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Folypolyglutamate synthetase from beef liver: assay, stabilization, and characterization
Canadian Journal of Biochemistry and Cell Biology = Revue Canadienne De Biochimie Et Biologie Cellulaire
|June 1, 1984
Summary
Researchers developed a new assay for folylpolyglutamate synthetase, enabling partial purification and characterization of the enzyme from beef liver. This assay also allowed for the detection of folate polyglutamates up to hexaglutamates.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Folylpolyglutamate synthetase (FPGS) is crucial for folate metabolism.
- Understanding FPGS activity is vital for comprehending cellular folate homeostasis and antifolate drug mechanisms.
Purpose of the Study:
- To develop and validate a reliable assay for folylpolyglutamate synthetase.
- To partially purify and characterize FPGS from beef liver.
- To investigate the substrate specificity and kinetic properties of FPGS.
Main Methods:
- Development of a [3H]glutamate incorporation assay.
- DEAE-cellulose chromatography for separation of substrates and products.
- Enzyme kinetic studies to determine Km values.
- Analysis of folate polyglutamate chain lengths using chromatography.
Main Results:
- A reliable FPGS assay was established, allowing complete separation of [3H]glutamate.
- FPGS was partially purified from beef liver, and its kinetic parameters were determined.
- Folate polyglutamates up to hexaglutamates were detected, with aminopterin identified as an active antifolate substrate.
Conclusions:
- The developed assay is effective for studying FPGS activity and folate polyglutamate synthesis.
- Kinetic characterization provides insights into FPGS function.
- Detection of long-chain polyglutamates highlights their biological significance.