A microassay for mammalian folylpolyglutamate synthetase

A new assay for the enzyme folylpoly-gamma-glutamate synthetase (FPGS) that offers significant advantages over other published procedures has been developed. This assay is based on the addition of high specific activity [3H]glutamic acid to (6-S)-tetrahydrofolate followed by trapping of the labeled tetrahydropteroyldiglutamate product as a covalently bound macromolecular complex by the addition of formaldehyde, fluorodeoxyuridylate, and pure bacterial thymidylate synthase. This complex is then separated from excess labeled glutamic acid by centrifugal elution of a 1-ml Sephadex G-50 column. The assay was found to be useful for the measurement of FPGS on small tissue samples and is amenable with the assay of FPGS in cell sonicates. Typically, blank values of 100-200 cpm are seen with a signal normally more than 10 times higher. Analysis of 20-30 samples can be accomplished in less than 90 min. As a result, this assay has proven useful for detection of enzyme in elution fractions from chromatographic columns.