Purification and properties of Neurospora folylpolyglutamate synthetase

Chan, P.Y.; Dale, P.L.; Cossins, E.A.

Purification and properties of Neurospora folylpolyglutamate synthetase

1991

Chan, P.Y. | Dale, P.L. | Cossins, E.A.

Folylpolyglutamate synthetase (FPGS) of Neurospora crassa, wild type (FGSC 853) was purified over 4000-fold and partially stabilized in the presence of glycerol. The purification protocol involved (NH4)2S04 fractionation and chromatography on DE-52 cellulose, Sephacryl S 200, phenylagarose and hydroxylapatite, Enzyme activity was assayed by incorporation of [3H]glutamate into polyglutamates of tetrahydrofolate and 5,10-methylenetetrahydrofolate. The enzyme had properties like those previously reported for the synthetases of bacterial and mammalian origins. Thus, Neurospora FPGS had an apparent Mr of ca 65 000, polyglutamate synthesis was favoured at alkaline pH and the reaction required ATP (Km 286 micromoles) and magnesium. A variety of folate substrates were utilized with 5,10-methylenetetrahydrofolate monoglutamate being the most effective (Km 27 micromoles). The antifolates aminopterin and methotrexate, supplied at 5-100 millimoles, were not substrates of the enzyme. HPLC analysis of the folate reaction products showed that methylene folate was converted to polyglutamates that were more highly conjugated when the FPGS reaction time was extended to 12 hr. Based on carboxypeptidase treatment of these products and their behaviour on HPLC it is concluded that purified Neurospora FPGS synthesizes a number of folyloligo-gamma-polyglutamates from methylenetetrahydrofolate monoglutamate.

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