Activity of poly(gamma-glutamylcysteinyl)glycine synthesis in crude extract of fission yeast, Schizosaccharomyces pombe.

Peptides, the structures of which are indicated by (y-Glu-Cys)n-Gly (n^.2), were first reported from the fission yeast Schizosaccharomyces pombe}] When the yeast cells were cultured in a Cd-containing growth medium, Cdbinding substances were formed,2) in which the peptides with ^-values of 2 and 3 were contained, and the peptides were named cadystin.1} These peptides occur in higher plants when they were exposed to heavy metals.3) In addition, the peptides with ^-values larger than 3 have also been found and the name phytochelatin has been proposed for this family of peptides. Manyreports have so far appeared for the occurrence of the peptides as a consequence of heavy metal challenge.4~6) The peptides have been thought to be involved in the detoxification and homeostasis of metal ions. Recently an enzyme that catalyzes the synthesis of the peptides has been isolated from Silene cucubalus.7'8) According to these reports (y-Glu-Cys)2-Gly is synthesized by transpeptidation of the y-glutamylcysteine moiety of glutathione to an acceptor glutathione molecule. Cadmium ion is needed for the activation of the enzymeand other metal ions such as Cu and Zn are also responsible for the activation. In this paper weshow that a crude extract of S. pombecells can synthesize the peptides. S. pombe (972h~) was cultured in 500ml of growth mediumcontaining 2%tryptone, 2% glucose, and 1% yeast extract. The cells were harvested at the late logarithmic phase, washed with buffer A (10 mMTris-HCl, pH 7.5, containing 1.5m sorbitol and 5mMMgCl2) and suspended in 40ml of buffer A, to which 5ml of enzyme solution (4.5 mg/ml Novozyme 234 containing 0.9 mg/ml Zymolyase-IOOT and 18mM 2-mercaptoethanol) were added. The mixture was incubated at 30°C for 1 hr and centrifuged. The cells that sedimented were washed with buffer A and resuspended in 1ml of buffer B (200mM Tris-HCl, pH 8.0, containing 1 mMsodium dithionite and 1 mMphenylmethanesulfonyl fluoride). The mixture was soaked in liquid nitrogen to freeze and then thawed to disrupt the cells. After centrifugation, the supernatant solution was collected and referred to as the crude extract. The concentrations of Cd, Cu, and Zn in the crude extract were 0.26,um, 4.8jam, and 0.93mM, respectively, by