Isolation and Characterization of Vacuoles from the Ergot Fungus Claviceps purpurea

Abstract

Protoplasts of Claviceps purpurea were prepared by treatment of mycelium with a lytic mixture of snail gut enzyme and cellulase from Trichoderma viride. Such protoplasts could be efficiently lysed by Triton X-100 treatment at high osmotic pressure without Ca2+ or Mg2+, allowing the release of intact vacuoles in high yields. Vacuoles obtained from cells grown in modified Vogel medium (vegetative-type cells not producing alkaloids) were isolated and purified by centrifugation from a 5% Ficoll 400 (wt/vol) phase into the interphase between two layers, one containing 0.25 M each of mannitol and sucrose, and one containing 0.5 M mannitol. Vacuoles derived from cells grown in a medium favoring ergot alkaloid synthesis (sclerotia-like cells) were isolated by gentle centrifugation of filtered protoplast lysates without addition of Ficoll 400. Biochemical analyses of the vacuole fraction isolated from either kind of cell revealed their function as compartments harboring several hydrolytic enzymes. However, the enrichment of free amino acids in vacuoles of sclerotia-like cells was less pronounced than that in vacuoles of vegetative-type cells, indicating a difference in metabolic compartmentation in the two types of cells.

D-Lysergic acid activation and cell-free synthesis of D-lysergyl peptides in an enzyme fraction from the ergot fungus Claviceps purpurea

“…Surprisingly, in the wild-type strain ATCC 20102 (producing only 5-10 mg of ergotamine/L) it was present at the same level. However, when all of these strains were grown in modified Vogel medium which does not support the formation of sclerotia-likc cells and alkaloid production (Keller et al, 1984b), the enzyme was nearly undetectable. This dependence on the growth conditions that favor a morphology characteristic for alkaloid production in C. purpurea provides additional evidence that the enzyme is linked to the secondary metabolism in the ergot fungus.…”

“…They produce 500-600 mg L'1 ergotamine after growth for 2 weeks in the inoculum medium of Amici et al (1966) in the additional presence of 3 mM tryptophan. The strains were maintained and grown as described previously (Keller et al, 1980(Keller et al, , 1984bKeller, 1983). Modified Vogel medium was as described (Keller et al, 1984b).…”

“…The strains were maintained and grown as described previously (Keller et al, 1980(Keller et al, , 1984bKeller, 1983). Modified Vogel medium was as described (Keller et al, 1984b). C. purpurea strain Ecc 93 was kindly provided by Dr.…”