Biosynthesis of poly(γ-glutamylcysteinyl)glycines in cadmium-tolerant Datura innoxia (Mill.) cells

Abstract

The presence of γ-carboxamide linkages in poly(γ-glutamylcysteinyl)glycines ((γ EC) n G) suggests that these polypeptides are likely to be the products of a biosynthetic pathway. However, the in vivo deamidation of proteins can result in the introduction of γ-carboxamide linkages. Synthetic oligodeoxynucleotide sequences were therefore used as probes to hydridize to any mRNA sequences which could encode such proteins. These probes did not hybridize to any mRNA sequences from cells grown either in the presence or absence of Cd, confirming that these metal-binding polypeptides are not directly encoded by structural genes. The kinetics of induction of synthesis of (γ EC) n G following exposure to Cd, was studied using reverse phase HPLC of extracts from cells grown in the presence of [ 35S]cysteine. These data indicate that the shorter polypeptides are substrates for the synthesis of longer forms. Induced synthesis of (γEC) 2G and (γEC) 3G was detected 5 min after exposure of cells to Cd. This rapid response implies that the pathway is regulated, at least initially, at a post-translational level. Observed insensitivity of this response to cycloheximide further supports this conclusion.