Cloning, Genomic Organization, and Characterization of a Human Cholinephosphotransferase

A cholinephosphotransferase activity catalyzes the final step in the de novo synthesis of phosphatidylcholine via the transfer of a phosphocholine moiety from CDP choline to diacylglycerol. Ethanolaminephosphotransferase activity catalyzes a similar reaction substituting CDP ethanolamine as the phosphobase donor. We report the identification and cloning of a human cDNA (human cholinephosphotransferase (hCPT1)) that codes for a cholinephosphotransferase-specific enzyme. This was demonstrated usingin vitro enzyme assays and in vivo measurement of the reconstitution of the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in yeast cells devoid of their own endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities. This contrasted with our previously cloned human choline/ethanolaminephosphotransferase cDNA that was demonstrated to code for a dual specificity choline/ethanolaminephosphotransferase. The hCPT1 and human choline/ethanolaminephosphotransferase (hCEPT1) predicted amino acid sequences possessed 60% overall identity and had only one variation in the amino acid residues within the CDP-alcohol phosphotransferase catalytic motif. In vitro assessment of hCPT1 and hCEPT1 derived cholinephosphotransferase activities also revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. Expression of the hCPT1 mRNA varied greater than 100-fold between tissues and was most abundant in testis followed by colon, small intestine, heart, prostate, and spleen. This was in marked contrast to the hCEPT1 mRNA, which has been found in similar abundance in all tissues tested to date. Both the hCPT1 and hCEPT1 enzymes were able to reconstitute the synthesis of PC in yeast to levels provided by the endogenous yeast cholinephosphotransferase; however, only hCEPT1-derived activity was able to complement the yeastCPT1 gene in its interaction with SEC14 and affect cell growth. A cholinephosphotransferase activity catalyzes the final step in the de novo synthesis of phosphatidylcholine via the transfer of a phosphocholine moiety from CDP choline to diacylglycerol. Ethanolaminephosphotransferase activity catalyzes a similar reaction substituting CDP ethanolamine as the phosphobase donor. We report the identification and cloning of a human cDNA (human cholinephosphotransferase (hCPT1)) that codes for a cholinephosphotransferase-specific enzyme. This was demonstrated usingin vitro enzyme assays and in vivo measurement of the reconstitution of the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in yeast cells devoid of their own endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities. This contrasted with our previously cloned human choline/ethanolaminephosphotransferase cDNA that was demonstrated to code for a dual specificity choline/ethanolaminephosphotransferase. The hCPT1 and human choline/ethanolaminephosphotransferase (hCEPT1) predicted amino acid sequences possessed 60% overall identity and had only one variation in the amino acid residues within the CDP-alcohol phosphotransferase catalytic motif. In vitro assessment of hCPT1 and hCEPT1 derived cholinephosphotransferase activities also revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. Expression of the hCPT1 mRNA varied greater than 100-fold between tissues and was most abundant in testis followed by colon, small intestine, heart, prostate, and spleen. This was in marked contrast to the hCEPT1 mRNA, which has been found in similar abundance in all tissues tested to date. Both the hCPT1 and hCEPT1 enzymes were able to reconstitute the synthesis of PC in yeast to levels provided by the endogenous yeast cholinephosphotransferase; however, only hCEPT1-derived activity was able to complement the yeastCPT1 gene in its interaction with SEC14 and affect cell growth. phosphatidylcholine phosphatidylethanolamine diacylglycerol platelet-activating factor dithiothreitol human choline/ethanolaminephosphotransferase human cholinephosphotransferase rapid amplification of cDNA ends kilobase polymerase chain reaction cholinephosphotransferase of S. cerevisiae choline/ethanolaminephosphotransferase of S. cerevisiae Phosphatidylcholine (PC)1 and phosphatidylethanolamine (PE) are the two most abundant phospholipids found in eukaryotic cells, generally comprising 50 and 25% of cellular phospholipid mass, respectively (1Raetz C.R.H. Annu. Rev. Genet. 1986; 20: 253-295Crossref PubMed Google Scholar). PC synthesis via the CDP choline pathway is responsible for essentially all de novo PC biosynthetic activity in all eukaryotic cell types thus far examined except (i) the liver where the methylation of phosphatidylethanolamine is predicted to contribute to 30% of net synthesis (2Ridgway N.D. Yao Z. Vance D.E. J. Biol. Chem. 1989; 264: 1203-1207Abstract Full Text PDF PubMed Google Scholar, 3DeLong C. Shen Y.