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• W2068013527 abstract "We have previously identified two enzyme activities that transfer the acetyl group from platelet-activating factor (PAF) in a CoA-independent manner to lysoplasmalogen or sphingosine in HL-60 cells, endothelial cells, and a variety of rat tissues. These were termed as PAF:lysoplasmalogen (lysophospholipid) transacetylase and PAF:sphingosine transacetylase, respectively. In the present study, we have solubilized and purified this PAF-dependent transacetylase 13,700-fold from rat kidney membranes (mitochondrial plus microsomal membranes) based on the PAF:lysoplasmalogen transacetylase activity. The mitochondria and microsomes were prepared and washed three times, then solubilized with 0.04% Tween 20 at a detergent/protein (w/w) ratio of 0.1. The solubilized fractions from mitochondria and microsomes were combined and subjected to sequential column chromatographies on DEAE-Sepharose, hydroxyapatite, phenyl-Sepharose, and chromatofocusing. The enzyme was further purified by native-polyacrylamide gel electrophoresis (PAGE) and affinity gel matrix in which the competitive inhibitor of the enzyme, 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphoethanolamine was covalently attached to the CH-Sepharose. On SDS-PAGE, the purified enzyme showed a single homogeneous band with an apparent molecular mass of 40 kDa. The purified enzyme catalyzed transacetylation of the acetyl group not only from PAF to lysoplasmalogen forming plasmalogen analogs of PAF, but also to sphingosine producing N-acetylsphingosine (C2-ceramide). In addition, this enzyme acted as a PAF-acetylhydrolase in the absence of lipid acceptor molecules. These results suggest that PAF-dependent transacetylase is an enzyme that modifies the cellular functions of PAF through generation of other diverse lipid mediators. We have previously identified two enzyme activities that transfer the acetyl group from platelet-activating factor (PAF) in a CoA-independent manner to lysoplasmalogen or sphingosine in HL-60 cells, endothelial cells, and a variety of rat tissues. These were termed as PAF:lysoplasmalogen (lysophospholipid) transacetylase and PAF:sphingosine transacetylase, respectively. In the present study, we have solubilized and purified this PAF-dependent transacetylase 13,700-fold from rat kidney membranes (mitochondrial plus microsomal membranes) based on the PAF:lysoplasmalogen transacetylase activity. The mitochondria and microsomes were prepared and washed three times, then solubilized with 0.04% Tween 20 at a detergent/protein (w/w) ratio of 0.1. The solubilized fractions from mitochondria and microsomes were combined and subjected to sequential column chromatographies on DEAE-Sepharose, hydroxyapatite, phenyl-Sepharose, and chromatofocusing. The enzyme was further purified by native-polyacrylamide gel electrophoresis (PAGE) and affinity gel matrix in which the competitive inhibitor of the enzyme, 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphoethanolamine was covalently attached to the CH-Sepharose. On SDS-PAGE, the purified enzyme showed a single homogeneous band with an apparent molecular mass of 40 kDa. The purified enzyme catalyzed transacetylation of the acetyl group not only from PAF to lysoplasmalogen forming plasmalogen analogs of PAF, but also to sphingosine producing N-acetylsphingosine (C2-ceramide). In addition, this enzyme acted as a PAF-acetylhydrolase in the absence of lipid acceptor molecules. These results suggest that PAF-dependent transacetylase is an enzyme that modifies the cellular functions of PAF through generation of other diverse lipid mediators. Platelet-activating factor (PAF), 1The abbreviations used are:PAF, platelet-activating factor; alkylacetyl-GPC, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; GPE, sn-glycero-3-phosphoethanolamine; PAF-AH, platelet-activating factor acetylhydrolase; BSA, bovine serum albumin; DTT, dithiothreitol; Pefabloc, p-aminoethyl benzenesulfonyl fluoride; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis; TLC, thin layer chromatography; LCAT, lecithin-cholesterol acyltransferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid.1The abbreviations