Abstract
This chapter discusses about a radioassay method for dihydrofolate synthetase activity in Escherichia coli. The method involves the measurement of the enzymic formation of dihydrofolate. Some of the characteristics of the enzyme were examined by the microbiological assay with Lactobacillus casei . This microbiological method was cumbersome and inaccurate. A simple, rapid, and accurate radioassay method, using L-[ 14 C]glutamic acid as substrate, is developed for a quantitative determination of dihydrofolate synthetase activity. Pteroic acid is synthesized, according to the method of Plante, and further purified by the method of Houlihan. The dihydro form used as substrate is prepared by reduction with sodium dithionite as described by Futterman. Chromatograms of reaction mixtures are analyzed for their ability to support the growth of L. casei . They are also carefully examined by serial sections and radioactive scans. The amount of product (dihydrofolic acid) formed is observed to increase linearly with time up to 50 min of incubation at 37° and to be directly proportional to the amount of protein added up to 2 mg/ml.
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