ISOLATION OF DIHYDROFOLATE AND FOLYLPOLYGLUTAMATE SYNTHETASE ACTIVITIES FROM NEUROSPORA

The possible association of dihydrofolate synthetase (DHFS) and folylp olyglutamate synthetase (FPGS) in Neurospora crassa (FGSC 853, wild ty pe) has been examined using mycelial extracts prepared and fractionate d in the presence of protease inhibitors. DHFS and FPGS were assayed b y following the incorporation of labelled glutamate into dihydrofolate and methylenetetrahydrofolate polyglutamate, respectively. Both of th ese activities were predominately cytosolic in mycelia that were harve sted 24 hr after sport inoculation of defined minimal medium. Relative ly small amounts of total mycelial DHFS activity were associated with mitochondrial fractions isolated by differential centrifugation. In co ntrast, ca 20% of the mycelial FPGS activity was mitochondrial. Treatm ent of the mitochondrial fractions with Triton X-100 suggested that th ese activities were not latent under the assay conditions employed. Se parate peaks of DHFS and FPGS activity were observed when (NH4)(2)SO4- fractionated protein was desalted and chromatographed on columns of ei ther Mono Q HR, DEAE-cellulose, heparin agarose, Matrex Green A or Rea ctive Green 5. Gel filtration indicated average M(r) values of 52 and 66 x 10(3) for DHFS and FPGS protein, respectively. Dihydrofolate synt hetase protein was purified over 1000-fold by a protocol that included chromatography on DEAE-cellulose, DEAE-Sephacel, heparin agarose and Matrex Green A. The isolated protein lacked ability to glutamyl conjug ate 5,10-methylene tetrahydrofolate. SDS-polyacrylamide gel electropho resis of the Matrex Green A peak fractions revealed a major protein ba nd of average M(r) 52 x 10(3) whose concentration appeared to parallel DHFS activity. FPGS protein (average M(r) 66 x 10(3)), which lacked a bility to glutamyl conjugate dihydropteroate, was recovered by a simil ar protocol. The reaction catalysed by DHFS protein displayed ATP depe ndency, was stabilized by glycerol, and product formation was favoured under alkaline conditions. The major catalytic properties of Neurospo ra DHFS are compared with those of other species.