Abstract
NAD+ glycohydrolase (NADase) activity and ADP-ribosylation have been proposed to be involved in the pro-oxidant induced calcium efflux from mitochondria. It
has been suggested that NADase activity results in the generation of free ADP-ribose from NAD+, which in turn would lead to non-enzymatic covalent modification
of mitochondrial proteins related to the calcium release system. Such a mechnism would assign hydrolysis of NAD+ by NADase a key step in the calcium efflux from
these organelles.
However, the present work describes the identification of bovine liver mitochondrial NAD+ glycohydrolase as a member of the class of multifunctional ADP-ribosyl
cyclases/cyclic ADP-ribose hydrolases, known to be potential second messenger enzymes. These enzymes catalyze the synthesis and degradation of cyclic
ADP-ribose (cADP-ribose), a potent intracellular calcium-mobilizing agent. The results support a new model for the pro-oxidant-induced calcium release from
mitochondria involving cADP-ribose as a specific messenger, rather than the non-enzymatic modification of proteins by ADP-ribose.
Moreover, it was found that under several conditions, which strongly inhibit the NADase, the extent of ADP-ribosylation in bovine liver mitochondria with NAD+ as
substrate was only slightly diminished, if at all. The reaction led to the specific modification of two proteins with apparent molecular masses of approx. 26 and 53
kDa, respectively. Under these selected conditions mitochondrial ADP-ribosylation seems to occur as an enzymatic reaction, rather than a non-enzymatic transfer of
ADP-ribose previously liberated from NAD+ by NADase activity.
The chemical stability of the protein-ADP-ribose bonds in mitochondria indicated that cysteine residues are the predominant acceptors. Furthermore, yeast aldehyde
dehydrogenase was efficiently ADP-ribosylated utilizing the mitochondrial activity and NAD+ as substrate. Pretreatment of this acceptor with N-ethylmaleimide, a
thiol-specific reagent, substantially decreased its modification. It is, therefore, concluded that bovine liver mitochondria contain a cysteine-specific
ADP-ribosyltransferase.
Besides its function in energy-conversion reactions, the results presented establish an additional role of the mitochondrial NAD+-pool - the transduction of signals. |