used are:PAF, platelet-activating factor; alkylacetyl-GPC, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; GPE, sn-glycero-3-phosphoethanolamine; PAF-AH, platelet-activating factor acetylhydrolase; BSA, bovine serum albumin; DTT, dithiothreitol; Pefabloc, p-aminoethyl benzenesulfonyl fluoride; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis; TLC, thin layer chromatography; LCAT, lecithin-cholesterol acyltransferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid.1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC), is a potent lipid mediator with a wide variety of biological activities related to physiological and pathological phenomena (1Snyder F. Biochim. Biophys. Acta. 1995; 1254: 231-249Crossref PubMed Scopus (184) Google Scholar, 2Izumi T. Shimizu T. Biochim. Biophys. Acta. 1995; 1259: 317-333Crossref PubMed Scopus (208) Google Scholar). PAF is produced by either de novo or remodeling pathway (1Snyder F. Biochim. Biophys. Acta. 1995; 1254: 231-249Crossref PubMed Scopus (184) Google Scholar). In addition, we found the PAF could also be metabolized by a novel pathway catalyzed by membrane-associated transacetylase that transfers the acetate group of PAF to lysoplasmalogen in HL 60 cells (3Lee T. Uemura Y. Snyder F. J. Biol. Chem. 1992; 267: 19992-20001Abstract Full Text PDF PubMed Google Scholar). This enzyme is CoA-independent and transfers the acetyl group from PAF to a variety of lysophospholipids acceptors in the order of radyl-GPC > radyl-glycerophosphoethanolamine (GPE) > acyl-glycerophosphoserine > acyl-glycerophosphoinositol > acyl-glycerophosphate > alkyl-glycerophosphate > fatty alcohol, whereas alkylglycerol, acylglycerol, or cholesterol are inactive as acceptors. This PAF-dependent transacetylase is participated in the biosynthesis of acyl analog of PAF (4Balestrieri M.L. Servillo L. Lee T. J. Biol. Chem. 1997; 272: 17431Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), which is the predominant molecular species of PAF in hematopoietic cells including endothelial cells, mast cells, and basophils, etc. (5Triggiani M. Schleimer R.P. Warner J.A. Chilton F.H. J. Immunol. 1991; 147: 660-666PubMed Google Scholar). In endothelial cells, PAF-dependent transacetylase activity is regulated by phosphorylation/dephosphorylation (4Balestrieri M.L. Servillo L. Lee T. J. Biol. Chem. 1997; 272: 17431Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Recently, we have demonstrated that a similar PAF-dependent transacetylase transfers the acetyl group of PAF to sphingosine in HL 60 cells (6Lee T. Ou M. Shinozaki K. Malone B. Snyder F. J Biol. Chem. 1996; 271: 209-217Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 7Lee T. Nigam S. Kumkel G. Prescott S.M. Platelet-activating Factor and Related Lipid Mediators in Health and Disease. Plenum Publishing Co., New York1996: 113-119Google Scholar). This enzyme activity appears to be responsible for the presence of acetylsphingosine (C2-ceramide) in the biological systems. For instance, C2-ceramide occurred in the micromolar range in undifferentiated and differentiated HL-60 cells (6Lee T. Ou M. Shinozaki K. Malone B. Snyder F. J Biol. Chem. 1996; 271: 209-217Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This is the concentration range that C2-ceramide has been shown to exert as a second messenger and a lipid mediator by many investigators (8Hannum Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1491) Google Scholar, 9Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Halmovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature. 1996; 380: 75-79Crossref PubMed Scopus (1711) Google Scholar). Since C2-ceramide has many biological activities that differ from PAF and sphingosine, therefore, this enzyme may serve as a modifier for the functions of PAF and sphingosine (6Lee T. Ou M. Shinozaki K. Malone B. Snyder F. J Biol. Chem. 1996; 271: 209-217Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Both enzyme activities are also found in rat tissues (6Lee T. Ou M. Shinozaki K. Malone B. Snyder F. J Biol. Chem. 1996; 271: 209-217Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Rat kidney membrane fractions have the highest PAF:sphingosine transacetylase activity, while both rat