Purification of guanosine triphosphate cyclohydrolase I and dihydrofolate reductase on a dihydrofolate-sepharose affinity column
✍ Scribed by Rudolf L. Then
- Publisher
- Elsevier Science
- Year
- 1979
- Tongue
- English
- Weight
- 575 KB
- Volume
- 100
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Folate was coupled t,o AH-Sepharose 4B, the gel poured into small columns, and the Sepharose-bound folate reduced in situ to dihydrofolate by dithionite/ascorbate at pH 6 to 7. The dihydrofolate-Sepharose column was used to purify guanosine triphosphate cyclohydrolase I (EC 3.5.4.16) and dihydrofolate reductase (EC 1.5.1.3). All steps were carried out in the cold and in the presence of 20 mM mercaptoethanol. GTP cyclohydrolase I bound strongly to the dihydrofolate-Sepharose column and was purified several-hundred-fold in a single step. It did not bind to folate-Sepharose. Binding to dihydrofolate-Sepharose is assumed to reflect a physiological role of dihydrofolate. GTP cyclohydrolase II did not bind to either folate-or dihydrofolate-Sepharose.
Dihydrofolate reductase from Escherichia coli B and from rat liver did not bind to folate-Sepharose under the test conditions, but could be well purified on the dihydrofolate-Sepharose column. This column is judged to be useful for the purification of other folate-converting enzymes.