Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536
Received on September 8, 2004; accepted on October 6, 2004
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Abstract |
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Key words: dimannosyldiacylglycerol / lipomannan / [3H]mannose-suicide / temperature-sensitive
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Introduction |
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To investigate the precise role(s) of the mannolipid intermediates, Man-P-Und and Man2-DAG, a [3H]mannose-suicide procedure was applied to isolate temperature-sensitive (ts) mutants that are defective for growth and LM synthesis by adapting the approach used for the selection of asparagine-linked glycosylation (alg) mutants in Saccharomyces cerevisiae by Huffaker and Robbins (1982, 1983)
. The rationale is that the mutants survive the radiation damage sustained by the wild-type cells because ts defects in individual steps in the biosynthetic pathway block the accumulation of [3H]mannose in the membrane-anchored LM. Thus the defects prevent the mutant cells from sustaining lethal radiation damage.
Approximately 26 ts micrococcal mutants were identified after replica-plating the survivors on Luria Bertani (LB) agar by comparing the colonial patterns at the permissive (30°C) and nonpermissive temperatures (37°C). Cells that fail to form colonies at 37°C were isolated and screened for biochemical alterations in all of the steps shown in Figure 1. Two mutants with altered levels of Man2-DAG and LM were selected and characterized here. One mutant, designated mms1, is defective in Man2-DAG synthesis, and the second mutant (mms2) appears to have a ts defect in the mannosyltransferase synthesizing Man-P-Und. In vivo and in vitro studies with the mutant deficient in Man2-DAG (mms1) provide the first solid evidence that the mannolipid serves as the lipid anchor precursor for LM. The results of in vitro experiments with membrane fractions from mms2 confirm the obligatory role of Man-P-Und in the elongation of Man2-DAG. The potential of the [3H]mannose-suicide selection procedure for identifying genes encoding other key membrane proteins involved in LM assembly, including mannolipid flippases, and the possibility that Man2-DAG is required for normal growth of M. luteus cells are discussed.
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Results |
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Micrococcal mutants mms1 and mms2 are temperature-sensitive for growth
Based on the assumption that ts defects in Man12-DAG and LM biosynthesis could affect cell viability, ethyl methanesulfonate (EMS)-treated cells were subjected to two rounds of [3H]mannose-suicide selection, replica plated (Miller, 1972) on LB agar, and grown at 30°C (permissive temperature) and 37°C (nonpermissive temperature). Two ts mutants, mms1 and mms2, were isolated by this procedure, and the enzymatic mutations have been characterized.
After 30 h of incubation at 30°C, all strains showed normal growth on LB agar (Figure 2A). In contrast to the wild-type cells, mms1 and mms2 cells failed to produce visible colonies at 37°C (Figure 2B). To determine if incubation at the nonpermissive temperature would lead to lethality, mms1 and mms2 cells were incubated at 37°C for 30 h in LB agar (no visible colonies formed), and then shifted to 30°C for an additional 30 h. Under these conditions, both mutants resumed normal growth (Figure 2C), indicating that the temperature-sensitive growth defects are reversible.
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Microscopy of NAO or FITC-Con A-stained protoplasts
CL-specific 10-N-nonyl-acridine orange (NAO) staining has been widely utilized to evaluate the level of CL in cell membranes (Mileykovskaya and Dowhan, 2000). Previously, it was shown that FITC-conjugated Con A binds to LM present on the outer surface of intact protoplasts of M. luteus (Pakkiri et al., 2004
). To corroborate that CL levels are apparently normal but the LM levels are reduced in the two ts mutants, protoplasts from wild-type, mms1, and mms2 cells were exposed to NAO (Figure 4af) and FITC-conjugated Con A (Figure 4gl). When exposed to NAO, protoplasts from all strains exhibited the same fluorescence intensity (Figure 4df). In contrast to wild-type protoplasts, the fluorescence intensity from mms1 and mms2 was severely reduced when exposed to FITC-conjugated Con A (Figure 4jl), indicative of lower amounts of LM present on the outer surface of the micrococcal protoplasts. These results confirm that the phospholipid levels are unaffected, but the LM level is drastically reduced in mms1 and mms2 cells at the nonpermissive temperature.
