The Experts below are selected from a list of 132 Experts worldwide ranked by ideXlab platform
Luciano Binaglia - One of the best experts on this subject based on the ideXlab platform.
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Purification of ethanolaminephosphotransferase from bovine liver microsomes.
Biochimica et biophysica acta, 1999Co-Authors: Alessandra Mancini, Francesca Del Rosso, Rita Roberti, Pierluigi Orvietani, Lucia Coletti, Luciano BinagliaAbstract:CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (EC 2. 7.8.1) has been purified to electrophoretic homogeneity and in a catalytically active form from bovine liver microsomes. The purification method is based on the high hydrophobicity of the protein whose charged sites appear to be masked from the interaction with the chromatographic stationary phases when membranes are solubilized with an excess of non-ionic detergent. The isolated protein has a molecular mass of about 38 kDa, as estimated by SDS-PAGE mobility, and exhibits both ethanolaminephosphotransferase and Cholinephosphotransferase activities. Evidence is given that both activities are Mn2+-dependent and that the same catalytic site is involved in Cholinephosphotransferase and ethanolaminephosphotransferase reactions. Mg2+-dependent CDP-choline:diacylglycerol Cholinephosphotransferase (EC 2.7.8.2) is completely inactivated during the solubilization and purification steps.
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Reversibility of the reactions catalyzed by Cholinephosphotransferase and ethanolaminephosphotransferase solubilized from rat-brain microsomes.
Biochimica et biophysica acta, 1992Co-Authors: Rita Roberti, Alessandra Mancini, Louis Freysz, Luciano BinagliaAbstract:The incorporation of CMP into CDP-ethanolamine and CDP-choline, catalyzed by ethanolaminephosphotransferase (EC 2.7.8.1) and Cholinephosphotransferase (EC 2.7.8.2), respectively, has been studied in solubilized preparations of rat-brain microsomes. Mn2+ ions were required for the maximal activity of both enzymes. The CMP concentration needed to reach the half-maximal reaction rate was 1.6 microM for both activities. The rate of incorporation of CMP into CDP-choline and CDP-ethanolamine was increased by increasing the concentration of phosphatidylcholine and phosphatidylethanolamine, respectively, in detergent-phospholipid micellar systems. The rate of the reaction at pH 6.5 was comparable with that measured at pH 8.5, whereas the rate of synthesis of phosphatidylcholine and phosphatidylethanolamine, catalyzed by the same enzymes, increased with pH. Ethanolaminephosphotransferase, which catalyzes the synthesis of phosphatidylethanolamine from CDP-ethanolamine and diacylglycerol, was co-eluted with the enzyme activity catalyzing the reverse reaction, when solubilized microsomes were submitted to anion exchange chromatography on DEAE Bio-Gel A. Cholinephosphotransferase was inactivated during the chromatographic procedure.
Robert M. Bell - One of the best experts on this subject based on the ideXlab platform.
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CDP-choline:1,2-diacylglycerol Cholinephosphotransferase.
Biochimica et Biophysica Acta, 1997Co-Authors: Christopher R. Mcmaster, Robert M. BellAbstract:Cholinephosphotransferase transfers a phosphocholine moiety from CDP-choline to diacylglycerol thus forming phosphatidylcholine (PtdCho) and CMP. This reaction defines the ultimate step in the Kennedy pathway for the genesis of de novo synthesized PtdCho. Hence, the intracellular location of Cholinephosphotransferase identifies both the site from which de novo synthesized PtdCho is transported to other organelles and the site from which it is assembled with proteins and other lipids for secretion from the cell during the generation of lung surfactant, lipoproteins, and bile. Most subcellular fractionation studies observed the majority of Cholinephosphotransferase activity in the endoplasmic reticulum, although the method of subcellular fractionation was found to grossly affect these results with activity alternately dispersed within Golgi, nuclear, and mitochondrial fractions. Coupling subcellular fractionation results with immunofluorescence or electron microscopy studies would resolve the issue of the site of PtdCho synthesis. However, antibodies have yet to be generated to Cholinephosphotransferase since its integral membrane-bound nature has prevented its purification from any source and a mammalian Cholinephosphotransferase cDNA has also yet to be isolated. However, Cholinephosphotransferase genes have recently been isolated from the yeast Saccharomyces cerevisiae. Structure/function analysis of the S. cerevisiae Cholinephosphotransferase has allowed for an in depth molecular examination resulting in the identification of the catalytic site. In addition, this analysis has generated the predicted amino acid data necessary to produce antibodies to pursue the site of PtdCho synthesis in this organism, as well as to provide information that should allow for the isolation of mammalian Cholinephosphotransferase cDNA(s).
