Metabolism of exogenous succinate by Claviceps purpurea: activity of some Krebs cycle enzymes and anaplerotic-gluconeogenic enzymes

Abstract

Claviceps purpurea PRL 1980 was grown on various chemically defined media. The fungus was able to utilize glucose, succinate, and alpha-ketoglutarate as sole carbon sources. Most rapid growth as determined by dry weight was obtained on glucose whereas growth was slowest on alpha-ketoglutarate. Succinate medium supplemented with yeast extract, casein hydrolysate, Proflo oil, glycerol, or glucose supported faster and more growth than the unsupplemented medium. Neither acetate, citrate, malate, pyruvate, succinamide, nor dimethylsuccinate was utilized as a sole carbon source. Methods for making enzyme preparations from the fungus were investigated and optimum assay conditions were determined. Satisfactory sonicates could be prepared in 5 minutes using 20 mM Tris (pH 7.5) with 5 mM EDTA. Five μmoles malate per cuvette was the optimum substrate level for malate dehydrogenase (decarboxylating) from Claviceps. The optimum pH for assays of malate dehydrogenase (decarboxylating) was determined to be pH 9.6. The activity of malate dehydrogenase was high over a broad pH range with pH 9.6 being near the optimum. The activity of fumarase at pH 7.5 was higher than at pH 7.0 or 8.0. The presence of EDTA in assays of malate dehydrogenase, malate dehydrogenase (decarboxylating), fumarase, and succinate dehydrogenase was shown to be beneficial in the assays; however the concentrations of EDTA in the fumarase assay could not exceed 0.115 mM for optimum activity. ...