Ernst-L. Winnacker

Phytochelatins: the principal heavy-metal complexing peptides of higher plants

A set of novel heavy-metal complexing peptides was isolated from plant cell suspension cultures; the structure of the peptides was established as (γ-glutamic acid-cysteine)n-glycine (n = 3 to 7). These peptides appear upon induction of plant cells with heavy metals and represent the principal metal-binding activities in the cells. The name phytochelatin is proposed for this new class of natural products.

Termination of the phytochelatin synthase reaction through sequestration of heavy metals by the reaction product

The newly discovered enzyme γ‐glutamylcysteine dipeptidyl transpeptidase was purified to apparent homogeneity from cell suspension cultures of Silene cucubalus. The enzyme catalyzes, in the presence of heavy metal ions, the formation of metal chelating peptides, the phytochelatins, from glutathione. The stoichiometry of the transfer reaction for the first phytochelatin member was determined to be: 2 (γ‐Glu‐Cys)‐Gly → (γ‐Glu‐Cys)2‐Gly + Gly. The enzyme is self regulated in that the reaction products, the phytochelatins, chelate the enzyme activating metal, thus terminating the enzymatic reaction. The higher order phytochelatins have a higher relative complexing affinity than the lower ones, as judged from their ability to terminate the enzymatic reaction.

Homo-phytochelatins are heavy metal-binding peptides of homo-glutathione containing fabales

Exposure of several species of the order Fabales to Cd2+ results in the formation of metal chelating peptides of the general structure (γ‐Glu‐Cys) n ‐β‐Ala (n = 2–7). They are assumed to be formed from homo‐glutathione and are termed homo‐phytochelatins, as they are homologous to the recently discovered phytochelatins. These peptides are induced by a number of metals such as Cd2+, Zn2+, Hg2+, Pb2+, AsO4 − and others. They are assumed to detoxify poisonous heavy metals and to be involved in metal homeostasis.

Synthesis of seven different homologous phytochelatins in metal-exposed Schizosaccharomyces pombe cells

The family of heavy metal‐sequestering peptides with the formula (γ‐Glu‐Cys) n Gly; n = 2–8), the phytochelatins (PCs), are not restricted to higher plants. In Schizosaccharomyces pombe the occurrence of two of these peptides has previously been reported. Reinvestigation of the peptide pattern of the heavy metal‐exposed fission yeast led to the discovery of five additional peptides, a pattern which now corresponds qualitatively exactly to the one in higher plants. From the kinetics of induction we propose that PCs are synthesized by consumption of glutathione or its biosynthetic precursor.

Induction of heavy-metal binding phytochelatins by inoculation of cell cultures in standard media

A large increase in phytochelatin (PC) synthesis occurred when cell cultures of different plant species were transferred from spent medium to fresh standard media. Phytochelatin accumulation correlated with the initial concentration of zinc ions in the nutrient solution. After reaching stationary growth phase, phytochelatins had almost disappeared from the cells which indicates a high turnover of these molecules under normal conditions. No significant formation of the heavy-metal complexing phytochelatins was observed if the microelement ions zinc and copper were omitted from the nutrient solutions for plant cell cultures. Both the induction and degradation phenomena of these peptides indicate that phytochelatins are involved in metal ion homeostasis in plants.

HDF2, the Second Subunit of the Ku Homologue from Saccharomyces cerevisiae

The high affinity DNA binding factor (HDF) protein of Saccharomyces cerevisiae is composed of two subunits and specifically binds ends of double-stranded DNA. The 70-kDa subunit, HDF1, shows significant homology with the 70-kDa subunit of the human Ku protein. Like the Ku protein, HDF1 has been shown to be involved in recombination and double stranded DNA break repair. We have purified and cloned HDF2, the second subunit of the HDF protein. The amino acid sequence of HDF2 shows a 45.6% homology with the 80-kDa subunit of the Ku protein. HDF1 by itself does not bind DNA, while HDF2 protein on its own seems to displays DNA binding activity. Targeted disruption of the HDF2 gene causes a temperature-sensitive phenotype for growth comparable to the phenotype of hdf1− strains. The human Ku protein cannot complement this temperature-sensitive phenotype. hdf2− strains are sensitive to bleomycin and methyl methanesulfonate, but this sensitivity is reduced in comparison with hdf1− strains.

Reactivation of metal-requiring apoenzymes by phytochelatin—metal complexes

The enzymatically inactive, metal‐requiring apoforms of diamino oxidase and of carbonic anhydrase were reactivated by copper‐ and zinc—phytochelatin complexes, respectively. The level and the rate of reactivation effected by metal complexes consisting of poly(γ‐glutamylcysteinyl)glycine as well as by the respective free metal ion were compared. An efficient transfer of zinc and copper from phytochelatin‐complexes to apoenzymes was observed in vitro.

Occurrence of heavy metal binding phytochelatins in plants growing in a mining refuse area

Phytochelatins were identified in roots of heavy metal-sensitiveAcer pseudoplatanus and-resistantSilene cucubalus plants grown in zinc-rich soil of a mine dump. Both plants, when collected from a metal-uncontaminated stand, located nearby, revealed no phytochelatins. Thus, we concluded that metal-binding phytochelatins are specifically induced in plants of heavy metal enriched ecosystems.

Induction of heavy metal-sequestering phytochelatin by cadmium in cell cultures of Rauvolfia serpentina

zelle. In Tabelle 1 sind die Gitterkonstanten sowie die Dichtewerte f/Jr alle neu erhaltenen Verbindungen zusammengestellt. Zur Darstellung dieser Verbindungen wurden jeweils iiquimolare Gemenge der bin/iren Fluoride NaF bzw. AgF, MF 2 und MF 3 in Goldampullen gefiillt und diese 10-14 Tage auf Temperaturen zwischen 500-700 ~ erhitzt. Unter diesen Reaktionsbedingungen wurden jeweils mikrokristalline Proben der beschriebenen Verbindungen erhalten. Zur pyknometrischen Dichtebestimmung wurde wasserfreies Kerosin (Siedeintervall 200-246 ~ als Absperrflfissigkeit benutzt.

Isolation and characterization of the porcine nuclear factor I (NFI) gene.

This study describes the isolation of a major portion of the gene for nuclear factor I (NFI) including its 5′‐flanking region with transcriptional start sites. We screened a porcine liver, genomic DNA library in phage EMBL3A with synthetic oligonucleotides derived from tryptic and cyanogen‐bromide peptide sequences obtained from purified NFI protein. The NFI gene is present as a single copy in porcine DNA.