Effects of zinc and cadmium on apoptotic DNA fragmentation in isolated bovine liver nuclei.

Isolated nuclei from mammalian cells contain a calcium-dependent endonuclease. The produced DNA fragmentation is a necessary step in the sequence of events resulting in apoptosis (programmed cell death). We report here that zinc and cadmium inhibit the calcium-dependent endonuclease. The essential metal ion zinc may counterbalance the calcium-mediated apoptosis. In contrast to zinc, cadmium alone stimulates the endonuclease by replacing calcium. Thus cadmium exerts a dual effect: micromolar concentrations inhibit the apoptotic endonuclease in the presence but activate the enzyme in the absence of calcium.

Cadmium and zinc mediated changes of the Ca(2+)-dependent endonuclease in apoptosis.

Isolated nuclei from mammalian cells contain a Ca(2+)-dependent endonuclease [1]. The produced DNA fragmentation is a necessary step in the sequence of events resulting in apoptosis [2]. We report here that zinc inhibits the DNA fragmentation in dependence of the free Ca2+ concentrations, suggesting that a balance between zinc and calcium might regulate the Ca(2+)-dependent endonuclease. Incubation of nuclei with different free calcium concentrations combined with cadmium shows a stronger inhibition of the DNA fragmentation than zinc. Cadmium inhibits the endonuclease in a calcium-independent way. Surprisingly cadmium alone is able to stimulate the endonuclease, thus to replace Ca2+.

Probing of active site residues of the zinc enzyme 5-aminolevulinate dehydratase by spin and fluorescence labels.

5-Aminolevulinate dehydratase from bovine liver requires Zn(II) for its activity and is inhibited by micromolecular concentrations of Pb(II). To elucidate the structure of the active site and its interactions between the active site and the metal binding site we labeled the active site for fluorescence studies and ESR spectroscopy. o-Phthalaldehyde reacted with active site lysyl and cysteinyl residues to form a fluorescent isoindole derivative. The fluorescence energy was independent of the deprivation of Zn(II) and of its substitution by the inhibitory Pb(II). For ESR-studies five iodoacetamide and four isothiocyanate pyrrolidine-N-oxyl derivatives with various spacer lengths were used to label the active site cysteinyl and lysyl residues, respectively. The ESR spectra of the modified enzyme preparations exhibited a significant immobilization of all labels, even with the longest spacers employed. Obviously the reactive cysteine is buried more than 12 A, and the active site lysine more than 11 A in a cleft of the enzyme structure. Zn(II) deprivation from the iodoacetamide spin-labeled enzyme caused a marked reversible increase in label mobility, whereas the Pb(II) substituted enzyme exhibited a smaller mobilization of the label. These results are interpreted by a model of the active site where the reactive cysteinyl and the lysyl side groups are close enough to be crosslinked by o-phthalaldehyde within a distance of 3 A. A structural role is assigned to Zn(II) in the enzyme, since Zn(II) deprivation does not alter the fluorescence of the isoindole derivative and increases the mobility of the cysteine-bound spin labels in the active site cleft.