-J. Thomas M.J. Cui Z. J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar) and (ii) yeast where the supply of exogenous choline dictates the relative contribution of the two PC biosynthetic pathways (4McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 14776-14783Abstract Full Text PDF PubMed Google Scholar, 5McGee T.P. Skinner H.B. Bankaitis V.A. J. Bacteriol. 1994; 176: 6861-6868Crossref PubMed Google Scholar). PE can also be synthesized by alternate routes with the CDP ethanolamine pathway supplemented by the decarboxylation of phosphatidylserine to PE, with the relative contribution of each route dependent on cell type (6Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 7Kuge O. Hasegawa K. Saito K. Nishijima M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4199-4203Crossref PubMed Scopus (52) Google Scholar, 8McMaster C.R. Choy P.C. J. Biol. Chem. 1992; 267: 14586-14591Abstract Full Text PDF PubMed Google Scholar, 9Trotter P.J. Pedretti J. Yates R. Voelker D.R. J. Biol. Chem. 1995; 270: 6071-6080Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar).The final step in the CDP alcohol pathways for the synthesis of PC and PE is catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase enzyme activities, respectively. Cholinephosphotransferase catalyzes the transfer of phosphocholine from CDP choline to diacylglycerol (DAG) with the release of CMP and the formation of PC (10McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 100-110Crossref PubMed Scopus (66) Google Scholar, 11McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 12Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar, 13Weiss S.B. Smith S.W. Kennedy E.P. J. Biol. Chem. 1958; 231: 53-64Abstract Full Text PDF PubMed Google Scholar), whereas the ethanolaminephosphotransferase reaction catalyzes a similar transfer substituting CDP ethanolamine as the phosphobase donor resulting in the formation of PE (14McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 117-123Crossref PubMed Scopus (31) Google Scholar, 15Mancini A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar, 16Ford D.A. Rosenbloom K.B. Gross R.W. J. Biol. Chem. 1992; 267: 11222-11228Abstract Full Text PDF PubMed Google Scholar). A cholinephosphotransferase has also been implicated in de novo synthesis of platelet-activating factor (PAF) (17Snyder F. Biochim. Biophys. Acta. 1997; 1348: 111-116Crossref PubMed Scopus (33) Google Scholar, 18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar). This putative PAF-specific cholinephosphotransferase was identified based on DTT susceptibility of cholinephosphotransferase activities measured in microsomal membranes, with PAF-specific activity being DTT resistant and PC-specific activity being DTT sensitive (18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar).The relative contribution of the CDP choline versus PE methylation pathways for the synthesis of PC have been implicated in regulating cell growth in the mammalian liver. PC synthesis through the CDP choline pathway favored proliferation, whereas PC synthesized by PE methylation inhibited cell growth and was negatively associated with the induction of liver tumor formation (19Cui Z. Houweling M. Vance D.E. J. Biol. Chem. 1994; 269: 24531-24533Abstract Full Text PDF PubMed Google Scholar, 20Walkey C.J., Yu, L. Agellon L.B. Vance D.E. J. Biol. Chem. 1998; 273: 27043-27046Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 21Tessitore L. Sesca E. Vance D.E. Int. J. Cancer. 2000; 86: 362-367Crossref PubMed Google Scholar). PC molecules synthesized by these two pathways have different fatty acyl chain compositions and this has been postulated to be the major contributor to the observed effects on cell growth (3DeLong C. Shen Y.-J. Thomas M.J. Cui Z. J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). The diacylglycerol specificity of cholinephosphotransferase regulates the fatty acyl composition of de novo PC synthesized through the CDP choline pathway and thus would be predicted to participate in the regulation of PC pathway-dependent liver tumor development. We recently cloned and characterized a human dual specificity choline/ethanolaminephosphotransferase (hCEPT1) capable of using both CDP choline and CDP ethanolamine as substrates in vitro(22Henneberry A.L. McMaster C.R. Biochem. J. 1999; 339: 291-298Crossref PubMed Scopus (90) Google Scholar). Metabolic labeling experiments demonstrated this enzyme was able to reconstitute the synthesis of both PC and PE when expressed in a yeast strain