Purification and NH2-Terminal Amino Acid Sequence of Human Thymidylate Synthase in an Overproducing Transformant of Mouse FM3A Cells

• Published in: Journal of biochemistry (Tokyo) Vol. 97; no. 3; pp. 845 - 850
• Main Authors: SHIMIZU, Kimiko, AYUSAWA, Dai, TAKEISHI, Keiichi, SENO, Takeshi
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.01.1985
• Subjects: 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; Human; Cell culture; Vertebrata; Mammalia; Cell line; Purification; Mouse; Rodentia; N terminal-Sequence; Isolation; Primary structure; Thymidylate synthase
• DOI: 10.1093/oxfordjournals.jbchem.a135125
• ISSN: 0021-924X

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-