Purification and cDNA cloning of human red cell pyrimidine 5'-nucleotidase isozymes
| Project/Area Number |
04671405
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Human genetics
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| Research Institution | Okinaka Memorial Institute for medical Research |
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Principal Investigator |
HIRONO Akira Okinaka Memorial Institute for medical Research, 専任研究員 (90181221)
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| Co-Investigator(Kenkyū-buntansha) |
FUJII Hisaichi 東京女子医科大学, 輸血部, 助教授 (70107762)
MIWA Shiro (財)冲中記念成人病研究所, 所長 (40034954)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1993: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1992: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | Pyrimidine 5'-nucleotidase / Isozymes / Pyrimidine 5'-nucleotidase deficiency / Purification / Hemolytic anemia |
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| Research Abstract |
Pyrimidine 5'-uncleotidase (P5N) is important in clinical medicine because the deficiency of the enzyme causes a hereditary nonspherocytic hemolytic anemia. We have been carrying out the structural analysis of P5N isozymes for the purpose of elucidating the primary cause of P5N deficiency. For the first step, we have tried the purification of P5N-II isozyme, Using standard chromatographical techniques, ninety micro grams of P5N-II were purified about 134,000-fold from 2,000 ml of human red blood cells. The purified enzyme had a specific activity of 44,130 mU/mg and gave a main band on SDS polyacry lamide gel electrophoresis. Kinetic studies using the purified P5N-II revealed that the enzyme had a unique property of using a suger phosphate ester 6-phosphogluconate as one of its potent substrates. This might be important in elucidating the physiological function of this isozyme. Although we had performed the amino acid compsition analysis and the partial amino acid sequencing of the enzyme, the N-terminal amino acid residue was found to be blocked. We have already constructed a cDNA library from human reticulocytes, and deblocking and further sequencing of amino acid residues N-terminal region for cDNA cloning is in progress.
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Report
(3 results)
Research Products
(14 results)