in which the endogenous cholinephosphotransferase and ethanolaminephosphotransferase genes were inactivated. In the work presented here we de novo cloned two cDNAs, each coding for a splice variant of a human cholinephosphotransferase (hCPT1). The cDNA product was expressed for characterization in vitroand in vivo and was found to code for a cholinephosphotransferase-specific enzyme.DISCUSSIONThe final steps in the Kennedy pathways for the synthesis of PC and PE are catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase activities, respectively (10McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 100-110Crossref PubMed Scopus (66) Google Scholar, 11McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 12Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar, 13Weiss S.B. Smith S.W. Kennedy E.P. J. Biol. Chem. 1958; 231: 53-64Abstract Full Text PDF PubMed Google Scholar, 14McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 117-123Crossref PubMed Scopus (31) Google Scholar, 15Mancini A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar, 16Ford D.A. Rosenbloom K.B. Gross R.W. J. Biol. Chem. 1992; 267: 11222-11228Abstract Full Text PDF PubMed Google Scholar). The hCPT1 cDNA described in this study codes for an enzyme that demonstrated cholinephosphotransferase activity in in vitroenzyme assays, and in yeast devoid of their endogenous cholinephosphotransferase activity, hCPT1 was able to reconstitute the PC biosynthetic pathway in vivo. The hCPT1 enzyme was unable to use CDP ethanolamine as a in vitro and was unable to reconstitute the PE biosynthetic pathway in vivo. This is in contrast to our previously human hCEPT1 which an activity capable of using both CDP choline and CDP ethanolamine as substrates in vitro and in A.L. McMaster C.R. Biochem. J. 1999; 339: 291-298Crossref PubMed Scopus (90) Google Scholar). has been that Kennedy pathways for the synthesis of PC and PE are in mammalian cells C. Annu. Rev. Biochem. 1995; PubMed Scopus Google Scholar, D.E. Vance D.E. Vance J.E. of and however, the of cholinephosphotransferase activity in both the hCPT1 and hCEPT1 enzymes and the dual specificity of for the synthesis of PC and PE that these two pathways be the hCEPT1 mRNA was similar in all tissues whereas the hCPT1 mRNA varied greater than 100-fold between the of with different for hCPT1 and hCEPT1 was the of and to complement the yeastCPT1 in cell both hCEPT1 and hCPT1 both in vivo PC synthesis to levels similar to provided by the yeast gene In is that PC and PE are synthesized by a dual specificity choline/ethanolaminephosphotransferase 1994; Google Scholar, C. A. P. Biochim. Biophys. Acta. Scopus Google Scholar), and be to hCEPT1 can both in mammalian A cell had been by with a in ethanolaminephosphotransferase activity C.R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and would have been to the hCPT1 and hCEPT1 in this cell this cell is with our that hCPT1 and hCEPT1 be was an of the expressed which to mammalian with to hCPT1 This the two to the complement of human and activities. This would be similar to the demonstrated gene complement in S. cerevisiae McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), and an of the C. also revealed only two genes coding for enzymes with to hCPT1 and hCEPT1 Genet. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). with the that hCEPT1 in both the CDP choline and CDP ethanolamine pathways in mammalian cells is the report of an enzyme to from liver A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar). be to the ethanolaminephosphotransferase is for by the of the PAF-specific cholinephosphotransferase activity had been previously from the PC-specific cholinephosphotransferase enzyme based on DTT (17Snyder F. Biochim. Biophys. Acta. 1997; 1348: 111-116Crossref PubMed Scopus (33) Google Scholar, 18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar). a PAF-specific cholinephosphotransferase activity that was by DTT and a PC-specific cholinephosphotransferase activity that was inhibited by We found that the cloned hCPT1 and hCEPT1 were both able to synthesize in The hCPT1 activity was and dependent on whereas the hCEPT1 activity was by hCPT1 hCEPT1 PC biosynthetic activity was inhibited by the hCPT1 the cholinephosphotransferase activities previously observed in mammalian for the synthesis of and yeast the in vivo specificity for the synthesis of PC molecules by hCPT1 and hCEPT1 the of mammalian for we are to hCPT1 and hCEPT1 specificities as the cholinephosphotransferase step is in the and thus have to these are development. Phosphatidylcholine (PC)1 and phosphatidylethanolamine (PE) are the two most abundant phospholipids found in eukaryotic cells, generally comprising 50 and 25% of cellular phospholipid mass, respectively (1Raetz C.R.H. Annu. Rev. Genet. 