kidneys and lung have the highest PAF:lysoplasmalogen transacetylase activity. To elucidate the relationship of both enzyme activities, we attempted to purify the transacetylase from rat kidney membranes, in which the highest enzyme activity toward both lipid acceptors (6Lee T. Ou M. Shinozaki K. Malone B. Snyder F. J Biol. Chem. 1996; 271: 209-217Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 7Lee T. Nigam S. Kumkel G. Prescott S.M. Platelet-activating Factor and Related Lipid Mediators in Health and Disease. Plenum Publishing Co., New York1996: 113-119Google Scholar). In the present report, we have achieved purification of the transacetylase to homogeneity and shown that this enzyme possesses three catalytic activities, namely, PAF-acetylhydrolase, PAF:lysophospholipid transacetylase, and PAF:sphingosine transacetylase. l-O-Hexadecyl-2-acetyl-GPC was obtained from Sigma. 1-O-Hexadecyl-2-N-methylcarbamyl-GPC and C2-ceramide were purchased from Biomol. 1-O-Alkyl-2-acetyl-GPC was from Avanti. Sphingosine was bought from Matreya, Inc. Alkenyllyso-GPE was a product from Serdary Research Lab. 1-O-hexadecyl-2-[3H]acetyl-GPC was the product of NEN Life Science Products. DEAE-Sepharose, phenyl-Sepharose, activated CH-Sepharose, PBE 94, and Polybuffer 74 were from Pharmacia Fine Chemicals (Uppsala, Sweden). Hydroxyapatite was obtained from Bio-Rad. Adult male Sprague-Dawley rats were from Taconic. PAF:lysoplasmalogen transacetylase and PAF:sphingosine transacetylase activities were determined according to our previously described methods (3Lee T. Uemura Y. Snyder F. J. Biol. Chem. 1992; 267: 19992-20001Abstract Full Text PDF PubMed Google Scholar, 6Lee T. Ou M. Shinozaki K. Malone B. Snyder F. J Biol. Chem. 1996; 271: 209-217Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The assay system of PAF:lysoplasmalogen transacetylase consisted of 50 μm1-O-hexadecyl-2-[3H]acetyl-GPC (0.3 μCi), 300 μm lysoplasmalogen (suspended in 0.1% bovine serum albumin (BSA)-saline), 100 mm Tris-HCl (pH 7.4), 2 mm sodium acetate, and 10 mm EDTA in a total volume of 250 μl. Incubations were carried out at 37 °C for 15 min. The lipids were extracted by the method of Bligh and Dyer (10Bligh E.G. Dyer W.T. Can. J. Biochem. Physiol. 1959; 261: 911-917Crossref Scopus (42411) Google Scholar). The lipids were separated by thin layer chromatography (TLC) using a solvent system of CHCl3/CH3OH/CH3COOH/H2O (50:25:8:4, v/v/v/v), and the radioactivities of the areas corresponding to PAF and alk-1-enylacetyl-GPE were counted using liquid scintillation fluid. The assay system of PAF:sphingosine transacetylase contained 15 μm1-O-hexadecyl-2-[3H]acetyl-GPC (1 μCi), 50 μm sphingosine (suspended in equal molar ratio of BSA), 100 mm Tris-HCl (pH 7.4), 2 mm sodium acetate, and 10 mm EDTA in a total volume of 250 μl. Incubations were carried out at 37 °C for 30 min. The lipids were separated by TLC using a solvent system of CHCl3/CH3OH (90:10, v/v). The radioactivities of the areas corresponding to PAF and C2-ceramide were measured. PAF-AH activity was assayed according to the method previously described (11Blank M.L. Lee T. Fitzgerald V. Snyder F. J. Biol. Chem. 1981; 256: 175-178Abstract Full Text PDF PubMed Google Scholar). The assay system of PAF-AH was composed of 20 μm1-O-hexadecyl-2-[3H]acetyl-GPC (0.1 μCi), 1 mm EDTA, and 100 μm potassium phosphate (pH 8.0) in a total volume of 500 μl. Incubations were carried out at 37 °C for 10 min. The reaction was stopped by sequential additions of 1 ml of CHCl3, 1 ml of CH3OH, and 0.5 ml of 10% sodium bicarbonate. The upper phase was washed with 1 ml of CHCl3 three times, and the radioactivities in an aliquot of 0.4 ml were determined. The kidneys were dissected out from male rats weighing 150–250 g and homogenized with four volumes of 0.25 m sucrose, 20 mm Tris-HCl (pH 7.4), 1 mm DTT, 1 mm EDTA, and 1 μg/ml leupeptin. The homogenates were centrifuged at 440 × gfor 10 min; the supernatant was collected as postnuclear fraction. The postnuclear fraction was centrifuged at 15,000 × g for 10 min to isolate the mitochondrial pellets. The postmitochondrial fractions were centrifuged at 100,000 × g for 1 h to obtain the microsomal fractions. Both mitochondrial and microsomal