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Affinity chromatography of dihydrofolate reductase
The Biochemical Journal
|August 1, 1971
Summary
Dihydrofolate reductase (DHFR) was purified using affinity chromatography. Methotrexate affinity columns effectively purified DHFR from Lactobacillus casei, revealing enzyme interconversion and stabilization effects.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- Dihydrofolate reductase (DHFR) is a crucial enzyme in folate metabolism.
- Understanding DHFR's properties is vital for developing targeted therapies.
- Affinity chromatography offers a powerful method for enzyme purification.
Purpose of the Study:
- To purify dihydrofolate reductase (DHFR) from Lactobacillus casei MTX/R.
- To investigate DHFR's behavior on affinity columns with folic acid and methotrexate.
- To elucidate the role of cofactors like NADPH in enzyme stability and activity.
Main Methods:
- Preparation of affinity columns using cyanogen bromide-activated agarose coupled with 1,6-diaminohexane.
- Covalent attachment of stable folate derivatives via carbodi-imide condensation.
- Purification and characterization of DHFR using folic acid and methotrexate affinity chromatography.
Main Results:
- Methotrexate affinity columns at pH 6.0 effectively retained DHFR.
- Folic acid columns only retarded the enzyme, indicating selective binding.
- NADPH stabilized DHFR and influenced its inactivation by methotrexate, especially with glycine.
Conclusions:
- Affinity chromatography with methotrexate is a highly effective method for purifying DHFR.
- DHFR exists in interconvertible forms, influenced by substrates and cofactors.
- NADPH plays a critical role in modulating DHFR stability and methotrexate sensitivity.