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Assay of micrococcal mannosyltransferases in vitro in crude homogenates from wild-type and mutant cells
To examine the biochemical defects in mms1 and mms2 in more detail, the rates of synthesis of the mannolipids and LM were compared in vitro with homogenates from wild-type and mutant cells at 37°C. In contrast to homogenates from wild type cells, [3H]mannose accumulated in [3H]Man-DAG when homogenates from mms1 cells were incubated with GDP-[3H]Man (Table II). The synthesis of [3H]Man2-DAG and [3H]LM was markedly lower in mms1 cells, indicating that the mannosyltransferase catalyzing the transfer of the second mannosyl unit to Man2-DAG is defective at the nonpermissive temperature. The rates of [3H]Man-P-Und synthesis in wild-type and mms1 cell homogenates were virtually identical. However, the synthesis of [3H]Man-P-Und by homogenates from mms2 cells was drastically reduced (Table II). Consequently, the synthesis of [3H]LM was also reduced, and the transfer of [3H]mannosyl units from GDP-[3H]Man was diverted to the synthesis of [3H]Man12DAG.
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Man-P-Und-mediated transfer of mannosyl units to LM by membranes from mms2 cells
To characterize the enzymatic defect in mms2 cells further, exogenous Man-P-Und was added to membranes from mms2 containing endogenous, prelabeled [3H]Man2-DAG. The putative mannolipid anchor precursor was converted to [3H]LM (Figure 7) in a concentration-dependent manner, and the [3H]LM formed under these conditions also appeared to be chromatographically identical to glycerol-(Man)4850 following deacylation (Figure 6C). These results confirm that mms2 cells are defective for the synthesis of the lipophilic mannosyl donor, Man-P-Und, and that the elongation of the lipid anchor precursor, Man2-DAG, requires Man-P-Und as the mannosyl donor.
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Discussion |
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In vivo and in vitro biochemical studies establish that mms1 cells have a ts defect in the mannosyltransferase(s) catalyzing the conversion of Man-DAG to Man2-DAG (Figure 1, step 2), and consequently accumulate the precursor intermediate, Man-DAG. The ts growth defect in this mutant strongly suggests that Man2-DAG synthesis is required for the normal growth of these micrococcal cells.
In a key direct experiment, membranes from mms1 cells are able to efficiently convert exogenous [3H]Man2-DAG to [3H]LM by a process dependent on Man-P-Und formation due to the deficiency of endogenous Man2-DAG. The radiolabeled LM formed from either exogenous [3H]Man2-DAG and unlabeled GDP-Man or from exogenous Man2-DAG and GDP-[3H]Man by membranes from mms1 cells appear to be virtually identical in size (4850 residues) to LM formed by membranes from wild-type cells as assessed by gel filtration analysis of the deacylated products onBio-Gel P-10. The role of Man-P-Und as the mannosyl donor for the elongation process is confirmed by the inhibitory effect of amphomycin.
The second mutant characterized in this study, mms2, has a temperature-sensitive defect in the synthesis of Man-P-Und (Figure 1, step 3), as determined by in vitro studies. The inability to synthesize Man-P-Und, the obligatory mannosyl donor, blocked the elongation of the membrane-anchored precursor, Man2-DAG, in mms2 cells on the outer surface of the cytoplasmic membrane (Pakkiri et al., 2004), resulting in the accumulation of Man2-DAG. In accord with the proposed pathway in Figure 1, Man2-DAG could be readily converted to LM when exogenous Man-P-Und was added in vitro.
The data from these studies clearly indicate that the synthesis of the lipid intermediates, Man2-DAG and Man-P-Und, is essential for LM formation and normal cell growth as indicated by growth defects at the restrictive temperature (37°C). The mutant cells are also partially defective in these biosynthetic steps under normal growth condition (data not shown), suggesting that residual enzyme activities may be sufficient for the viability and propagation of these mutant cells. It has been reported that some ts mutants of S. cerevisiae showed similar phenotypes and are also partially defective at the nonrestrictive temperature (Huffaker and Robbins, 1983; Runge and Robbins, 1986
).