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Choline- and ethanolaminephosphotransferases from Saccharomyces cerevisiae.
Methods in enzymology, 1992Co-Authors: Russell H. Hjelmstad, Robert M. BellAbstract:Publisher Summary Phosphatidylcholine (PC) and phosphatidylethanolamine (PE)—the principal phospholipids of eukaryotic membranes—are synthesized from the common precursor sn -1,2-diacylglycerol and CDPcholine or CDPethanolamine, respectively, in reactions catalyzed by membrane-bound amino alcohol phosphotransferases. Comparative enzymological studies in Saccharomyces cerevisiae (S. cerevisiae) and higher eukaryotic cells have suggested the presence of distinct microsomal Cholinephosphotransferase and ethanolaminephosphotransferase enzymes. In addition, a distinct Cholinephosphotransferase may function in 1-alkyl-2-acetyloglycerophosphocholine (platelet-activating factor (PAF)) synthesis. The yeast S. cerevisiae provides an excellent genetic system in which to pursue in-depth studies on the structure, function, and regulation of choline- and ethanolaminephosphotransferases ept mutants activities can be isolated. Mutants defective in Cholinephosphotransferase (cpt mutants) and ethanolaminephosphotransferase (ept mutants) activities can also be isolated, and the corresponding structural genes for two amino alcohol phosphotransferases can be cloned, sequenced, and used to generate chromosomal null mutations. The mixed micellar assays described in the chapter have been developed to perform detailed enzymological studies of the individual CPT1 and EPTI gene products. Both the Cholinephosphotransferase (CPT1) and ethanolaminephosphotransferase (EPT1) gene products exhibit Michaelis-Menten kinetics with respect to the mole fraction of dioleoylglycerol present in the micellar phase. The properties of the CPT1 and EPT1 gene product-dependent Cholinephosphotransferase activities and the EPT1 gene product-dependent ethanolaminephosphotransferase activity are summarized.
Rita Roberti - One of the best experts on this subject based on the ideXlab platform.
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Purification of ethanolaminephosphotransferase from bovine liver microsomes.
Biochimica et biophysica acta, 1999Co-Authors: Alessandra Mancini, Francesca Del Rosso, Rita Roberti, Pierluigi Orvietani, Lucia Coletti, Luciano BinagliaAbstract:CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (EC 2. 7.8.1) has been purified to electrophoretic homogeneity and in a catalytically active form from bovine liver microsomes. The purification method is based on the high hydrophobicity of the protein whose charged sites appear to be masked from the interaction with the chromatographic stationary phases when membranes are solubilized with an excess of non-ionic detergent. The isolated protein has a molecular mass of about 38 kDa, as estimated by SDS-PAGE mobility, and exhibits both ethanolaminephosphotransferase and Cholinephosphotransferase activities. Evidence is given that both activities are Mn2+-dependent and that the same catalytic site is involved in Cholinephosphotransferase and ethanolaminephosphotransferase reactions. Mg2+-dependent CDP-choline:diacylglycerol Cholinephosphotransferase (EC 2.7.8.2) is completely inactivated during the solubilization and purification steps.
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Reversibility of the reactions catalyzed by Cholinephosphotransferase and ethanolaminephosphotransferase solubilized from rat-brain microsomes.
