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• W1979156768 abstract "The variant CHO-K1 cell line, NRel-4, is unable to synthesize plasmalogens because of a severe reduction in dihydroxyacetonephosphate acyltransferase (DHAPAT) activity (Nagan, N., A. K. Hajra, L. K. Larkins, P. Lazarow, P. E. Purdue, W. B. Rizzo, and R. A. Zoeller. 1998. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol. Biochem. J. 332: 273–279). Northern analysis demonstrated that the loss of this activity was attributable to a severe reduction in mRNA levels for DHAPAT. Transfection of NRel-4 cells with a plasmid bearing the human DHAPAT cDNA recovered DHAPAT activity and plasmalogen biosynthesis. Examination of clonal isolates from the transfected population showed that recovery of as little as 10% of wild-type DHAPAT activity restored plasmalogen levels to 55% of normal, whereas in one isolate, NRel-4.15, which overexpressed DHAPAT activity by 6-fold over wild-type cells, plasmalogen levels were returned only to wild-type values. Although the rate of plasmenylethanolamine biosynthesis was restored in NRel-4.15, the biosynthesis of nonether glycerolipids was either decreased or unaffected, suggesting that peroxisomal DHAPAT does not normally contribute to nonether glycerolipid biosynthesis.These data demonstrate that a defect in the gene that codes for peroxisomal DHAPAT is the primary lesion in the NRel-4 cell line and that the peroxisomal DHAPAT is essential for the biosynthesis of plasmalogens in animal cells. The variant CHO-K1 cell line, NRel-4, is unable to synthesize plasmalogens because of a severe reduction in dihydroxyacetonephosphate acyltransferase (DHAPAT) activity (Nagan, N., A. K. Hajra, L. K. Larkins, P. Lazarow, P. E. Purdue, W. B. Rizzo, and R. A. Zoeller. 1998. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol. Biochem. J. 332: 273–279). Northern analysis demonstrated that the loss of this activity was attributable to a severe reduction in mRNA levels for DHAPAT. Transfection of NRel-4 cells with a plasmid bearing the human DHAPAT cDNA recovered DHAPAT activity and plasmalogen biosynthesis. Examination of clonal isolates from the transfected population showed that recovery of as little as 10% of wild-type DHAPAT activity restored plasmalogen levels to 55% of normal, whereas in one isolate, NRel-4.15, which overexpressed DHAPAT activity by 6-fold over wild-type cells, plasmalogen levels were returned only to wild-type values. Although the rate of plasmenylethanolamine biosynthesis was restored in NRel-4.15, the biosynthesis of nonether glycerolipids was either decreased or unaffected, suggesting that peroxisomal DHAPAT does not normally contribute to nonether glycerolipid biosynthesis. These data demonstrate that a defect in the gene that codes for peroxisomal DHAPAT is the primary lesion in the NRel-4 cell line and that the peroxisomal DHAPAT is essential for the biosynthesis of plasmalogens in animal cells. Plasmalogens represent a subset of phospholipids that differ from other glycerolipids in that, attached to the sn-1 position of the glycerol backbone is an alkyl chain attached through a vinyl ether linkage (1Nagan N. Zoeller R.A. Plasmalogens: biosynthesis and functions.Prog. Lipid Res. 2001; 40: 199-229Crossref PubMed Scopus (469) Google Scholar). The vinyl ether gives plasmalogens their unique physical and chemical properties. Every mammalian cell contains plasmalogens. In most cells, plasmalogens are represented as a subclass of ethanolamine phospholipids, in which they can represent a major portion of that head group class (2Horrocks L.A. Sharma M. Plasmalogens and O-alkylglycerophospholipids.in: Neuberger A. van Deenen L.L.M. New Comprehensive Biochemistry. Elsevier Biomedical Press, Amsterdam1982: 51-95Crossref Scopus (118) Google Scholar). Typically less abundant are choline plasmalogens, although in certain tissues, such as heart and muscle, they are found at relatively high levels. Plasmalogens have been proposed to be important for the stimulated release of arachidonic acid from phospholipid pools (3Ford D.A. Gross R.W. Plasmenylethanolamine is the major storage depot for arachidonic acid in rabbit vascular smooth muscle and is rapidly hydrolyzed after angiotensin II stimulation.Proc. Natl. Acad. Sci. USA. 1989; 86: 3479-3483Crossref PubMed Scopus (109) Google Scholar, 4Yang H.C. Farooqui A.A. Horrocks L.A. Characterization of plasmalogen-selective phospholipase A2 from bovine brain.Adv. Exp. Med. Biol. 1996; 416: 309-313Crossref PubMed Google Scholar, 5Creer M.H. McHowat J. Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation.Am. J. Physiol. 1998; 275: C1498-C1507Crossref PubMed Google Scholar), the fusion of biological membranes (6Glaser P.E. Gross R.W. Plasmenylethanolamine facilitates rapid membrane fusion: a stopped-flow kinetic investigation correlating the propensity of a major plasma membrane constituent to adopt an HII phase with its ability to promote membrane fusion.Biochemistry. 1994; 33: 5805-5812Crossref PubMed Scopus (176) Google Scholar), and the protection against reactive oxygen species (7Zoeller R.A. Morand O.H. Raetz C.R.H. A possible role for plasmalogens in protecting animal cells against photosensitized killing.J. Biol. Chem. 1988; 263: 11590-11596Abstract Full Text PDF PubMed Google Scholar, 8Morand O.H. Zoeller R.A. Raetz C.R.H. Disappearance of plasmalogens from membranes of animal cells subjected to photosensitized oxidation.J. Biol. Chem. 1988; 263: 11597-11606Abstract Full Text PDF PubMed Google Scholar, 9Zoeller R.A. Lake A.C. Nagan N. Gaposchkin D.P. Legner M.A. Lieberthal W.W. Plasmalogens as endogenous antioxidants: somatic cell mutants reveal the importance of the vinyl ether.Biochem. J. 1999; 338: 769-776Crossref PubMed Scopus (192) Google Scholar, 10Engelmann B. Brautigam C. Thiery J. Plasmalogen phospholipids as potential protectors against lipid peroxidation of low density lipoproteins.Biochem. Biophys. Res. Commun. 1994; 204: 1235-1242Crossref PubMed Scopus (115) Google Scholar). The mechanism for the maintenance of plasmalogen levels and the rate-limiting factor(s) for plasmalogen biosynthesis have not been identified. Although the pathway for plasmenylethanolamine biosynthesis has been established, the exact biosynthetic pathway for plasmenylcholine has not. Unlike plasmenylethanolamine, the saturated ether lipid species, plasmanylcholine is not a precursor for plasmenylcholine (11Lee T.C. Qian C.G. Snyder F. Biosynthesis of choline plasmalogens in neonatal rat myocytes.Arch. Biochem. Biophys. 1991; 286: 498-503Crossref PubMed Scopus (24) Google Scholar). Plasmenylcholine appears to be synthesized from plasmenylethanolamine through the replacement of ethanolamine with choline (11Lee T.C. Qian C.G. Snyder F. Biosynthesis of choline plasmalogens in neonatal rat myocytes.Arch. Biochem. Biophys. 1991; 286: 498-503Crossref PubMed Scopus (24) Google Scholar, 12Ford D.A. Gross R.W. Identification of endogenous 1-O-alk-1′-enyl-2-acyl-sn-glycerol in myocardium and its effective utilization by choline phosphotransferase.J. Biol. Chem. 1988; 263: 2644-2650Abstract Full Text PDF PubMed Google Scholar) or possibly through the methylation of plasmenylethanolamine (13Mozzi R. Gramignani D. Andriamampandr C. Freysz L. Massarelli R. Choline plasmalogen synthesis by the methylation pathway in chick neurons in culture.Neurochem. Res. 1989; 14: 579-583Crossref PubMed Scopus (10) Google Scholar). Our lack of knowledge in this area is attributable, in part, to the lack of molecular tools for studying the system. Only recently have any of the enzymes that participate in the biosynthetic pathway for plasmalogens been purified or the genes that code for them cloned (14Thai T.P. Heid H. Rackwitz H.R. Hunziker A. Gorgas K. Just W.W.W. Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase.FEBS Lett. 1997; 420: 205-211Crossref PubMed Scopus (57) Google Scholar, 15de Vet E.C. Zomer A.W. Lahaut G.J. van den Bosch H. Polymerase chain reaction-based cloning of alkyl-dihydroxyacetonephosphate synthase complementary DNA from guinea pig liver.J. Biol. Chem. 1997; 272: 798-803Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Although early steps in any biosynthetic pathway are commonly candidates as control points, this paradigm may not apply to plasmalogen biosynthesis. The regulation of plasmalogen biosynthesis is likely to be complex and linked to the biosynthesis of nonether glycerolipids, because later steps in the pathway are also used in the synthesis of diacyl phospholipids and triglycerides (1Nagan N. Zoeller R.A. Plasmalogens: biosynthesis and functions.Prog. Lipid Res. 2001; 40: 199-229Crossref PubMed Scopus (469) Google Scholar). Recent evidence also suggests that dihydroxyacetonephosphate acyltransferase (DHAPAT), which catalyzes the first step in plasmalogen biosynthesis, may play a significant role in nonether glycerolipid biosynthesis as well; this activity is increased several-fold during the differentiation of 3T3-L1 cells to adipocytes (16Hajra A.K. Larkins L.K. Das A.K. Hemati N. Erickson R.L. MacDougald O.A.A. Induction of the peroxisomal glycerolipid-synthesizing enzymes during differentiation of 3T3-L1 adipocytes. Role in triacylglycerol synthesis.J. Biol. Chem. 2000; 275: 9441-9446Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Therefore, the role of DHAPAT in the synthesis of this latter group of glycerolipids (nonether) remains to be determined. We have described the isolation of variants of the fibroblast-like cell line CHO-K1 that are deficient in plasmalogens as a result of the loss of various steps along the biosynthetic pathway (17James P.F. Lake A.C. Hajra A.K. Larkins L.K. Robinson M. Buchanan F.G. Zoeller R.A.A. An animal cell mutant with a deficiency in acyl/alkyl-dihydroxyacetone-phosphate reductase activity. Effects on the biosynthesis of ether-linked and diacyl glycerolipids.J. Biol. Chem. 1997; 272: 23540-23546Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 18Nagan N. Hajra A.K. Das A.K. Moser H.W. Moser A. Lazarow P. Purdue P.E. Zoeller R.A.A. A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis.Proc. Natl. Acad. Sci. USA. 1997; 94: 4475-4480Crossref PubMed Scopus (36) Google Scholar, 19Nagan N. Hajra A.K. Larkins L.K. Lazarow P. Purdue P.E. Rizzo W.B. Zoeller R.A.A. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.Biochem. J. 1998; 332: 273-279Crossref PubMed Scopus (34) Google Scholar). These cell lines can be useful in identifying the factors that determine plasmalogen levels as well as in defining the relationship between ether lipids and diacyl lipid synthesis. The mutant CHO cell line NRel-4 displays greatly reduced plasmalogen levels because of a loss of DHAPAT (19Nagan N. Hajra A.K. Larkins L.K. Lazarow P. Purdue P.E. Rizzo W.B. Zoeller R.A.A. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.Biochem. J. 1998; 332: 273-279Crossref PubMed Scopus (34) Google Scholar). In this report, we show that the loss of this activity is attributable to reduced levels of the message for DHAPAT, making NRel-4 a useful null cell model for examining the role of DHAPAT in the regulation of plasmalogen levels. We have expressed the human DHAPAT gene in NRel-4 cells to demonstrate that this can recover plasmalogen biosynthesis in these cells and to examine what effect varied levels of DHAPAT activity have on the synthesis of both ether and nonether glycerolipids. [1-3H]ethanolamine (30 Ci/mmol; 1 Ci = 37 gigabecquerel) was obtained from Amersham (Arlington Heights, IL). [γ-32P]ATP, [32P]inorganic phosphate (32Pi), [9,10-3H]oleic acid, and EN3 HANCE spray were obtained from Perkin-Elmer/New England Nuclear (Boston, MA). 32P-labeled dihydroxyacetonephosphate (DHAP) and glycerol-3-phosphate (Gro-3-P) were synthesized by enzymatic phosphorylation of dihydroxyacetone or glycerol using [γ-32P]ATP and glycerol kinase (20Schlossman D.M. Bell R.M. Microsomal sn-glycerol 3-phosphate and dihydroxyacetone phosphate acyltransferase activities from liver and other tissues. Evidence for a single enzyme catalyzing both reactions.Arch. Biochem. Biophys. 1977; 182: 732-742Crossref PubMed Scopus (60) Google Scholar). Ecoscint A liquid scintillation fluid was obtained from National Diagnostics (Atlanta, GA). Silica gel 60 TLC plates (Merck) were purchased from American Scientific Products (McGaw Park, IL). Tissue culture dishes (Corning) were obtained from VWR Scientific (Boston, MA). All other reagents, unless specified, were purchased from Sigma (St. Louis, MO). CHO-K1 cells were obtained from the American Type Culture Collection (Manassas, VA). Plasmenylethanolamine is the dominant plasmalogen species, constituting ∼35–40% of the ethanolamine phospholipids; there is almost no plasmenylcholine. NRel-4 is a plasmalogen-deficient CHO-K1-derived cell line with a severe deficiency in DHAPAT activity (19Nagan N. Hajra A.K. Larkins L.K. Lazarow P. Purdue P.E. Rizzo W.B. Zoeller R.A.A. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.Biochem. J. 1998; 332: 273-279Crossref PubMed Scopus (34) Google Scholar). NZel-1 is a plasmalogen-deficient variant of CHO-K1 defective in alkyl-DHAP synthase (18Nagan N. Hajra A.K. Das A.K. Moser H.W. Moser A. Lazarow P. Purdue P.E. Zoeller R.A.A. A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis.Proc. Natl. Acad. Sci. USA. 1997; 94: 4475-4480Crossref PubMed Scopus (36) Google Scholar). Cells were grown in Ham's F12 medium supplemented with 10% fetal bovine serum (Atlas Biologicals, Denver, CO) at 37°C in 5% CO2. The human DHAPAT cDNA (14Thai T.P. Heid H. Rackwitz H.R. Hunziker A. Gorgas K. Just W.W.W. Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase.FEBS Lett. 1997; 420: 205-211Crossref PubMed Scopus (57) Google Scholar) was removed from the pBluescript SK phagemid vector and inserted into the shuttle vector pBK-CMV (Stratagene, La Jolla, CA) using the EcoRI (5′) and XhoI (3′) restriction sites. The construct was used to transform XL1-Blue MRF′, which was then grown on kanamycin-containing agar to form colonies. Selected white colonies were expanded and cDNA preparations were analyzed by restriction analysis, and those strains whose cDNA demonstrated the appropriate agarose electrophoresis patterns were further expanded for large-scale DNA preparation using an endotoxin-free plasmid kit supplied by Qiagen (Valencia, CA). The cDNA inserts were sequenced to ensure that DHAPAT cDNA was unaltered and properly inserted. NRel-4 cells were transfected using SuperFect transfection reagent (Qiagen), and the transfected populations were grown for 6 weeks in G418 (500 μg/ml) to develop a stably transfected population. To generate clonal isolates, a cell suspension was diluted to a concentration of 2 cells/ml, and 200 μl aliquots were transferred to the wells of a 96-well plate. After 10 days of growth at 37°C, wells were examined for the formation of a single colony. The cells in these wells were harvested with trypsin and expanded for further study. Conditioned medium (medium in which cells had been previously grown) was filtered using a sterile 0.22 μm filter and used for the dilutions and transfer to the 96-well plates. This was necessary to ensure that the cells would grow at very low density. Glycerol-3-phosphate acyltransferase (Gro-3-PAT) and DHAPAT activities were measured using whole-cell homogenates that were prepared as described previously (18Nagan N. Hajra A.K. Das A.K. Moser H.W. Moser A. Lazarow P. Purdue P.E. Zoeller R.A.A. A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis.Proc. Natl. Acad. Sci. USA. 1997; 94: 4475-4480Crossref PubMed Scopus (36) Google Scholar). DHAPAT and Gro-3-PAT activities were measured as described by Jones and Hajra (21Jones C.L. Hajra A.K. Properties of guinea pig liver peroxisomal dihydroxyacetone phosphate acyltransferase.J. Biol. Chem. 1980; 255: 8289-8295Abstract Full Text PDF PubMed Google Scholar) using palmitoyl-CoA and either 32P-labeled Gro-3-P or DHAP as the acyl acceptor. Protein content was determined using the method of Lowry et al. (22Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Detection of DHAPAT in immobilized colonies (colony autoradiography) was performed as described previously (23Zoeller R.A. Raetz C.R.H. Strategies for isolating somatic cell mutants defective in lipid biosynthesis.Methods Enzymol. 