These studies establish that the [3H]mannose-suicide selection procedure is an effective approach to isolating specific micrococcal mutant variants with defects in the enzymes catalyzing various steps in LM assembly. Based on the potential biochemical defect(s) in the LM assembly steps, the mutants can be categorized into three classes that would ostensibly allow the mutant cells to survive [3H]mannose-suicide. Class I micrococcal mutants are those that are blocked in mannose transport/entry or the biosynthesis of GDP-Man. Several class 1 mutants were identified after metabolic labeling with [3H]mannose in vivo (data not shown). The in vivo results in this study firmly establish that mms1 and mms2 cells do not belong to this category because [3H]mannose is incorporated into [3H]Man1-2DAG (Figure 5). Micrococcal mutants that are defective in the synthesis of mannolipid intermediates, Man1-2-DAG and Man-P-Und from GDP-Man or the exoplasmic Man-P-Und-mediated transfer of mannosyl units to LM (Figure 1, steps 67), are classified as class II.
Finally, class III mutants, which are potentially the most interesting, are defined as mutants that are impaired in the transverse diffusion of Man-P-Und or Man2-DAG (Figure 1, steps 45) from the inner leaflet to outer monolayer of the cytoplasmic membrane. Prospective class III mutants with defects in membrane proteins mediating the transbilayer movement of Man-P-Und and Man2-DAG will be based on assaying the loss of transport of the chemoenzymatically synthesized, water-soluble analogs, Man-P-nerol (Man-P-Und) and sn-1,2-diC4-DAG-[3H]Man2 (Man2-DAG) into intact protoplasts or sealed micrococcal membrane vesicles. This approach has successfully implicated endoplasmic reticulum proteins in the transbilayer movement of Man-P-dolichol (Rush and Waechter, 1995, 2004
) and Glc-P-dolichol (Rush and Waechter, 1998
) in liver and brain microsomes, and Fuc4NAc-ManNAcA-GlcNAc-P-P-Und (Lipid III), the trisaccharyl donor in the biosynthesis of enterobacterial common antigen in Escherichia coli (Rick et al., 2003
).
In summary, the in vitro and in vivo results with the two ts mutants strongly suggest that mms1 cells have a defect in the mannosyltransferase forming Man2-DAG and mms2 cells have a defect in the enzyme synthesizing Man-P-Und. The results also suggest that Man2-DAG may be essential for normal growth of M. luteus cells. To obtain definitive evidence for these conclusions, it will be necessary to identify the genes encoding the mannosyltransferases by complementing the ts defects. Work is in progress to identify the micrococcal genes encoding the mutations in mms1 and mms2 by complementing the ts phenotype with a wild-type genomic DNA library, similar to the cloning of the alg genes (Aebi et al., 1996; Couto et al., 1984
; Jackson et al., 1993
) and other genes related to the dolichol pathway (Shridas et al., 2003
). In any case, the deficiencies in Man2-DAG and LM in mms1 cells have provided an excellent model system for establishing for the first time that Man2-DAG functions as the lipid anchor precursor for LM in M. luteus cells. Another future goal in the micrococcal system will be to elucidate the mechanism and topology of the formation of the succinyl ester linkages on the LM chains as well as their physiological function(s). M. luteus and the related micrococcal species, Micrococcus flavus and Micrococcus sodonensis, contain succinylated mannosyl residues in LM. The micrococcal LM is proposed to bind Mg2+ (Powell et al., 1975
) essential for functional DD-carboxypeptidases, transpeptidases, and autolytic enzymes involved in peptidoglycan remodeling during cell growth and division (Salton, 1980
). It remains to be established if the loss of the succinylated LM and consequently the inability to bind Mg2+ is related to the growth sensitivity of the LM-deficient mms1 and mms2 cells at the restrictive temperature.