Biochimica et biophysica acta, 1992Co-Authors: Rita Roberti, Alessandra Mancini, Louis Freysz, Luciano BinagliaAbstract:The incorporation of CMP into CDP-ethanolamine and CDP-choline, catalyzed by ethanolaminephosphotransferase (EC 2.7.8.1) and Cholinephosphotransferase (EC 2.7.8.2), respectively, has been studied in solubilized preparations of rat-brain microsomes. Mn2+ ions were required for the maximal activity of both enzymes. The CMP concentration needed to reach the half-maximal reaction rate was 1.6 microM for both activities. The rate of incorporation of CMP into CDP-choline and CDP-ethanolamine was increased by increasing the concentration of phosphatidylcholine and phosphatidylethanolamine, respectively, in detergent-phospholipid micellar systems. The rate of the reaction at pH 6.5 was comparable with that measured at pH 8.5, whereas the rate of synthesis of phosphatidylcholine and phosphatidylethanolamine, catalyzed by the same enzymes, increased with pH. Ethanolaminephosphotransferase, which catalyzes the synthesis of phosphatidylethanolamine from CDP-ethanolamine and diacylglycerol, was co-eluted with the enzyme activity catalyzing the reverse reaction, when solubilized microsomes were submitted to anion exchange chromatography on DEAE Bio-Gel A. Cholinephosphotransferase was inactivated during the chromatographic procedure.
Alessandra Mancini - One of the best experts on this subject based on the ideXlab platform.
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Purification of ethanolaminephosphotransferase from bovine liver microsomes.
Biochimica et biophysica acta, 1999Co-Authors: Alessandra Mancini, Francesca Del Rosso, Rita Roberti, Pierluigi Orvietani, Lucia Coletti, Luciano BinagliaAbstract:CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (EC 2. 7.8.1) has been purified to electrophoretic homogeneity and in a catalytically active form from bovine liver microsomes. The purification method is based on the high hydrophobicity of the protein whose charged sites appear to be masked from the interaction with the chromatographic stationary phases when membranes are solubilized with an excess of non-ionic detergent. The isolated protein has a molecular mass of about 38 kDa, as estimated by SDS-PAGE mobility, and exhibits both ethanolaminephosphotransferase and Cholinephosphotransferase activities. Evidence is given that both activities are Mn2+-dependent and that the same catalytic site is involved in Cholinephosphotransferase and ethanolaminephosphotransferase reactions. Mg2+-dependent CDP-choline:diacylglycerol Cholinephosphotransferase (EC 2.7.8.2) is completely inactivated during the solubilization and purification steps.
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Reversibility of the reactions catalyzed by Cholinephosphotransferase and ethanolaminephosphotransferase solubilized from rat-brain microsomes.
Biochimica et biophysica acta, 1992Co-Authors: Rita Roberti, Alessandra Mancini, Louis Freysz, Luciano BinagliaAbstract:The incorporation of CMP into CDP-ethanolamine and CDP-choline, catalyzed by ethanolaminephosphotransferase (EC 2.7.8.1) and Cholinephosphotransferase (EC 2.7.8.2), respectively, has been studied in solubilized preparations of rat-brain microsomes. Mn2+ ions were required for the maximal activity of both enzymes. The CMP concentration needed to reach the half-maximal reaction rate was 1.6 microM for both activities. The rate of incorporation of CMP into CDP-choline and CDP-ethanolamine was increased by increasing the concentration of phosphatidylcholine and phosphatidylethanolamine, respectively, in detergent-phospholipid micellar systems. The rate of the reaction at pH 6.5 was comparable with that measured at pH 8.5, whereas the rate of synthesis of phosphatidylcholine and phosphatidylethanolamine, catalyzed by the same enzymes, increased with pH. Ethanolaminephosphotransferase, which catalyzes the synthesis of phosphatidylethanolamine from CDP-ethanolamine and diacylglycerol, was co-eluted with the enzyme activity catalyzing the reverse reaction, when solubilized microsomes were submitted to anion exchange chromatography on DEAE Bio-Gel A. Cholinephosphotransferase was inactivated during the chromatographic procedure.
Yasuo Nakazawa - One of the best experts on this subject based on the ideXlab platform.
