Purification and NH2-Terminal Amino Acid Sequence of Human Thymidylate Synthase in an Overproducing Transformant of Mouse FM3A Cells
Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following facts: i) The source of the enzyme was a transformant of mouse FM3A mutant cells which lacks mouse thymidylate synthase but overproduces huma...
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Published in | Journal of biochemistry (Tokyo) Vol. 97; no. 3; pp. 845 - 850 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
Oxford
Oxford University Press
01.01.1985
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Subjects | |
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Abstract | Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following facts: i) The source of the enzyme was a transformant of mouse FM3A mutant cells which lacks mouse thymidylate synthase but overproduces human thymidylate synthase. ii) The enzyme could be purified on two kinds of affinity column, Cibacron blue dye-bound agarose and methotrexate-bound Sepharose. iii) The enzyme could finally be separated from a trace of impurities by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The purified human thymidylate synthase had a subunit with a molecular weight of 33,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was subjected to Edman degradation and the NH2-terminal 24 amino acids were sequenced by successive use of a high-sensitivity gas-phase protein sequencer and high performance liquid chromatography to be as follows: Pro 1 -Val-Ala-Gly-Ser-Glu-Leu-Pro-Arg- Arg 10 - -Pro11-Leu-Pro-Pro-Ala-Ala-Glu-Arg-Asp20- -Ala21-Glu-Pro-Arg- |
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AbstractList | Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following facts: i) The source of the enzyme was a transformant of mouse FM3A mutant cells which lacks mouse thymidylate synthase but overproduces human thymidylate synthase. ii) The enzyme could be purified on two kinds of affinity column, Cibacron blue dye-bound agarose and methotrexate-bound Sepharose. iii) The enzyme could finally be separated from a trace of impurities by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The purified human thymidylate synthase had a subunit with a molecular weight of 33,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was subjected to Edman degradation and the NH2-terminal 24 amino acids were sequenced by successive use of a high-sensitivity gas-phase protein sequencer and high performance liquid chromatography to be as follows: Pro-Val-Ala-Gly-Ser-Glu-Leu-Pro-Arg-Arg-Pro-Leu-Pro-Pro-Ala-Ala-Gln-Glu- Arg-Asp -Ala-Glu-Pro-Arg-. Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following facts: i) The source of the enzyme was a transformant of mouse FM3A mutant cells which lacks mouse thymidylate synthase but overproduces human thymidylate synthase. ii) The enzyme could be purified on two kinds of affinity column, Cibacron blue dye-bound agarose and methotrexate-bound Sepharose. iii) The enzyme could finally be separated from a trace of impurities by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The purified human thymidylate synthase had a subunit with a molecular weight of 33,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was subjected to Edman degradation and the NH2-terminal 24 amino acids were sequenced by successive use of a high-sensitivity gas-phase protein sequencer and high performance liquid chromatography to be as follows: Pro 1 -Val-Ala-Gly-Ser-Glu-Leu-Pro-Arg- Arg 10 - -Pro11-Leu-Pro-Pro-Ala-Ala-Glu-Arg-Asp20- -Ala21-Glu-Pro-Arg- |
Author | TAKEISHI, Keiichi AYUSAWA, Dai SENO, Takeshi SHIMIZU, Kimiko |
Author_xml | – sequence: 1 givenname: Kimiko surname: SHIMIZU fullname: SHIMIZU, Kimiko organization: Department of Immunology and Virology, Saitama Cancer Center Research Institute, Ina-machi, Saitama 362 – sequence: 2 givenname: Dai surname: AYUSAWA fullname: AYUSAWA, Dai organization: Department of Immunology and Virology, Saitama Cancer Center Research Institute, Ina-machi, Saitama 362 – sequence: 3 givenname: Keiichi surname: TAKEISHI fullname: TAKEISHI, Keiichi organization: Department of Immunology and Virology, Saitama Cancer Center Research Institute, Ina-machi, Saitama 362 – sequence: 4 givenname: Takeshi surname: SENO fullname: SENO, Takeshi organization: Department of Immunology and Virology, Saitama Cancer Center Research Institute, Ina-machi, Saitama 362 |
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Keywords | Human Cell culture Vertebrata Mammalia Cell line Purification Mouse Rodentia N terminal-Sequence Isolation Primary structure Thymidylate synthase |
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Notes | ArticleID:97.3.845 1 This work was supported in part by grants for cancer research from the Ministry of Education, Science and Culture of Japan, and the Society for Promotion of Cancer Research of Japan. istex:540B4ADD811C6912ACBDBF5D951A7BC28FC372AE 2 To whom correspondence should be addressed. ark:/67375/HXZ-PP5H1PKK-X ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
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Snippet | Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following... |
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SubjectTerms | Amino Acid Sequence Analytical, structural and metabolic biochemistry Animals Biological and medical sciences Cell Line Chromatography, Affinity Chromatography, High Pressure Liquid Cloning, Molecular DNA Electrophoresis, Polyacrylamide Gel Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Gene Expression Regulation Humans Methyltransferases - isolation & purification Mice Mutation Thymidylate Synthase - genetics Thymidylate Synthase - isolation & purification Transferases |
Title | Purification and NH2-Terminal Amino Acid Sequence of Human Thymidylate Synthase in an Overproducing Transformant of Mouse FM3A Cells |
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