This proposal describes experiments designed to investigate the regulation and enzymology of one-carbon metabolism mediated by folate coenzymes. Specifically, the regulation of C1-THF synthase, the central enzyme in folate interconversions, will be investigated in the rat as a mammalian model. Regulation of the enzyme will be examined under four experimental paradigms, chosen because they all involve perturbations of the one-carbon pathways. These paradigms include nutrition (vitamin B12 and folate deficiencies, methanol toxicity, altered thyroid function, and fructose-induced hyperuricemia), development, organ regeneration, and neoplasia. C1-THF synthase specific activities, concentration, and mRNA levels will be determined in an effort to characterize not only the spectrum of regulatory responses, but also the level and potential mechanism(s) of those responses. The enzyme will be purified and characterized from rat liver and a cDNA clone will be isolated and sequenced to generate structural information on the enzyme. The final phase of the project is directed at relating the findings in the rat model to human disease states by purifying and characterizing the enzyme from human tissue and obtaining a cDNA clone for the human enzyme to aid in structural analysis of that protein. It is hoped that these studies will add to our understanding of the regulation of this central area of metabolism and allow more rational basis for the treatment and maintenance of human disorders related to one-carbon metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK036913-01
Application #
3235502
Study Section
Biochemistry Study Section (BIO)
Project Start
1986-04-01
Project End
1989-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
1
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Texas Austin
Department
Type
Schools of Arts and Sciences
DUNS #
City
Austin
State
TX
Country
United States
Zip Code
78713
Howard, Katherine M; Muga, Stephanie J; Zhang, Liwen et al. (2003) Characterization of the rat cytoplasmic C1-tetrahydrofolate synthase gene and analysis of its expression in liver regeneration and fetal development. Gene 319:85-97
West, M G; Barlowe, C K; Appling, D R (1993) Cloning and characterization of the Saccharomyces cerevisiae gene encoding NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase. J Biol Chem 268:153-60
Garcia-Martinez, L F; Appling, D R (1993) Characterization of the folate-dependent mitochondrial oxidation of carbon 3 of serine. Biochemistry 32:4671-6
Pasternack, L B; Laude Jr, D A; Appling, D R (1992) 13C NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiae. Biochemistry 31:8713-9
Appling, D R (1991) Compartmentation of folate-mediated one-carbon metabolism in eukaryotes. FASEB J 5:2645-51
Barlowe, C K; Appling, D R (1990) Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme. Mol Cell Biol 10:5679-87
Thigpen, A E; West, M G; Appling, D R (1990) Rat C1-tetrahydrofolate synthase. cDNA isolation, tissue-specific levels of the mRNA, and expression of the protein in yeast. J Biol Chem 265:7907-13
Barlowe, C K; Appling, D R (1990) Isolation and characterization of a novel eukaryotic monofunctional NAD(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase. Biochemistry 29:7089-94
Appling, D R; Ayo, S H; Kreisberg, J I (1990) Immunolocalization of C1-tetrahydrofolate synthase in the rat kidney. Biochem Biophys Res Commun 168:625-30
Barlowe, C K; Williams, M E; Rabinowitz, J C et al. (1989) Site-directed mutagenesis of yeast C1-tetrahydrofolate synthase: analysis of an overlapping active site in a multifunctional enzyme. Biochemistry 28:2099-106

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