fractions were washed with the same buffer solution twice, and the washed mitochondria and microsomes were used as a source for enzyme purification. Mitochondrial fractions were suspended in 20 mm Tris-HCl (pH 7.4) containing 0.04% Tween 20, 1 μg/ml leupeptin, 1 mm EDTA, and 1 mm DTT at a detergent/protein (w/w) ratio of 0.1. The mixture was gently stirred at 0 °C for 1 h and then centrifuged at 100,000 × g for 1 h. The enzyme activity was twice extracted from microsomal fractions by detergent in the same way as described for the mitochondria. High salt wash (0.5 mNaCl) without the presence of detergent (data not shown) could not further elute the enzyme activity from mitochondria or microsomes. We have observed that the mitochondrial fraction had the highest specific activity and the microsomal fraction contained the highest total activity of transacetylase among the subcellular fractions isolated from HL-60 cells (7Lee T. Nigam S. Kumkel G. Prescott S.M. Platelet-activating Factor and Related Lipid Mediators in Health and Disease. Plenum Publishing Co., New York1996: 113-119Google Scholar). Therefore, we decided to combine both solubilized mitochondria and microsomes as the starting materials for column chromatography. The solubilized enzyme was applied onto a column of DEAE-Sepharose (2.5 × 6.11 cm, 30 ml), which was equilibrated with 20 mm Tris-HCl (pH 7.4), 1 mm DTT, 1 μg/ml leupeptin, and 0.02% Tween 20. The column was washed with the same buffer solution and was then eluted with 0.15 m NaCl in 20 mm Tris-HCl (pH 7.4), 1 mm DTT, and 0.02% Tween 20. During initial experiments, the solubilized enzyme bound to the DEAE-Sepharose column was eluted by a linear gradient of NaCl. There was only one major eluted peak of enzyme activity that started to come out at 0.15 m NaCl from the column. Based on this information, stepwise elution of the enzyme activity with 0.15m NaCl from the DEAE-Sepharose column was performed subsequently in order to speed up the purification process. Active fractions of DEAE-Sepharose were applied onto a column of hydroxyapatite (1.5 × 8.49 cm, 15 ml), which was equilibrated with 20 mm Tris-HCl (pH 7.4), 1 mm DTT, 1 μg/ml leupeptin, and 0.02% Tween 20. The column was washed with the same buffer solution, and the enzyme activity was eluted with 50 mm potassium phosphate (pH 7.0) containing 1 mmDTT, 1 μg/ml leupeptin, and 0.02% Tween 20. The rationale to use the stepwise elution from hydroxyapatite with 50 mm potassium phosphate was similar to that of the experiments with DEAE-Sepharose column chromatography. We had previously found that the most of the enzyme activities was eluted from hydroxyapatite column starting at 50 mm potassium phosphate during a linear gradient run of the chromatography. The active fractions isolated from hydroxyapatite column chromatography were applied onto a column of phenyl-Sepharose (1.0 × 6.37 cm, 5 ml), which was equilibrated with 5% (w/v) ammonium sulfate in 20 mm Tris-HCl (pH 7.4), 1 mm EDTA, 1 mm DTT, 1 μg/ml leupeptin, and 0.02% Tween 20. The column was washed with the same solution, and the enzyme activity was eluted with 20 mm Tris-HCl (pH 7.4), 1 mm EDTA, 1 mm DTT, 1 μg/ml leupeptin and 0.02% Tween 20. The active fractions pooled from phenyl-Sepharose were applied onto a column of PBE94 (1.0 × 6.37 cm, 5 ml), which was equilibrated with 20 mm Tris-HCl (pH 7.4), 1 mm DTT, 1 mm EDTA, 1 μg/ml leupeptin, and 0.02% Tween 20. The column was washed with 25 mm histidine-HCl (pH 6.2), 1 mm DTT, 1 mm EDTA, 1 μg/ml leupeptin, and 0.02% Tween 20, and the enzyme activity was eluted by decreasing pH with Polybuffer 74 (pH 4.0) in 1 mm DTT and 0.02% Tween 20. Five-ml fractions were collected into the tube containing 0.7 ml of 1 m Tris-HCl (pH 7.4) to neutralize the pH. The enzyme activity was unstable at pH 5.0, and 88% of enzyme activity was lost 16 h thereafter. It was necessary to neutralize the pH of the sample solution soon after the enzyme was eluted from the column. The active fractions from chromatofocusing were concentrated into 4.5 ml by using a small size of hydroxyapatite column (1 × 0.27 cm, 1 ml). The enzyme was further purified by native-PAGE according to the method of Ornstein and Davis (12Ornstein