1992; 209: 34-51Crossref PubMed Scopus (15) Google Scholar). Briefly, transfected cells were plated onto a 100 mm diameter tissue culture dish at 300 cells per dish and allowed to attach overnight. Cells were then overlaid with a sterile polyester cloth. The cells were allowed to grow and form colonies within the polyester matrix for 8–10 days at 37°C. The polyester bearing the colonies was removed from the dish, washed once with PBS, and placed at −20°C overnight. The polyester cloth was thawed and placed in a 100 mm tissue culture dish containing 3 ml of DHAPAT assay mix at pH 5.5 (4 μCi [32P]DHAP/ml) and incubated for 15 min at 37°C. The reaction was stopped by the addition of 3 ml of ice-cold 20% TCA. The cloth was then placed on a Buchner funnel and rinsed five times with 25 ml portions of ice-cold 3% TCA to remove TCA-soluble substrate; the TCA-insoluble product, acyl-[32P]DHAP, remained associated with the colony. After drying for 1 h, the cloth was exposed to X-ray film overnight at −80°C. To visualize the colonies, the cloth was placed in 0.5% (w/v) Coomassie blue in methanol-water-acetic acid (45:45:10, v/v) for 30 min followed by destaining with methanol-water-acetic acid (45:45:10, v/v). Cells (2.5 × 105) were grown for 18 h in sterile glass scintillation vials at 37°C in growth medium containing [1-3H]ethanolamine (1 μCi/ml). The medium was removed, the cells were washed once with 2 ml of PBS, and the lipids were extracted in 3.8 ml of CHCl3/methanol/PBS (1:2:0.8) containing 300 μg of a carrier lipid (beef heart lipids). After transferring to a test tube, 1 ml of CHCl3 and 1 ml of PBS were added to form a two-phase Bligh and Dyer system (24Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar), and the lower (organic) phase was collected after centrifugation. Solvent was removed using a stream of nitrogen, and the labeled phospholipids were separated by two-stage, single-dimension TLC (23Zoeller R.A. Raetz C.R.H. Strategies for isolating somatic cell mutants defective in lipid biosynthesis.Methods Enzymol. 1992; 209: 34-51Crossref PubMed Scopus (15) Google Scholar). The labeled species were visualized by exposure to X-ray film at −80°C after spraying the plates with EN3HANCE. Labeled bands were scraped into scintillation vials containing 1 ml of methanol, 8 ml of scintillation fluid was added, and radioactivity was quantitated by liquid scintillation spectrometry. A 454 bp fragment of the 5′ end of CHO-K1 DHAPAT was generated by polymerase chain reaction using primers 5′-ATGGACGTTCCTAGCTCCTN-3′ for the 5′ end and 5′-AGCAGGACTACAGGGTGCTC-3′ for the 3′ end. The primers were designed based on the nucleotide sequence of the CHO-K1 DHAPAT gene (D. Liu, unpublished data). Probe (100 ng) and 50 ng of pTRI-B-Actin-Mouse (Ambion) were labeled with [α-32P]ATP using the RadPrime kit (Invitrogen, Carlsbad, CA). Total RNA was isolated from cells using Trizol reagent (Invitrogen). RNA was fractionated using a denaturing 0.8% agarose-formaldehyde gel, transferred to nylon membranes (Schleicher and Schuell, Keene, NH), air-dried, and fixed by ultraviolet cross-linking at 1,200 joules. Nylon membranes were prehybridized in Church-Gilbert hybridization buffer (0.5 M sodium phosphate, pH 7.4, 7% sodium dodecylsulfate, 1% BSA, and 1.0 mM EDTA) for 4 h at 62°C. The radioactive probes were denatured by boiling for 5 min and added to the hybridization buffer. After overnight hybridization at 62°C, the blots were washed sequentially as follows: 2× saline-sodium citrate buffer [1× saline-sodium citrate buffer (SSC) = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0] containing 0.5% SDS for 15 min at 42°C, 0.5× SSC and 0.5% SDS at 42°C for 15 min, and 0.1× SSC and 0.5% SDS for 30 min at 42°C. The blots were then exposed to Kodak X-AR film at −80°C for 2 days. Immunoblotting using the anti-DHAPAT antibody was carried out as described previously (14Thai T.P. Heid H. Rackwitz H.R. Hunziker A. Gorgas K. Just W.W.W. Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase.FEBS Lett. 1997; 420: 205-211Crossref PubMed Scopus (57) Google Scholar). Briefly, confluent monolayers were scraped and pelleted, solubilized in Solu-CHAPS (1 mM dithiothreitol, 1 mM EDTA, 15 mM CHAPS, 50 mM Tris-HCl, and 150 mM NaCl, pH 7.0), and proteins were precipitated using 10% TCA. Proteins were separated using 12% SDS-PAGE and subsequently transblotted into polyvinylidene difluoride membranes. After blocking with PBS and 5% nonfat dry milk, blots were processed with anti-DHAPAT antibody. Horseradish peroxidase-conjugated secondary antibody and chemiluminescence (ECL kit; Amersham, San Francisco, CA) were used to visualize the bands. Immunofluorescence