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CDPcholine:1,2-diacylglycerol Cholinephosphotransferase from rat liver microsomes. I. Solubilization and characterization of the partially purified enzyme and the possible existence of an endogenous inhibitor
Lipids, 1993Co-Authors: Kozo Ishidate, Ritsuko Matsuo, Yasuo NakazawaAbstract:The solubilization and partial purification of Cholinephosphotransferase (CDPcholine:1,2-diacylglycerol Cholinephosphotransferase, EC 2.7.8.2) from rat liver microsomes were examined in the presence of ionic (sodium deoxycholate), nonionic (Triton X-100, n -octylglycoside), or zwitter ionic (CHAPS) detergents. Among the four detergents tested, only sodium deoxycholate was found to be an efficient solubilizer of Cholinephosphotransferase activity from microsomal membranes, whereas the other three detergents caused irreversible inactivation of the enzyme at the solubilization step. Addition of phospholipids at the solubilization step, or after solubilization of the membrane proteins, could not preserve or reconstitute activity to any extent. The sodium deoxycholate-solubilized activity was partially purified by gel permeation chromatography (Superose 12HR). The partially purified preparation appeared to consist of a large aggregate containing phospholipids; further dissociation of the protein-phospholipid complex caused complete inactivation of the enzyme. The partially purified Cholinephosphotransferase showed a specific activity of 100–130 nmol/min/mg protein, which is the highest activity reported to date from any tissue source; this amounts to a 4-fold enrichment of Cholinephosphotransferase activity from the original KCl-washed rat liver microsomes. Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) activity was copurified and 6-fold enriched with a total recovery of 60%. During the purification of Cholinephosphotransferase activity, a putative endogenous inhibitor of Cholinephosphotransferase was also solubilized and was isolated from the microsomal membranes. This heat-labile, nondialyzable inhibitor was shown to act specifically on Cholinephosphotransferase and not on ethanolaminephosphotransferase. Further characterization of the inhibitory activity revealed that it may act at the binding step of the Cholinephosphotransferase to its lipid substrate, diacylglycerol.
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CDP-choline: 1,2-diacylglycerol Cholinephosphotransferase from rat liver microsomes. II. Photoaffinity labeling by radioactive CDP-choline analogs.
Biochimica et biophysica acta, 1992Co-Authors: Kozo Ishidate, Ritsuko Matsuo, Yasuo NakazawaAbstract:Abstract Photoaffinity labeling of Cholinephosphotransferase from rat liver microsomes directly by its substrate, [32P]CDP-choline or by a synthetic photoreactive CDP-choline analog, 3′(2′)-0-(4-benzoyl)benzoyl [32P]CDP-choline (BB-[32P]CDP-choline), was examined for the possible identification of its molecular form on subsequent SDS-PAGE followed by 32P-autoradiography. When the partially purified Cholinephosphotransferase was photoirradiated in the presence of [32P]CDP-choline, a considerable amount of 32P-radioactivity was incorporated into the TCA-insoluble component. This incorporation was dependent on irradiation time, Mg2+ or Mn2+-requiring and inhibited strongly by the presence of Ca2+. Either CDP-choline or CDP-ethanolamine inhibited the ultraviolet irradiation-dependent incorporation of 32P-radioactivity into the TCA-insoluble component in a dose-dependent manner, whereas neither phosphocholine or 5'-CDP had any effect on this process. These results strongly suggested that the observed 32P-incorporation from [32P]CDP-choline into the protein component could be a consequence of the covalent interaction between Cholinephosphotransferase and its substrate, [32P]CDP-choline. Two polypeptides, 25 kDa and 18 kDa, with high 32P-radioactivity were clearly identified on a SDS gel after the direct photoaffinity labeling with [32P]CDP-choline for more than 5 min of ultraviolet irradiation. On the other hand, when BB-[32P]CDP-choline was used as a photoaffinity ligand, a single polypeptide with apparent molecular size of 55 kDa could be rapidly photolabeled within 2.5 min, then this band gradually lost its 32P-radioactivity with increasing time of ultraviolet irradiation. Thus, the overall results strongly indicated that Cholinephosphotransferase in rat liver microsomes exists most likely as a 55 kDa polypeptide (or subunit) and that 25 kDa and 18 kDa peptides identified after the direct photoaffinity labeling with [32P]CDP-choline were probably the photo-cleavage products of Cholinephosphotransferase during the prolonged ultraviolet irradiation, both of which could contain the catalytic domain of the original enzyme protein(s).