1986; 20: 253-295Crossref PubMed Google Scholar). PC synthesis via the CDP choline pathway is responsible for essentially all de novo PC biosynthetic activity in all eukaryotic cell types thus far examined except (i) the liver where the methylation of phosphatidylethanolamine is predicted to contribute to 30% of net synthesis (2Ridgway N.D. Yao Z. Vance D.E. J. Biol. Chem. 1989; 264: 1203-1207Abstract Full Text PDF PubMed Google Scholar, 3DeLong C. Shen Y.-J. Thomas M.J. Cui Z. J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar) and (ii) yeast where the supply of exogenous choline dictates the relative contribution of the two PC biosynthetic pathways (4McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 14776-14783Abstract Full Text PDF PubMed Google Scholar, 5McGee T.P. Skinner H.B. Bankaitis V.A. J. Bacteriol. 1994; 176: 6861-6868Crossref PubMed Google Scholar). PE can also be synthesized by alternate routes with the CDP ethanolamine pathway supplemented by the decarboxylation of phosphatidylserine to PE, with the relative contribution of each route dependent on cell type (6Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 7Kuge O. Hasegawa K. Saito K. Nishijima M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4199-4203Crossref PubMed Scopus (52) Google Scholar, 8McMaster C.R. Choy P.C. J. Biol. Chem. 1992; 267: 14586-14591Abstract Full Text PDF PubMed Google Scholar, 9Trotter P.J. Pedretti J. Yates R. Voelker D.R. J. Biol. Chem. 1995; 270: 6071-6080Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The final step in the CDP alcohol pathways for the synthesis of PC and PE is catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase enzyme activities, respectively. Cholinephosphotransferase catalyzes the transfer of phosphocholine from CDP choline to diacylglycerol (DAG) with the release of CMP and the formation of PC (10McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 100-110Crossref PubMed Scopus (66) Google Scholar, 11McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 12Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar, 13Weiss S.B. Smith S.W. Kennedy E.P. J. Biol. Chem. 1958; 231: 53-64Abstract Full Text PDF PubMed Google Scholar), whereas the ethanolaminephosphotransferase reaction catalyzes a similar transfer substituting CDP ethanolamine as the phosphobase donor resulting in the formation of PE (14McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 117-123Crossref PubMed Scopus (31) Google Scholar, 15Mancini A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar, 16Ford D.A. Rosenbloom K.B. Gross R.W. J. Biol. Chem. 1992; 267: 11222-11228Abstract Full Text PDF PubMed Google Scholar). A cholinephosphotransferase has also been implicated in de novo synthesis of platelet-activating factor (PAF) (17Snyder F. Biochim. Biophys. Acta. 1997; 1348: 111-116Crossref PubMed Scopus (33) Google Scholar, 18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar). This putative PAF-specific cholinephosphotransferase was identified based on DTT susceptibility of cholinephosphotransferase activities measured in microsomal membranes, with PAF-specific activity being DTT resistant and PC-specific activity being DTT sensitive (18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar). The relative contribution of the CDP choline versus PE methylation pathways for the synthesis of PC have been implicated in regulating cell growth in the mammalian liver. PC synthesis through the CDP choline pathway favored proliferation, whereas PC synthesized by PE methylation inhibited cell growth and was negatively associated with the induction of liver tumor formation (19Cui Z. Houweling M. Vance D.E. J. Biol. Chem. 1994; 269: 24531-24533Abstract Full Text PDF PubMed Google Scholar, 20Walkey C.J., Yu, L. Agellon L.B. Vance D.E. J. Biol. Chem. 1998; 273: 27043-27046Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 21Tessitore L. Sesca E. Vance D.E. Int. J. Cancer. 2000; 86: 362-367Crossref PubMed Google Scholar). PC molecules synthesized by these two pathways have different fatty acyl chain compositions and this has been postulated to be the major contributor to the observed effects on cell growth (3DeLong C. Shen Y.