microscopy was carried out as described previously (25Soto U. Pepperkok R. Ansorge W. Just W.W. Import of firefly luciferase into mammalian peroxisomes in vivo requires nucleoside triphosphates.Exp. Cell Res. 1993; 205: 66-75Crossref PubMed Scopus (46) Google Scholar). Briefly, cells were grown on glass coverslips, washed, and fixed with 3% paraformaldehyde. After 5 min of permeabilization in 1% Triton X-100, cells were washed and incubated for 1 h with anti-catalase (25Soto U. Pepperkok R. Ansorge W. Just W.W. Import of firefly luciferase into mammalian peroxisomes in vivo requires nucleoside triphosphates.Exp. Cell Res. 1993; 205: 66-75Crossref PubMed Scopus (46) Google Scholar) and anti-DHAPAT (14Thai T.P. Heid H. Rackwitz H.R. Hunziker A. Gorgas K. Just W.W.W. Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase.FEBS Lett. 1997; 420: 205-211Crossref PubMed Scopus (57) Google Scholar) antibodies that had been raised in rabbits. After washing, secondary goat anti-rabbit antibodies (Sigma) coupled to either FITC (for DHAPAT) or tetramethyl rhodamine isothiocyanate (for catalase) were used to detect the proteins. Cells were then mounted on slides using moviol/paraphenylendiamine, and images were taken with a Zeiss Axiovert microscope. Cells were plated into sterilized glass scintillation vials and grown for 72 h at 37°C in medium containing 32Pi (2.5 μCi/ml) to label the phospholipids to constant specific activity. The medium was removed, the cell monolayer was washed once with PBS, and cellular lipids were extracted as described above. Phospholipids were separated using two-dimensional TLC (26Esko J.D. Raetz C.R.H. Mutants of Chinese hamster ovary cells with altered membrane phospholipid composition. Replacement of phosphatidylinositol by phosphatidylglycerol in a myo-inositol auxotroph.J. Biol. Chem. 1980; 255: 4474-4480Abstract Full Text PDF PubMed Google Scholar). Between dimensions, the region containing lipids was sprayed with 10 mM HgCl2 in glacial acetic acid to cleave the vinyl ether bond of the plasmenyl species, allowing for the separation of the resulting sn-1-lysophospholipid and the unaffected diacyl species (27Owens K. A two-dimensional thin-layer chromatographic procedure for the estimation of plasmalogens.Biochem. J. 1966; 100: 354-361Crossref PubMed Scopus (113) Google Scholar). Plates were exposed to GBX-2 X-ray film at −80°C after preflash. 32Pi-labeled phospholipids were scraped from the TLC plates directly into scintillation vials for quantitation by liquid scintillation spectrometry. For short-term labeling, cells were incubated for 3 h in medium containing 32Pi (20 μCi/ml) and samples were processed as above. To measure the turnover of total phospholipid pools, cells were labeled for 72 h with 32Pi (2.5 μCi/ml). Cells were harvested with trypsin and plated into sterile glass scintillation vials (105 cells/vial) in 1 ml of medium containing 32Pi (2.5 μCi/ml) and allowed to attach overnight. Labeling medium was removed and replaced with 2 ml of unlabeled medium. Cellular phospholipids were extracted at 0, 24, and 48 h after removal of the labeled medium. Solvent was removed by evaporation under a nitrogen stream, samples were resuspended in CHCl3, and an aliquot was transferred to a scintillation vial. After allowing the solvent to evaporate, 1 ml of methanol was added, followed by 8 ml of scintillation fluid, and the amount of chloroform-soluble radioactivity was determined using liquid scintillation spectrometry. Cells were plated into sterilized glass scintillation vials and allowed to attach overnight. Medium was removed and replaced with 1 ml of growth medium containing 2 μM [9,10-3H]oleic acid. After 3 h at 37°C, medium was removed, the cell monolayer was washed once with 3 ml of growth medium, and the cellular lipids were extracted as described above. An aliquot of each sample was run on silica gel 60 using n-hexane-ethyl ether-acetic acid (70:30:1, v/v) to separate neutral lipids; another aliquot was separated on silica gel 60 using chloroform-methanol-acetic acid-water (25:15:3:1.5, v/v) to separate phospholipid head group classes. Plates were exposed to GBX-2 X-ray film at −80°C after spraying with