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Role of mitochondria in folate metab...
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Lin, Bi-Fong.
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Role of mitochondria in folate metabolism.
紀錄類型:
書目-電子資源 : Monograph/item
正題名/作者:
Role of mitochondria in folate metabolism./
作者:
Lin, Bi-Fong.
出版者:
Ann Arbor : ProQuest Dissertations & Theses, : 1991,
面頁冊數:
132 p.
附註:
Source: Dissertations Abstracts International, Volume: 54-03, Section: B.
Contained By:
Dissertations Abstracts International54-03B.
標題:
Livestock. -
電子資源:
https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=9228742
Role of mitochondria in folate metabolism.
Lin, Bi-Fong.
Role of mitochondria in folate metabolism.
- Ann Arbor : ProQuest Dissertations & Theses, 1991 - 132 p.
Source: Dissertations Abstracts International, Volume: 54-03, Section: B.
Thesis (Ph.D.)--University of California, Berkeley, 1991.
This item must not be sold to any third party vendors.
Folate coenzymes act as acceptors or donors of one carbon units in a variety of reactions involved in amino acid and nucleotide metabolism. Folylpolyglutamates are the preferred substrates for the enzymes of one carbon metabolism. Consequently, the enzyme folylpolyglutamate synthetase (FPGS), which catalyzes the addition of $\\gamma$-glutamyl residues to intracellular folates, plays a major role in folate homeostasis. A mutant Chinese hamster ovary cell (AUXB1), lacking FPGS activity, is auxotrophic for glycine, adenosine, thymidine and methionine. We have recently shown that AUXB1 cells transfected with the human FPGS gene (2$\\sp{\\rm o}$-2) support normal glycine biosynthesis. However, D5-3A8, an AUXB1 cell transfected with the E. coli FPGS gene and expressing E. coli FPGS activity, did not. The purpose of my study was to investigate the role of folylpolyglutamates in glycine synthesis within subcellular compartments, specifically in mitochondria. FPGS activity in WTT2 and 2$\\sp{\\rm o}$-2 cells was found in both mitochondria and cytosol. However, FPGS activity and cellular folate, primarily triglutamates, in D5-3A8 cells were only found in the cytosol. This showed that mitochondrial folate accumulation and metabolism are dependent on mitochondrial FPGS. The triglutamate derivatives that accumulate in the cytosol of cells expressing the E. coli FPGS enzyme appear to be as effective as the normally formed longer derivatives in the synthesis of thymidine and purines, but not glycine. A cell line expressing E. coli FPGS in mitochondria was made using molecular cloning techniques. A mammalian expression vector carrying the E. coli FPGS gene with a leader peptide was prepared and was used to transfect AUXB1 and D5-3A8 cell lines. Transfected cells were selected by hygromycin resistance, a selective marker cotransfected with the modified E. coli FPGS gene, and by the gly$\\sp{+}$ phenotype. Mitochondrial FPGS activity was found in these transfectants and folates, predominant folyltriglutamates, were found in both cytosol and mitochondria. A comparison of these transfectants with D5-3A8 and wildtype WTT2 cells was made to evaluate the role of mitochondria in folate metabolism. The data demonstrated that pteroyltriglutamates in the mitochondria are as effective as longer chain length derivatives as coenzymes in the synthesis of glycine.Subjects--Topical Terms:
539534
Livestock.
Subjects--Index Terms:
folylpolyglutamate synthetase
Role of mitochondria in folate metabolism.
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Folate coenzymes act as acceptors or donors of one carbon units in a variety of reactions involved in amino acid and nucleotide metabolism. Folylpolyglutamates are the preferred substrates for the enzymes of one carbon metabolism. Consequently, the enzyme folylpolyglutamate synthetase (FPGS), which catalyzes the addition of $\\gamma$-glutamyl residues to intracellular folates, plays a major role in folate homeostasis. A mutant Chinese hamster ovary cell (AUXB1), lacking FPGS activity, is auxotrophic for glycine, adenosine, thymidine and methionine. We have recently shown that AUXB1 cells transfected with the human FPGS gene (2$\\sp{\\rm o}$-2) support normal glycine biosynthesis. However, D5-3A8, an AUXB1 cell transfected with the E. coli FPGS gene and expressing E. coli FPGS activity, did not. The purpose of my study was to investigate the role of folylpolyglutamates in glycine synthesis within subcellular compartments, specifically in mitochondria. FPGS activity in WTT2 and 2$\\sp{\\rm o}$-2 cells was found in both mitochondria and cytosol. However, FPGS activity and cellular folate, primarily triglutamates, in D5-3A8 cells were only found in the cytosol. This showed that mitochondrial folate accumulation and metabolism are dependent on mitochondrial FPGS. The triglutamate derivatives that accumulate in the cytosol of cells expressing the E. coli FPGS enzyme appear to be as effective as the normally formed longer derivatives in the synthesis of thymidine and purines, but not glycine. A cell line expressing E. coli FPGS in mitochondria was made using molecular cloning techniques. A mammalian expression vector carrying the E. coli FPGS gene with a leader peptide was prepared and was used to transfect AUXB1 and D5-3A8 cell lines. Transfected cells were selected by hygromycin resistance, a selective marker cotransfected with the modified E. coli FPGS gene, and by the gly$\\sp{+}$ phenotype. Mitochondrial FPGS activity was found in these transfectants and folates, predominant folyltriglutamates, were found in both cytosol and mitochondria. A comparison of these transfectants with D5-3A8 and wildtype WTT2 cells was made to evaluate the role of mitochondria in folate metabolism. The data demonstrated that pteroyltriglutamates in the mitochondria are as effective as longer chain length derivatives as coenzymes in the synthesis of glycine.
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