L. Davis B.J. Ann. N. Y. Acad. Sci. 1964; 121: 321-349Crossref PubMed Scopus (3328) Google Scholar). Separating gel (2 ml) consisting of 7.5% polyacrylamide, 0.375 m Tris-HCl (pH 8.8), and 0.02% Tween 20 was prepared in a glass tube (15 × 0.5 cm). Stacking gel (0.1 ml) composed of 5% polyacrylamide, 0.125m Tris-H3PO4 (pH 6.8), and 0.02% Tween 20 were overlaid to the separating gel. The enzyme solution (0.4 ml) was added to each tube. Electrophoresis was carried out at a constant voltage of 80 V and 4 °C until the dye migrated toward the end of the gel. The gels were then removed from the glass tubes and horizontally sliced into fragments (2 mm distance). The individual pieces were transferred into microtiter plate wells, soaked in 50 μl of 25 mm Tris-HCl (pH 7.4), 1 mm DTT, 0.02% Tween 20 for 16 h, and the supernatants with the highest enzyme activities were collected. 1-O-Hexadecyl-2-N-methylcarbamyl-GPC (5 mg, 9.28 μmol) dissolved in 0.5 ml of 0.16 m acetate (pH 5.6) containing 80 mm CaCl2 was combined with 1 ml of cabbage phospholipase D and 0.5 ml of 20% ethanolamine. The reaction was carried out at room temperature for 16 h (similar to that described in Ref. 13Uemura Y. Lee T. Snyder F. J. Biol. Chem. 1991; 266: 8268-8272Abstract Full Text PDF PubMed Google Scholar), and the lipid was extracted by the method of Bligh and Dyer (10Bligh E.G. Dyer W.T. Can. J. Biochem. Physiol. 1959; 261: 911-917Crossref Scopus (42411) Google Scholar). The reaction product, 1-O-hexadecyl-2-N-methylcarbamyl-GPE, was purified by TLC using a solvent system of CHCl3/CH3OH/CH3COOH/H2O (50:25:8:4, v/v/v/v). The purified phospholipid (8.36 μmol) was dissolved in 2 ml of 50 mm borate (pH 8.0) in CH3OH and reacted with activated CH-Sepharose (2 ml) suspended in the same solution for 16 h at 22 °C. The resulting gel was washed with 50 mm borate (pH 8.0) in CH3OH thoroughly, and the remaining reactive sites were blocked with 1 m Tris-HCl (pH 8.0) for 16 h at room temperature. Based on results from phosphorus determinations (14Rouser G. Siakotos A.N. Fleischer S. Lipids. 1966; 1: 85-86Crossref PubMed Scopus (1314) Google Scholar), 0.98 μmol of ligand was bound to 1 ml of CH-Sepharose The active fractions of native-PAGE were combined with 50 μl of affinity gel matrix in a microcentrifuge tube, which was equilibrated with 20 mm Tris-HCl (pH 7.4) containing 0.1 m NaCl, 1 mm DTT, and 0.02% Tween 20 and mixed gently at 4 °C for 30 min. The gel was washed with the same buffer and followed by the same solution without NaCl. The washed gel was combined with 5 mm alkylacetyl-GPC dissolved in 20 mm Tris-HCl (pH 7.4) in 1 mm DTT, and 0.02% Tween 20 and incubated for 1 h at 4 °C. The mixture was centrifuged for 5 min at 10,000 × g, and the supernatant with enzyme activity was transferred into another tube. PAF in this enzyme preparation was removed by hydroxyapatite (0.5 ml) column chromatography. The resulting enzyme solution was dialyzed against 20 mm Tris-HCl (pH 7.4) containing 40% glycerol, 1 mm DTT, and 0.02% Tween 20. The purified preparation was stored at −20 °C, and no significant decrease in enzyme activity was observed at least for 1 month. SDS-PAGE was carried out according to the method of Laemmli (15Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206658) Google Scholar) using 10% polyacrylamide gel. The proteins were visualized by silver staining using a silver staining kit for protein (Pharmacia Fine Chemicals). The sequencing of the protein was carried out at Harvard Microchemistry Facility (Boston, MA) by tandem mass spectrometry and Edman degradation analysis. The purified protein was subjected to electrophoresis on 10% SDS-PAGE gel. The protein band on the gel was visualized by Coomassie Brilliant Blue. The sliced gel was subjected to digestion with trypsin and microsequencing. In most instances, protein was measured by a protein assay kit (Bio-Rad) using BSA as the standard. However, when the protein concentrations in the samples were low (e.g. Table III, steps 3–7), a series of one to one (1:1) dilutions of 1 μg/ml BSA (ranging from 31.25 ng to 1 μg per spot) and the samples in duplicates were spotted on a 3MM filter paper. The spots were allowed to air-dry and were stained with 