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 | |
Online Access | Get full text |
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Summary: | 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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Bibliography: | 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 |
ISSN: | 0021-924X 1756-2651 |
DOI: | 10.1093/oxfordjournals.jbchem.a135125 |