-J. Thomas M.J. Cui Z. J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). The diacylglycerol specificity of cholinephosphotransferase regulates the fatty acyl composition of de novo PC synthesized through the CDP choline pathway and thus would be predicted to participate in the regulation of PC pathway-dependent liver tumor development. We recently cloned and characterized a human dual specificity choline/ethanolaminephosphotransferase (hCEPT1) capable of using both CDP choline and CDP ethanolamine as substrates in vitro(22Henneberry A.L. McMaster C.R. Biochem. J. 1999; 339: 291-298Crossref PubMed Scopus (90) Google Scholar). Metabolic labeling experiments demonstrated this enzyme was able to reconstitute the synthesis of both PC and PE when expressed in a yeast strain in which the endogenous cholinephosphotransferase and ethanolaminephosphotransferase genes were inactivated. In the work presented here we de novo cloned two cDNAs, each coding for a splice variant of a human cholinephosphotransferase (hCPT1). The cDNA product was expressed for characterization in vitroand in vivo and was found to code for a cholinephosphotransferase-specific enzyme. final steps in the Kennedy pathways for the synthesis of PC and PE are catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase activities, respectively (10McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 100-110Crossref PubMed Scopus (66) Google Scholar, 11McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 12Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar, 13Weiss S.B. Smith S.W. Kennedy E.P. J. Biol. Chem. 1958; 231: 53-64Abstract Full Text PDF PubMed Google Scholar, 14McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 117-123Crossref PubMed Scopus (31) Google Scholar, 15Mancini A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar, 16Ford D.A. Rosenbloom K.B. Gross R.W. J. Biol. Chem. 1992; 267: 11222-11228Abstract Full Text PDF PubMed Google Scholar). The hCPT1 cDNA described in this study codes for an enzyme that demonstrated cholinephosphotransferase activity in in vitroenzyme assays, and in yeast devoid of their endogenous cholinephosphotransferase activity, hCPT1 was able to reconstitute the PC biosynthetic pathway in vivo. The hCPT1 enzyme was unable to use CDP ethanolamine as a in vitro and was unable to reconstitute the PE biosynthetic pathway in vivo. This is in contrast to our previously human hCEPT1 which an activity capable of using both CDP choline and CDP ethanolamine as substrates in vitro and in A.L. McMaster C.R. Biochem. J. 1999; 339: 291-298Crossref PubMed Scopus (90) Google Scholar). has been that Kennedy pathways for the synthesis of PC and PE are in mammalian cells C. Annu. Rev. Biochem. 1995; PubMed Scopus Google Scholar, D.E. Vance D.E. Vance J.E. of and however, the of cholinephosphotransferase activity in both the hCPT1 and hCEPT1 enzymes and the dual specificity of for the synthesis of PC and PE that these two pathways be the hCEPT1 mRNA was similar in all tissues whereas the hCPT1 mRNA varied greater than 100-fold between the of with different for hCPT1 and hCEPT1 was the of and to complement the yeastCPT1 in cell both hCEPT1 and hCPT1 both in vivo PC synthesis to levels similar to provided by the yeast gene In is that PC and PE are synthesized by a dual specificity choline/ethanolaminephosphotransferase 1994; Google Scholar, C. A. P. Biochim. Biophys. Acta. Scopus Google Scholar), and be to hCEPT1 can both in mammalian A cell had been by with a in ethanolaminephosphotransferase activity C.R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and would have been to the hCPT1 and hCEPT1 in this cell this cell is with our that hCPT1 and hCEPT1 be was an of the expressed which to mammalian with to hCPT1 This the two to the complement of human and activities. This would be similar to the demonstrated gene complement in S. cerevisiae McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), and an of the C. also revealed only two genes coding for enzymes with to hCPT1 and hCEPT1 Genet. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). with the that hCEPT1 in both the CDP choline and CDP ethanolamine pathways in mammalian cells is the report of an enzyme to from liver A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar). be to the ethanolaminephosphotransferase is for by the of the PAF-specific cholinephosphotransferase activity had been previously from the PC-specific cholinephosphotransferase enzyme based on DTT (17Snyder F. Biochim. Biophys. Acta. 1997; 1348: 111-116Crossref PubMed Scopus (33) Google Scholar, 18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar). a PAF-specific cholinephosphotransferase activity that was by DTT and a PC-specific cholinephosphotransferase activity that was inhibited by We found that the cloned hCPT1 and hCEPT1 were both able to synthesize in The hCPT1 activity was and dependent on whereas the hCEPT1 activity was by hCPT1 hCEPT1 PC biosynthetic activity was inhibited by the hCPT1 the cholinephosphotransferase activities previously observed in mammalian for the synthesis of and yeast the