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Materials and methods |
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Preparation of cell homogenates and membranes
Cells were grown by shaking liquid cultures at 30°C on LB media consisting of 1% bactopeptone, 1% NaCl, and 0.5% yeast extract. During exponential growth (A600 1.2), the cells were shifted to 37°C for 6 h. These were then sedimented by centrifugation (3000 x g, 10 min), and washed twice with distilled water and once with 50 mM TrisHCl (pH 7.6) containing 5 mM EDTA (buffer A) before resuspension in the same buffer (20 ml). Lysozyme (150 µg/ml), DNase 1 (10 µg/ml), and RNase (5 µg/ml) were added to a stirred cell suspension for 45 min at 4°C, and the cells were disrupted by passage through a French pressure cell (18,000 psi). This procedure was repeated three times at 4°C before placing the crude cell lysate on ice for an additional 15 min until a decrease in viscosity was observed. Unbroken cells were sedimented by centrifugation (3000 x g, 10 min) and discarded. The resulting lysate was homogenized by 810 strokes with a Dounce homogenizer using pestle B and stored at 20°C at a final protein concentration of 1015 mg/ml. Crude membranes were sedimented by centrifugation of cell homogenates at 4°C (50,000 x g, 1 h). The supernatant fluid was removed, and the membrane pellet washed four times with buffer A. The membrane pellet was resuspended at a final protein concentration of 810 mg/ml and stored at 20°C until further use.
Preparation of intact protoplasts
Cells harvested during exponential growth were sedimented by centrifugation (3000 x g, 10 min), washed with distilled water as described, and resuspended in 50 mM TrisHCl (pH 7.6) containing 0.8 M sucrose, 150 mM NaCl, and 10 mM MgCl2 (buffer B). Following incubation for 20 min at 22°C with lysozyme (150 µg/ml) and DNase 1 (1 µg/ml), protoplasts were sedimented by centrifugation (20,000 x g, 20 min). The protoplasts were washed once with buffer B, resuspended at a final protein concentration of 10 mg/ml, and stored at 20°C. The intactness of the protoplasts was assessed by incubating with GDP-[3H]Man as described (Pakkiri et al., 2004).
Staining of protoplasts with NAO and FITC-Con A conjugate
Protoplasts (15 mg) were washed twice with a buffer containing 25 mM HEPES (pH 7.4), 0.8 M sucrose, 150 mM NaCl, 1 mM CaCl2, 1 mM MnCl2, and 0.5 mM MgCl2 (buffer C). The washed protoplasts were resuspended and incubated with buffer C (0.2 ml) containing 2% bovine serum albumin (BSA) at 30°C. After 1 h, the protoplasts were sedimented by centrifugation (20,000 x g, 20 min) to remove the BSA and resuspended in buffer C containing 20 µg of either NAO or FITC-conjugated Con A in a final volume of 0.2 ml. After 1 h at 30°C, the incubation mixture was centrifuged to remove unbound fluorescent conjugates, washed three times, and resuspended in 0.05 ml of buffer C. Samples (1 µl) were placed on a microscope slide and visualized by light microscopy (Nikon Eclipse E600) under bright field or fluorescence to assess the surface exposure of CL and LM. Images were recorded with a SPOT camera (Diagnostic Instruments) and manipulated with Adobe Photoshop 7.0.
Isolation of ts mutants
A [3H]mannose-suicide selection was performed essentially by the procedure of Huffaker and Robbins (1982) with minor modifications. Cells were grown exponentially in 10 ml LB media at 30°C and sedimented by centrifugation (3000 x g, 10 min). The cell pellet was resuspended in 0.5 ml of 100 mM sodium phosphate (pH 7.0) containing 3% EMS. After 2 h at 30°C, the mutagenic reaction was terminated by diluting with 40 vols of 5% sodium thiosulfate, and the cells were sedimented by centrifugation (3000 x g, 10 min) and washed three times with ice-cold distilled water (autoclaved). The survival rate of the EMS-treated cells was
40% as determined by plating on LB agar at 30°C. The mutagenized cells were grown in 5 ml of LB media by shaking at 30°C for 16 h (A600 1.2), shifted to 37°C for 30 min, and sedimented and resuspended in 0.3 ml LB media containing 0.5 mCi of [2-3H]mannose. In control experiments, nonradioactive mannose was used. After 1 h at 37°C, the cells were sedimented by centrifugation (3000 x g, 10 min), washed three times with sterile ice-cold distilled water, and resuspended in 2.0 ml of 25% glycerol and 50% LB. Aliquots of the suspensions were stored at 80°C.
