases, for the fatty acid analyses, and the Department of Biochemistry (Core Pathology), Westmead Hospital for the neutral lipid analyses. At the time of re-submission of this manuscript, we discovered that A. Ferretti et al. have quite independently and simultaneously come to similar conclusions, namely that NMR-visible lipid is present in DRMs. Their work is now available in the European Biophysical Journal online, DOI 10.1007/s00249–002-0273-8 as of January 2003. References 1. Hakuma ¨ ki, J.M. & Kauppinen, R.A. (2000) 1 HNMRvisible lipids in the life and death of cells. TIBS 25, 357–362. 2. Ferretti,A.,Knijn,A.,Iorio,E.,Pulciani,S.,Giambenedetti,M., Molinari, A., Meshini, S., Stringaro, A., Calcabrini, A., Freitas, I., Strom, R., Arancia, G. & Podo, F. (1999) Biophysical and structural characterization of 1 H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts. Biochim. Biophys. Acta 1438, 329–348. 3. King, N.J.C., Delikatny, E.J. & Holmes, K.T. (1994) 1 H Magnetic resonance spectroscopy of primary human and murine cells of the myeloid lineage. Immunomethods 4, 188–198. 4. Veale, M.F., Roberts, N.J., King, G.F. & King, N.J.C. (1997) The generation of 1 H-NMR-detectable mobile lipid in stimulated lymphocytes: relationship to cellular activation, the cell cycle, and phosphatidylcholine-specific phospholipase C. Biochem. Biophys. Res. Commun. 239, 868–874. 5.May,G.L.,Wright,L.C.,GrootObbink,K.,Byleveld,P.M., Garg, M.L., Ahmad, Z.I. & Sorrell, T.C. (1997) Increased satu- rated triacylglycerol levels in plasma membranes of human neutrophils stimulated by lipopolysaccharide. J. Lipid Res. 38, 1562–1570. 6. Mountford, C.E. & Wright, L.C. (1988) Organization of lipids in the plasma membranes of malignant and stimulated cells: a new model. TIBS 13, 172–177. 7. Roman,S.K.,Jeitner,T.M.,Hancock,R.,Cooper,W.A.,Ride- out, D.C. & Delikatny, E.J. (1997) Induction of magnetic resonance-visible lipid in a transformed human breast cell line by tetraphenylphosphonium chloride. Int. J. Cancer 73, 570–579. 8.Wright,L.C.,GrootObbink,K.,Delikatny,E.J.,Santangelo, R.T. & Sorrell, T.C. (2000) The origin of 1 H NMR-visible tri- acylglycerol in human neutrophils. High fatty acid environments result in preferential sequestration of palmitic acid into plasma membrane triacylglycerol. Eur. J. Biochem. 267, 68–78. 9. Di Vito, M., Lenti, L., Knijn, A., Iorio, E., D’Agostino, F., Molinari, A., Calcabrini, A., Stringaro, A., Meschini, S., Arancia, G.,Bozzi,A.,Strom,R.&Podo,F.(2001) 1 H NMR-visible mobile lipid domains correlate with cytoplasmic lipid bodies in apoptotic T-lymphoblastoid cells. Biochim. Biophys. Acta 1530, 47–66. 10. Brown, D.A. & London, E. (1998) Functions of lipid rafts in biological membranes. Ann. Rev. Cell Dev. Biol. 14, 111–136. 11. Parolini,I.,Topa,S.,Sorice,M.,Pace,A.,Ceddia,P.,Montesoro, E., Pavan, A., Lisanti, M.P., Peschle, C. & Sargiacomo, M. (1999) Phorbol ester-induced disruption of the CD4-Lck complex occurs within a detergent-resistant microdomain of the plasma mem- brane. Involvement of the translocation of activated protein kinase C isoforms. J. Biol. Chem. 274, 14176–14187. 12. Stefanova,I.,Horejsi,V.,Ansotegui,I.J.,Knapp,W.&Stock- inger, H. (1991) GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science 254, 1016–1091. 13. Pol,A.,Luetterforst,R.,Lindsay,M.,Heino,S.,Ikonen,E.& Parton, R.G. (2001) A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance. J. Cell Biol. 152, 1057–1070. 14. Prattes, S., Ho ¨ rl, G., Hammer, A., Blaschitz, A., Graier, W., Sattler, W., Zechner, R. & Steyrer, E. (2000) Intracellular dis- tribution and mobilization of unesterified cholesterol in adipo- cytes: triglyceride droplets are surrounded by cholesterol-rich ER-like surface layer structures. J. Cell Sci. 113, 2977–2989. 15. Brown, D.A. & London, E. (2000) Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J. Biol. Chem. 275, 17221–17224. 16. Pralle,A.,Keller,P.,Florin,E L.,Simons,K.&Ho ¨ rber, J.K.H. (2000) Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. J. Cell Biol. 148, 997–1007. 17. Montixi,C.,Langlet,C.,Bernard,A M.,Thimonier,J.,Dubois, C.,Wurbel,M A.,Chauvin,J P.,Pierres,M.&He,H T.(1998) Engagement of T cell receptor triggers its recruitmen to low-den- sity detergent-insoluble membrane domains. EMBO J. 17, 5334– 5348. 18. Braun, S., Kalinowski, H O. & Berger, S. (1998) In: 150 and More Basic NMR Experiments,p.473.Wiley-VCH,NY.,NY. 19. Ellgaard, L. & Helenius, A. (2001) ER quality control: towards an understanding at the molecular level. Curr. Opin. Cell Biol. 13, 431–437. 20. May,G.L.,Wright,L.C.,Holmes,K.T.,Williams,P.G.,Smith, I.C.P.,Wright,P.E.,Fox,R.M.&Mountford,C.E.(1986) Assignment of methylene proton resonances in NMR spectra of embryonic and transformed cells to plasma membrane trigly- ceride. J. Biol. Chem. 261, 3048–3053. 21. MacKinnon, W.B., May, G.L. & Mountford, C.E. (1992) Esteri- fied cholesterol and triglyceride are present in plasma membranes of Chinese hamster ovary cells. Eur. J. Biochem. 205, 827–839. 22. Davis, R.A. (1991) Lipoprotein structure and secretion. In: Biochemistry of Lipids, Lipoproteins and Membranes (Vance, D.E. & Vance, J., eds), Vol. 20 p. 419. Elsevier, Amsterdam. 23. Hamilton, J.A. (1989) Interactions of triglycerides with phos- pholipids: incorporation into the bilayer structure and formation of emulsions. Biochemistry 28, 2514–2520. 24. Gorrissen, H., Tulloch, A.P. & Cushley, R.J. (1982) Deuterium magnetic resonance of triacylglycerols in phospholipid bilayers. Chem. Phys. Lipids 31, 245–255. 25. Murphy, D.J. (2001) The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog. Lipid Res. 40, 325– 438. 26. Ostermeyer,A.G.,Paci,J.M.,Zeng,Y.,Lublin,D.M.,Munro,S. & Brown, D.A. (2001) Accumulation of caveolin in the endoplasmic reticulum redirects the protein to lipid storage drop- lets. J. Cell Biol. 152, 1071–1078. 27. Murphy, D.J. & Vance, J. (1999) Mechanisms of lipid-body for- mation. TIBS 24, 109–115. 2100 L. C. Wright et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . neither the choline headgroups of PtdCho and sphingomyelin (except for a small peak in THP -1) nor the sphingosine chain of sphingomyelin were visible in the. present in the DRM fractions containing the NMR-visible lipid. The DRM fractions contained about 2% of the total membrane protein content. THP -1 cells and membranes.