Precursor-product relationships of Poly(γ-glutamylcysteinyl)glycine biosynthesis in Datura innoxia

Abstract

Suspension cultures of Datura innoxia cells were pulse-labeled with [³⁵S]cysteine, then exposed to Cd to determine whether there is a direct precursor-product relationship amongst the different forms of the Cd-induced polypeptides, poly(γ-glutamylcysteinyl)glycines [(γEC)_nG, n=2 to 5]. Degradation of the polypeptides and possible regeneration of the [³⁵S]-labeled glutathione and cysteine pools were also examined. After 2 h of exposure to [³⁵S]cysteine, about 70% of the [³⁵S]cysteine in the soluble fraction of the cell was incorporated into [³⁵S]glutathione before exposure of the cells to Cd. One h after Cd exposure, most of the cellular [³⁵S]glutathione was depleted and label was incorporated into (γEC)_nG. Analysis of [³⁵S](γEC)_nG by reverse phase HPLC showed no direct precursor-product relationship between the synthesis of the shorter and longer chain forms. However, the rate of synthesis of the different polypeptides was linear for 32 h after Cd exposure. There was no evidence of degradation of [³⁵S](γEC)_nG nor was it excreted into the medium within this period. From these results it is suggested that in the presence of Cd, a large pool of (γEC)_nG is unavailable for elongation to (γEC)_n+1G.