Growth of the radiation-exposed mutants was monitored by periodically thawing an aliquot and scoring for growth on LB agar at 30°C. After 35 days of storage at 80°C, cell viability was reduced to less than 1% of the unexposed cell population. The surviving cells were subjected to a second round of [3H]mannose-suicide selection and were stored at 80°C. After 35 days, the cells were serially diluted, plated on LB agar (150200 colonies/plate) and incubated at 30°C for 30 h. This master plate was replica-plated onto LB agar and incubated at either 30°C (permissive) or 37°C (nonpermissive). After 30 h, the growth of the colonies on each plate was compared with the master plate. Micrococcal strains that were able to grow at 30°C, but not at 37°C were isolated and analyzed further for ts defects in mannolipid and LM biosynthesis.
Extraction and purification of DAG and Man12DAG
Cells were grown exponentially at 30°C, shifted to 37°C for 6 h, and sedimented by centrifugation (3000 x g, 10 min). Crude lipids (0.5 g) were extracted from membranes with 20 vols of CHCl3/CH3OH (2:1). The lipid extracts were then washed sequentially with 1/5 vol of 0.9% NaCl and CHCl3/CH3OH/H2O (3:48:47). The washed lower (organic) phases were concentrated by rotary evaporation under reduced pressure at 30°C (Buchi Rotavapor). The lipid extracts were redissolved in CHCl3 (0.3 ml) and applied to a silicic acid column (2.5 x 15 cm) preequilibrated with CHCl3. The column was sequentially eluted with 5 column vols of CHCl3 to recover free fatty acids and DAG, 20 column vols of acetone to elute neutral mannolipids (Man12-DAG), and 5 column vols of CHCl3/CH3OH (1:1) to elute phospholipids. The partially purified fractions were concentrated to dryness by rotary evaporation and resuspended in 100150 µl of CHCl3/CH3OH (2:1). Mannolipids were analyzed by thin-layer chromatography on silica gel plates by developing with CHCl3/CH3OH/H2O (65:25:4) and detected by spraying with an orcinol/sulfuric acid solution (Churms and Zweig, 1982). Phospholipids are detected by spraying with a phosphate-specific reagent (Dittmer and Lester, 1964
). For further purification, the lipid fractions were redissolved in CHCl3 (0.3 ml) and applied to a DEAE-cellulose column (2.5 x 10 cm) preequilibrated with CHCl3. Neutral lipids were eluted with 8 column vols of CHCl3, Man-DAG was eluted with 8 column vols of CHCl3/CH3OH (98:2), and Man2-DAG was eluted with 10 column vols of CHCl3/CH3OH (90:10) (Kates, 1972
). Highly purified samples were obtained by preparative thin-layer chromatography on silica plates developed with petroleum ether/ethyl ether/acetic acid (80:20:1) for DAGs or CHCl3/CH3OH/H2O (65:25:4) for Man12-DAG.
Radiolabeled Man12-DAG were synthesized by metabolically labeling wild-type cells grown to A600 1.2 on 50 ml LB media, and sedimented by centrifugation (3000 x g, 10 min). The cells were resuspended in fresh LB media (1 ml) containing 50100 µCi of [2-3H]mannose and incubated for 3 h at 22°C. The metabolically labeled [3H]Man12-DAGs were extracted and purified by chromatography on DEAE-cellulose and silica plates as described. Man-P-Und was enzymatically synthesized and purified from partially purified preparations of Man-P-Und synthase (Rush et al., 1993).
In vitro assay for mannosyltransferase activities
The micrococcal mannosyltransferases were assayed in vitro by procedures described previously (Pakkiri et al., 2004). Typical reaction mixtures contained cell homogenates (1.9 mg protein), 5080 mM TrisHCl (pH 7.68.0),20 mM MgCl2, and 15 µM GDP-[3H]Man (150 cpm/pmol) in a total volume of 0.15 ml. After incubation at 37°C, the enzymatic reactions were terminated by the addition of 20 vols of CHCl3/CH3OH (2:1). The lipid extracts containing the [3H]mannolipids were saved, and the delipidated membrane residues were extracted twice with 1 ml CHCl3/CH3OH (2:1). The lipid extracts (5 ml) were pooled and washed with 1/5 volume of 0.9% NaCl to remove unreacted GDP-[3H]Man. The organic (lower) phase was washed twice with CHCl3/CH3OH/H2O (3:48:47), and the organic solvent was evaporated to dryness under N2. An aliquot was taken to determine the total amount of [3H]mannose incorporated into the [3H]mannolipids, and another aliquot was used to analyze the reaction products by chromatography on thin-layer silica gel plates developed with CHCl3/CH3OH/H2O (65:25:4). The plates were dried at room temperature, and the radiolabeled products were located with an AR-2000 Imaging Scanner (BioScan, Washington, DC) to determine the distribution of [3H]Man-DAG, [3H]Man2-DAG, and [3H]Man-P-Und. The rate of synthesis of each mannolipid was determined by multiplying the percentage of each radiolabeled mannolipid by the total amount of [3H]mannolipid formed. The incorporation of [3H]mannose into the membrane-associated LM was determined by washing the delipidated membrane residue with 3 ml CH3OH-0.9% NaCl (1:1) and then twice with 3 ml CH3OH/H2O (1:1) to remove residual unreacted GDP-[3H]Man. The delipidated membrane residue was solubilized in 0.5 ml of 1% Sodium dodecyl sulfate (100°C, 5 min) and mixed with 4 ml scintillation cocktail. The amount of [3H]LM formed was determined by liquid scintillation spectrometry.