0.25% Coomassie Brilliant Blue R-250 (Bio-Rad), 45% CH3OH, 10% CH3COOH for 30 min. The paper was destained with 45% CH3OH, 10% CH3COOH until the background was nearly white. Then, the protein amount was determined by comparing the intensities of the spots from samples with those from BSA standards. The amount of protein in the final enzyme preparation (Table III, step 8) was estimated by comparing the intensity of the protein band with those of standard proteins after SDS-PAGE and silver staining.Table IIIPurification of PAF-dependent transacetylase from rat kidney membranesStepsProteinTotal activitySpecific activityPurificationRecoverymgnmol/minnmol/min·mg-fold%1Mitochondria82117042.101100Microsome58034086.02Solubilization from mitochondria (1st)98.4140414.75.968.9Solubilization from Microsome (1st)73.2171623.6Solubilization from Microsome (2nd)69.64045.93DEAE-Sepharose36.1375810428.573.54Hydroxyapatite9.93318932188.062.45Phenyl-Sepharose4.50274260916753.66Chromatofocusing1.131823161044135.77Native PAGE0.025668926900737013.58Affinity gel matrix0.0044622350000137004.4 Open table in a new tab Table IISolubilization of PAF:lysoplasmalogen transacetylase from mitochondrial and microsomal fractions of rat kidneysProteinTotal activitySpecific activityRecoverymgnmol/minnmol/min·mg%Mitochondria68.4 ± 1.2142 ± 18.52.1 ± 0.3100Microsome48.3 ± 3.2284 ± 13.16.0 ± 0.4100First solubilization from mitochondria8.2 ± 1.0117 ± 3.914.7 ± 1.282.3Second solubilization from mitochondria10.4 ± 0.811.6 ± 2.21.2 ± 0.38.2First solubilization from microsome6.1 ± 0.4143 ± 8.923.6 ± 1.750.4Second solubilization from microsome5.8 ± 0.633.7 ± 2.25.9 ± 0.511.9Four groups of mitochondrial and microsomal fractions were independently prepared from 21 rats as described under “Experimental Procedures.” The solubilization of enzyme was carried out with 0.04% Tween 20 at a detergent/protein (w/w) ratio of 0.1. Values were represented as means ± S.E. (n = 4) per kidney. Open table in a new tab Table IEffects of Tween 20 concentrations and Tween 20: protein ratios on the solubilization of PAF:lysoplasmalogen transacetylase from the membrane fraction of rat kidneysTween 20 concentrationTween 20/proteinActivity in supernatantProtein in supernatantPurification%w/w%%-fold0.020.141.39.34.20.021.041.319.92.10.022.045.413.93.10.040.137.710.63.60.050.143.29.54.5Rat kidneys were homogenized in buffer and the membrane fraction was isolated as described (13Uemura Y. Lee T. Snyder F. J. Biol. Chem. 1991; 266: 8268-8272Abstract Full Text PDF PubMed Google Scholar). The membrane fraction suspended in 0.25m sucrose and 10 mm Hepes, (pH 7.0) was mixed with 0.2% Tween 20 to attain the desired Tween 20 concentration and Tween 20/protein ratio. The mixture was stirred at 4 °C for 30 min and then centrifuged at 100,000 × g for 60 min to obtain the supernatant and pellet fraction. Transacetylase activities and protein concentrations in both fractions were measured to ensure proper recovery. Data represent the average of at least duplicate determinations. Open table in a new tab Four groups of mitochondrial and microsomal fractions were independently prepared from 21 rats as described under “Experimental Procedures.” The solubilization of enzyme was carried out with 0.04% Tween 20 at a detergent/protein (w/w) ratio of 0.1. Values were represented as means ± S.E. (n = 4) per kidney. Rat kidneys were homogenized in buffer and the membrane fraction was isolated as described (13Uemura Y. Lee T. Snyder F. J. Biol. Chem. 1991; 266: 8268-8272Abstract Full Text PDF PubMed Google Scholar). The membrane fraction suspended in 0.25m sucrose and 10 mm Hepes, (pH 7.0) was mixed with 0.2% Tween 20 to attain the desired Tween 20 concentration and Tween 20/protein ratio. The mixture was stirred at 4 °C for 30 min and then centrifuged at 100,000 × g for 60 min to obtain the supernatant and pellet fraction. Transacetylase activities and protein concentrations in both fractions were measured to ensure proper recovery. Data represent the average of at least duplicate determinations. An essential step in purifying the membrane enzyme is to first solubilize the enzyme from the membrane. Although there are assortments of detergents (ionic and non-ionic) and