EN3HANCE. Labeled lipids were scraped from the TLC plates directly into scintillation vials containing 1 ml of methanol followed by the addition of 8 ml of liquid scintillation cocktail. Radioactivity was quantitated by liquid scintillation spectrometry. To determine the relative levels of the different subclasses (diacyl, plasmanyl, and plasmenyl) within a head group class, lipids were extracted from unlabeled cells as above and isolated by development on silica gel G plates using chloroform-methanol-acetic acid-water (25:15:3:1.5, v/v). The choline and ethanolamine phospholipids were recovered from the plate and treated with phospholipase C followed by benzoylation of the resulting diradylglycerols (28Blank M.L. Cress E.A. Lee P. Stephens N. Piantadosi C. Snyder F.F. Quantitative analysis of ether-linked lipids as alkyl- and alk-1-enyl-glycerol benzoates by high-performance liquid chromatography.Anal. Biochem. 1983; 133: 430-436Crossref PubMed Scopus (46) Google Scholar). Diacyl, alkylacyl, and alkenylacyl benzoylated derivatives were separated by TLC (silica gel G) using benzene-hexane-ethyl ether (50:45:5, v/v), visualized by spraying the TLC plate with water, and recovered from the plate using chloroform-methanol (2:1). Solvent was removed by evaporation under a nitrogen stream, and samples were resuspended in 100% ethanol and quantitated by absorbance at 230 nm. Multiple groups were analyzed by ANOVA, followed by Tukey's honestly significant difference multiple comparison procedure. The data in Table 2 were analyzed with Student's t-test.TABLE 2Subclass distribution within the ethanolamine and choline phospholipids in CHO-K1 and isolate 4.15Ethanolamine PhospholipidsCholine PhospholipidsStrainPlasmenylPlasmanylDiacylPlasmenylPlasmanylDiacyl% of head group classCHO-K145.8 ± 2.05.6 ± 2.948.6 ± 4.00.4 ± 0.525.4 ± 0.674.2 ± 1.2Isolate 4.1541.8 ± 1.54.3 ± 3.053.9 ± 4.4ND17.5 ± 0.7aValue versus CHO-K1 (P ⩽ 0.001) as determined by Student's t-test. All other comparisons yielded P values >0.05.82.5 ± 0.7aValue versus CHO-K1 (P ⩽ 0.001) as determined by Student's t-test. All other comparisons yielded P values >0.05.Cellular lipids were extracted and choline and ethanolamine phospholipids were isolated as described in Materials and Methods. Phospholipids were treated with phospholipase C, and the resulting diradylglycerols were benzoylated using benzoic anhydride. Benzoylated derivatives of the different radyl species, diacyl, alkylacyl (plasmanyl), and alkenylacyl (plasmenyl), were separated and quantitated as described in Materials and Methods. All values represent averages ± SD of three samples within one experiment. ND, not detected.a Value versus CHO-K1 (P ⩽ 0.001) as determined by Student's t-test. All other comparisons yielded P values >0.05. Open table in a new tab Cellular lipids were extracted and choline and ethanolamine phospholipids were isolated as described in Materials and Methods. Phospholipids were treated with phospholipase C, and the resulting diradylglycerols were benzoylated using benzoic anhydride. Benzoylated derivatives of the different radyl species, diacyl, alkylacyl (plasmanyl), and alkenylacyl (plasmenyl), were separated and quantitated as described in Materials and Methods. All values represent averages ± SD o" @default.
• W1979156768 created "2016-06-24" @default.
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• W1979156768 creator A5058505761 @default.
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• W1979156768 date "2005-04-01" @default.
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• W1979156768 title "Role of dihydroxyacetonephosphate acyltransferase in the biosynthesis of plasmalogens and nonether glycerolipids" @default.
• W1979156768 cites W1500405769 @default.
• W1979156768 cites W1504597376 @default.
• W1979156768 cites W1542916233 @default.
• W1979156768 cites W1559697816 @default.
• W1979156768 cites W1561532665 @default.
• W1979156768 cites W1574033922 @default.
• W1979156768 cites W1617550360 @default.
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• W1979156768 cites W2034384686 @default.
• W1979156768 cites W2035263095 @default.
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