Isolation of dihydrofolate and folylpolyglutamate synthetase activities from Neurospora

Abstract

The possible association of dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) in Neurospora crassa (FGSC 853, wild type) has been examined using mycelial pxtracts prepared and fractionated in the presence of protease inhibitors. DHFS and FPGS were assayed by following the incorporation of labelled glutamate into dihydrofolate and methylenetetrahydrofolate polyglutamate, respectively. Both of these activities were predominately cytosolic in mycelia that were harvested 24 hr after spore inoculation of defined minimal medium. Relatively small amounts of total mycelial DHFS activity were associated with mitochondrial fractions isolated by differential centrifugation. In contrast, ca 20% of the mycelial FPGS activity was mitochondrial. Treatment of the mitochondrial fractions with Triton X-100 suggested that these activities were not latent under the assay conditions employed. Separate peaks of DHFS and FPGS activity were observed when (NH 4) 2SO 4-fractionated protein was desalted and chromatographed on columns of either Mono Q HR, DEAE-cellulose, heparin agarose, Matrex Green A or Reactive Green 5. Gel filtration indicated average M r values of 52 and 66 × 10 3 for DHFS and FPGS protein, respectively. Dihydrofolate synthetase 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 conjugate 5,10-methylene tetrahydrofolate. SDS-polyacrylamide gel electrophoresis of the Matrex Green A peak fractions revealed a major protein band of average M r 52 × 10 3 whose concentration appeared to parallel DHFS activity. FPGS protein (average M r 66 × 10 3), which lacked ability to glutamyl conjugate dihydropteroate, was recovered by a similar protocol. The reaction catalysed by DHFS protein displayed ATP dependency, was stabilized by glycerol, and product formation was favoured under alkaline conditions. The major catalytic properties of Neurospora DHFS are compared with those of other species.