in vivo specificity for the synthesis of PC molecules by hCPT1 and hCEPT1 the of mammalian for we are to hCPT1 and hCEPT1 specificities as the cholinephosphotransferase step is in the and thus have to these are development. The final steps in the Kennedy pathways for the synthesis of PC and PE are catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase activities, respectively (10McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 100-110Crossref PubMed Scopus (66) Google Scholar, 11McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: 28010-28016Abstract Full Text PDF PubMed Google Scholar, 12Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar, 13Weiss S.B. Smith S.W. Kennedy E.P. J. Biol. Chem. 1958; 231: 53-64Abstract Full Text PDF PubMed Google Scholar, 14McMaster C.R. Bell R.M. Biochim. Biophys. Acta. 1997; 1348: 117-123Crossref PubMed Scopus (31) Google Scholar, 15Mancini A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar, 16Ford D.A. Rosenbloom K.B. Gross R.W. J. Biol. Chem. 1992; 267: 11222-11228Abstract Full Text PDF PubMed Google Scholar). The hCPT1 cDNA described in this study codes for an enzyme that demonstrated cholinephosphotransferase activity in in vitroenzyme assays, and in yeast devoid of their endogenous cholinephosphotransferase activity, hCPT1 was able to reconstitute the PC biosynthetic pathway in vivo. The hCPT1 enzyme was unable to use CDP ethanolamine as a in vitro and was unable to reconstitute the PE biosynthetic pathway in vivo. This is in contrast to our previously human hCEPT1 which an activity capable of using both CDP choline and CDP ethanolamine as substrates in vitro and in A.L. McMaster C.R. Biochem. J. 1999; 339: 291-298Crossref PubMed Scopus (90) Google Scholar). has been that Kennedy pathways for the synthesis of PC and PE are in mammalian cells C. Annu. Rev. Biochem. 1995; PubMed Scopus Google Scholar, D.E. Vance D.E. Vance J.E. of and however, the of cholinephosphotransferase activity in both the hCPT1 and hCEPT1 enzymes and the dual specificity of for the synthesis of PC and PE that these two pathways be the hCEPT1 mRNA was similar in all tissues whereas the hCPT1 mRNA varied greater than 100-fold between the of with different for hCPT1 and hCEPT1 was the of and to complement the yeastCPT1 in cell both hCEPT1 and hCPT1 both in vivo PC synthesis to levels similar to provided by the yeast gene In is that PC and PE are synthesized by a dual specificity choline/ethanolaminephosphotransferase 1994; Google Scholar, C. A. P. Biochim. Biophys. Acta. Scopus Google Scholar), and be to hCEPT1 can both in mammalian A cell had been by with a in ethanolaminephosphotransferase activity C.R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and would have been to the hCPT1 and hCEPT1 in this cell this cell is with our that hCPT1 and hCEPT1 be was an of the expressed which to mammalian with to hCPT1 This the two to the complement of human and activities. This would be similar to the demonstrated gene complement in S. cerevisiae McMaster C.R. Bell R.M. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), and an of the C. also revealed only two genes coding for enzymes with to hCPT1 and hCEPT1 Genet. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). with the that hCEPT1 in both the CDP choline and CDP ethanolamine pathways in mammalian cells is the report of an enzyme to from liver A. Del Rosso F. Roberti R. Orvietani P. Coletti L. Binaglia L. Biochim. Biophys. Acta. 1999; 1437: 80-92Crossref PubMed Scopus (17) Google Scholar). be to the ethanolaminephosphotransferase is for by the of the A PAF-specific cholinephosphotransferase activity had been previously from the PC-specific cholinephosphotransferase enzyme based on DTT (17Snyder F. Biochim. Biophys. Acta. 1997; 1348: 111-116Crossref PubMed Scopus (33) Google Scholar, 18Woodard D.S. Lee T.-C. Snyder F. J. Biol. Chem. 1987; 262: 2520-2527Abstract Full Text PDF PubMed Google Scholar). a PAF-specific cholinephosphotransferase activity that was by DTT and a PC-specific cholinephosphotransferase activity that was inhibited by We found that the cloned hCPT1 and hCEPT1 were both able to synthesize in The hCPT1 activity was and dependent on whereas the hCEPT1 activity was by hCPT1 hCEPT1 PC biosynthetic activity was inhibited by the hCPT1 the cholinephosphotransferase activities previously observed in mammalian for the synthesis of and yeast the in vivo specificity for the synthesis of PC molecules by hCPT1 and hCEPT1 the of mammalian for we are to hCPT1 and hCEPT1 specificities as the cholinephosphotransferase step is in the and thus have to these are development. We and for the of this

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.