A study to examine the amino acids associated with the substrate binding sites in Lactobacillus casei thymidylate synthetase will be undertaken. Various folate analogues will be covalently fixed to the protein in the presence and absence of deoxyuridine 5'-phosphate and 5-fluoro-deoxyuridine 5'-phosphate to determine whether these nucleotides promote the specificity of binding. The peptides containing bound folate will be isolated and sequenced according to methods previously described by us. From this information it should be possible to locate the folate binding site(s) within the linear sequence of thymidylate synthetase and to compare its position within the sequence with what has already been established for 5-fluoro-deoxyuridine 5'-phosphate. Similar studies will be conducted with the folate analogue, methotrexate which will also be fixed to the synthetase and its location established by peptide isolation and sequencing. Treatment of the enzyme with amino acid modifying agents should also provide information on the relationship of specific amino acids to both the binding reaction and their involvement in the mechanistics of the reaction. A comparative study of thymidylate synthetase isolated from Lactobacillus casei, Eschericia coli B, T2 bacteriophage induced E. coli B, Ehrlich ascites cells, HeLa cells, and calf thymus will be conducted. This shall include molecular weight, amino acid analysis, peptide mapping and end group analysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026387-08
Application #
3273870
Study Section
Biochemistry Study Section (BIO)
Project Start
1979-04-01
Project End
1988-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
8
Fiscal Year
1987
Total Cost
Indirect Cost
Name
New York State Department of Health
Department
Type
DUNS #
002436061
City
Menands
State
NY
Country
United States
Zip Code
12204
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Maley, G F; Maley, F (1988) Properties of a defined mutant of Escherichia coli thymidylate synthase. J Biol Chem 263:7620-7
Chu, F K; Maley, F; Martinez, J et al. (1987) Interrupted thymidylate synthase gene of bacteriophages T2 and T6 and other potential self-splicing introns in the T-even bacteriophages. J Bacteriol 169:4368-75
Chu, F K; Maley, G F; Wang, A M et al. (1987) Localization of the T4 phage ribonucleotide reductase B1 subunit gene and the nucleotide sequence of its upstream and 5' coding regions. Gene 57:143-8
Chu, F K; Maley, G F; Maley, F (1987) Mechanism and requirements of in vitro RNA splicing of the primary transcript from the T4 bacteriophage thymidylate synthase gene. Biochemistry 26:3050-7
Maley, F; Maley, G F; West, D K et al. (1986) RNA processing in a structural gene from bacteriophage T4. Biochem Soc Trans 14:813-5
Belfort, M; Pedersen-Lane, J; Ehrenman, K et al. (1986) RNA splicing and in vivo expression of the intron-containing td gene of bacteriophage T4. Gene 41:93-102
Chu, F K; Maley, G F; West, D K et al. (1986) Characterization of the intron in the phage T4 thymidylate synthase gene and evidence for its self-excision from the primary transcript. Cell 45:157-66
Ehrenman, K; Pedersen-Lane, J; West, D et al. (1986) Processing of phage T4 td-encoded RNA is analogous to the eukaryotic group I splicing pathway. Proc Natl Acad Sci U S A 83:5875-9
West, D K; Belfort, M; Maley, G F et al. (1986) Cloning and expression of an intron-deleted phage T4 td gene. J Biol Chem 261:13446-50

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