1988 Fiscal Year Final Research Report Summary
Stracture and expression of genes of the enzymes involved in purine nucleotide catabolism
| Project/Area Number |
62480135
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Yokohama city university, School of medicine |
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Principal Investigator |
TSUSHIMA Keizo Yokohama city university, School of medicine,professor, 医学生化学, 教授 (20045917)
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| Co-Investigator(Kenkyū-buntansha) |
YAMAZAKI Kenichi Nagoya university, School of Agriculture, research associate, 農学部生化学制御研施設 (40182480)
NISHINO Tomoko Yokohama city university, School of medicine, research associate, 医学生化学, 助手 (80075613)
NISHINO Takeshi Yokohama city university, School of medicine, associate professor, 医学生化学, 助教授 (40094312)
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| Project Period (FY) |
1987 – 1988
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| Keywords | Purine nuceotide / Xanthine dehydrogenase / cDNA cloning / Structure and function of the enzyme / 反応速度論的解析 |
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| Research Abstract |
Previously we have purified the enzymes involved in purine catabolism to homogeneity from many sources. These enzymes are among these enzymes xanthine dehydrogenase responses most dramtaically, isolation of cDNA clone has been attempted. Furthermore, redox and kinetic properties of xanthine dehydrogenase were extensively studied.
(1) Screening of the cDNA library with rabbit antiserum to purified rat liver xanthine dehydrogenase yielded six immunologically positive clones. phage DNAs of two out of the six clones were isolated and the cDNA insert (1 kbp) of each clone was subjected to nucleotide sequence analysis. To clone longer cDNA, screening of the cDNA library were repeated.
(2) Proteolytic cleavage of the enzyme yielded several domain peptides. Most of the amino acids sequence of the smalleslt domain peptide and partial amino acid sequence were known. The NAD binding site of the enzyme was modified with 5'-FSBA. After proteolytic cleavage of the midified enzyme the modified peptide was isolated and sequenced.
(3) Kinetic studies of xanthine dehydrogenase elucidate the mechanism of the reaction of the enzyme with xanthine, NAD and oxygen as substrates.
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