Deacylation of [3H]LM from wild-type and mms1 cells by mild alkali treatment
[3H]LM from wild-type and mms1 cells was synthesized in vitro as described earlier by incubating membranes (1 mg protein) with 74 mM Tris-HCl (pH 8.0), 20 mM MgCl2, 1 mM GDP-Man, and purified exogenous [3H]Man2-DAG (50,000 cpm) dispersed in 0.5% sodium taurocholate (final conc. 0.05%, w/v) by ultrasonication, in a total volume of 0.15 ml for 1 h at 30°C. The enzymatically radiolabeled preparations were deacylated by mild alkali treatment (0.2 ml 0.1 N KOH in CH3OH/toluene, 3:1 for 1 h at 4°C) (Waechter and Lester, 1973). The reactions were neutralized by adding 0.2 ml 0.1 N acetic acid followed by 20 volumes of CHCl3/CH3OH (2:1) and 3 vols of 0.9% NaCl. The binary mixtures were mixed vigorously and placed on ice for 5 min before centrifugation (2000 x g, 1 min). The deacylated [3H]LM from wild-type and mms1 membranes were recovered from the upper (aqueous) phase and carefully separated from the lower (organic) phase. The molecular size of the deacylated LM was estimated by gel filtration chromatography on a Bio-Gel P-10 column (1.8 x 20 cm), equilibrated, and eluted with 0.1 N acetic acid. Fractions (1.4 ml) were collected using a Gilson (Middleton, WI) FC-80K microfractionator, and the amount of radioactivity in each fraction was determined by liquid scintillation spectrometry.
-Mannosidase treatment of deacylated [3H]LM from wild-type and mms1 cells
[3H]LM from wild-type and mms1 cells was deacylated as described and treated with jack bean -mannosidase (5 U) in 0.1 M sodium acetate (pH 6.0) in a total volume of 0.2 ml for 4 h at 30°C. The release of free [3H]mannose was assessed by gel filtration chromatography on Bio-Gel P-10.
Quantitative compositional analysis of phospholipids, mannolipids, and LM
Lipids extracted from membranes of wild-type, mms1, and mms2 cells grown exponentially at 30°C and shifted to 37°C for 6 h were sequentially purified by chromatography on silicic acid and DEAE-cellulose as described earlier. The phosphorus content in phospholipids (CL, PG, and PI) was determined by the method of Bartlett (1959) and the mannose content in Man12DAG and deacylated LM was determined by phenol-sulfuric method (Dubois et al., 1956
).
General analytical methods
Protein concentrations were estimated with the Pierce (Rockford, IL) BCA Protein Assay Reagent using BSA as standard. Und-P and Man-P-Und were detected by staining the thin-layer chromatography plates with phospholipid reagent (Dittmer and Lester, 1964), and Man12-DAG were detected by spraying with water, Rhodamine 6G (Kates, 1972
) or orcinol-sulfuric acid (Churms and Zweig, 1982
). The amount of radioactivity in the various samples was determined by scintillation counting in a Packard Tri-Card 2100TR liquid scintillation spectrometer (Packard Instrument, Meriden, CT) after the addition of Econosafe Liquid Scintillation Counting Cocktail.
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Acknowledgements |
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Abbreviations |
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References |
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