| dc.contributor.author | Henneberry, Annette Lisa. | en_US |
| dc.date.accessioned | 2014-10-21T12:35:58Z | |
| dc.date.available | 2001 | |
| dc.date.issued | 2001 | en_US |
| dc.identifier.other | AAINQ66672 | en_US |
| dc.identifier.uri | http://hdl.handle.net/10222/55803 | |
| dc.description | Phosphatidylcholine (PC) and phosphatidyletlianolamine (PE) are the most abundant phospholipids in eukaryotic cells, comprising 50% and 25% of membrane phospholipid mass, respectively. Cholinephosphotransferase catalyzes the final step in the de novo biosynthesis of PC through the Kennedy pathway via the transfer of phosphocholine from CDP-choline to diacylglycerol, with the release of CMP. Ethanolaminephosphotransferaser catalyzes an analogous reaction with CDP-ethanolamine as the phosphobase donor for the de novo biosynthesis of PE. Together, these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PC and PE synthesized de novo. Attempts to purify these enzymes have been largely unsuccessful due to their integral membrane bound nature, and thus, most of the studies to date have used microsomal membrane preparations to characterize these enzymes. The recent cloning of the Sacccharomyces cerevisiae choline- and ethanolaminephosphotransferases (CPT1 and EPT1) has facilitated the search for mammalian cDNAs encoding these enzyme activities. This work reports the cloning expression, and characterization of the first mammalian choline- and ethanolaminephosphotransferase cDNAs. Human choline/ethanolaminephosphotransferase 1 (hCEPT1) codes for a 416 amino acid protein that can catalyze the de novo biosynthesis of both PC and PE in vitro and in vivo. Human cholinephosphotransferase 1 (hCPT1) encodes a 406 amino acid protein specific for the enzymatic synthesis of PC in vitro and in vivo. In vitro assessment of hCEPT1 and hCPT1-derived cholinephosphotransferase activity revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. While hCEPT1 mRNA was found in similar abundance in all tissues tested, expression of hCPT1 mRNA varied greater than 100-fold between tissues, and was most abundant in testis, followed by colon, small intestine, heart, prostate, and spleen. Immunofluorescence studies revealed that hCEPT1p was localized to the ER and the nuclear membrane, while hCPT1p was localized to the Golgi apparatus. Site-directed mutagenesis of hCEPT1 demonstrated that glycine156 plays a role in CDP-ethanolamine binding, while threonine214, valine 216, and isoleucine221 appear to play a role in determining the specificity of DAG utilized by hCEPT1p. Mutation of glutamate215 suggested that it might play a role in both DAG and CDP-aminoalcohol binding, although the exact roles remain to be elucidated. | en_US |
| dc.description | This work has also investigated the role of PC in the diacylglycerol homeostasis required for Sec14p-dependent Golgi function and cell growth in yeast. We have shown that, in contrast to previous work. CPT1- and EPT1-derived cholinephosphotransferase activities can significantly overlap in vivo such that EPT1 can contribute 60% of net PC synthesis via the Kennedy pathway. Variation in the level of diacylglycerol consumption through alterations in PC synthesis directly correlated with the level of SEC14-dependent invertase secretion and affected cell viability. Administration of synthetic di8:0 diacylglycerol resulted in a partial rescue of cells from sec14-mediated cell death. The addition of di8:0 diacylglycerol did not alter endogenous phospholipid metabolic pathways, nor was it converted to di8:0 phosphatidic acid. In addition, only hCEPT1-derived activity, and not hCPT1, was able to complement the yeast CPT1 gene in its interaction with SEC14 and affect cell growth. | en_US |
| dc.description | Thesis (Ph.D.)--Dalhousie University (Canada), 2001. | en_US |
| dc.language | eng | en_US |
| dc.publisher | Dalhousie University | en_US |
| dc.publisher | | en_US |
| dc.subject | Biology, Cell. | en_US |
| dc.subject | Chemistry, Biochemistry. | en_US |
| dc.title | Molecular analysis of lipid synthesis and its role in vesicle trafficking: De novo cloning, expression, and characterization of two human cholinephosphotransferases. | en_US |
| dc.type | text | en_US |
| dc.contributor.degree | Ph.D. | en_US |
