Enzymic, cysteine-specific ADP-ribosylation in bovine liver mitochondria.
NAD+ glycohydrolase (NADase) and non-enzymic ADP-ribosylation have been thought to be involved in the regulation of mitochondrial Ca2+ fluxes. In this study it was found that several conditions (5 mM nicotinamide, 5 mM 3-aminobenzamide, 2 mM EDTA, 1 mM ATP, 10 mM dithiothreitol) known to strongly inhibit the NADase decreased ADP-ribosylation in bovine liver mitochondrial membranes with [32P]NAD+ as substrate to only a limited extent, if at all. The reaction led to the specific modification of two proteins with apparent molecular masses of approx. 26 and 53 kDa. An excess of added free ADP-ribose diminished the incorporation of label from [32P]NAD+ only slightly. Dithiothreitol inactivated the NADase, whereas ADP-ribosylation was unaffected. At low concentrations (25 microM) ADP-ribosylation was efficient with NAD+, but not ADP-ribose, as substrate. Under these conditions mitochondrial ADP-ribosylation seems to occur as an enzymic reaction rather than a non-enzymic transfer of ADP-ribose previously liberated from NAD+ by NAD+ glycohydrolase. The chemical stability of the protein-ADP-ribose bonds in the mitochondrial membranes indicated that cysteine residues are the predominant acceptors. Moreover, yeast aldehyde dehydrogenase, known to be a substrate for thiol-associated ADP-ribosylation, was efficiently ADP-ribosylated by using the mitochondrial activity and NAD+ as substrate. The modification of a cysteine residue in the aldehyde dehydrogenase was verified by the observation that pretreatment of this acceptor protein with N-ethylmaleimide substantially decreased its modification. It is therefore concluded that bovine liver mitochondria contain a cysteine-specific ADP-ribosyltransferase.
Jorcke
D
D
Institut für Biochemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Federal Republic of Germany.
Ziegler
M
M
Herrero-Yraola
A
A
Schweiger
M
M
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism
D004220:Disulfides; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D000444:Aldehyde Dehydrogenase; D011065:Poly(ADP-ribose) Polymerases; D009244:NAD+ Nucleosidase; D003545:Cysteine; D005033:Ethylmaleimide