chaotropic agents available commercially, the detergent selected will be the one that does not inhibit or inactivate enzyme activity, does not present the enzyme in an aggregated form, and has a high critical micellar concentration. Additionally, the ratio of protein to detergent concentration is important. In preliminary experiment, among the several ionic and non-ionic detergents we tested (such as CHAPSO, octylglucoside, and Triton X-100), PAF:lysoplasmalogen transacetylase activity was most effectively solubilized by Tween 20. Enzyme activity was solubilized from rat kidney membrane fractions (mitochondria plus microsomes, 100,000 × g pellets) by Tween 20 at a concentration of 0.02–0.05%, and the maximal specific activity was observed at a detergent/protein (w/w) ratio of 0.1 (TableI). When the efficiency of the solubilization was tested on mitochondrial and microsomal fractions separately instead of using total membrane fraction, it was found that substantial amounts of enzyme activity still remained in the 100,000 × g pellets, especially with that of the microsomal fractions (TableII). Therefore, a second attempt on the solubilization of the remaining pellets was carried out. Around 8% and 10% of transacetylase activities were obtained from the second solubilization of the mitochondrial and microsomal pellets, respectively. However, the second solubilization from the mitochondrial fraction decreased the specific activity of the enzyme because relatively large amounts of unspecific proteins were concomitantly released. Thus, solubilization procedures were performed twice for the microsomal fraction and once for the mitochondrial fraction hereon. Overall, Tween 20 (0.02%) was included in all purification steps because it maintained the stability of enzyme activity. The apparent isoelectric point of the enzyme was estimated to be 5.0 by chromatofocusing (data not shown). Native-PAGE was the crucial step for the purification of this enzyme. The specific activity was increased 17-fold, as compared with that in the previous step (TableIII). It should be noted that the enzyme activity was easily extracted from the polyacrylamide gel by soaking the gel slice in the buffer solution without squeezing the gel or the aid of electricity, as commonly performed as a method to extract the proteins from the polyacrylamide gel. 1-O-Hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (data not shown) and 1-O-hexadecyl-2-N-methylcarbamyl-GPE, which are structurally related to PAF, competitively inhibit PAF:lysoplasmalogen transacetylase activity in Tween 20 (0.05%) solubilized membrane fraction from rat kidneys with K m = 9.1 μm, and K i = 10.1 μm for hexadecylacetyl-GPC and hexadecyl-N-methylcarbamyl-GPE, respectively (Fig. 1). Thus, hexadecyl-N-methylcarbamyl-GPE is expected to be useful as a specific ligand for the affinity gel matrix. This lipid has the advantage of being resistant to enzymatic hydrolysis, while acetyl ester of PAF is easily degraded by PAF-acetylhydrolase(s) in the cells. Additionally, the amino group of hexadecylmethylcarbamyl-GPE can be linked to the carboxyl-terminal group of CH-Sepharose covalently. All the enzyme activity was adsorbed to the resulting gel matrix, while enzyme activity was not adsorbed to the CH-Sepharose without ligand at all (data not shown). The enzyme activity was not eluted from the gel matrix by 0.5% CHAPS, 1% Triton X-100, 50% ethylene glycol, or 0.5m NaCl. In order to elute the enzyme activity from the column with a reasonable recovery, more than 5 mm PAF was required. The concentration of the ligand in the gel matrix was estimated to be 0.98 mm; therefore, a relatively high concentration of PAF was needed for replacement. These results also indicate that the enzyme specifically recognizes the ligand at the site, which is necessary for the interaction with the substrate. Starting from rat kidney membrane, 13,700-fold purification of the enz" @default.
• W2068013527 created "2016-06-24" @default.
• W2068013527 creator A5009523207 @default.
• W2068013527 creator A5020286965 @default.
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• W2068013527 date "1999-03-01" @default.
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