Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in mitochondria.
Mitochondrial ADP-ribosylation leads to modification of two proteins of approximately 26 and 53 kDA: The nature of these proteins and, hence, the physiological consequences of their modification have remained unknown. Here, a 55 kDa protein, glutamate dehydrogenase (GDH), was established as a specific acceptor for enzymatic, cysteine-specific ADP-ribosylation in mitochondria. The modified protein was isolated from the mitochondrial preparation and identified as GDH by N-terminal sequencing and mass spectrometric analyses of tryptic digests. Incubation of human hepatoma cells with [14C]adenine demonstrated the occurrence of the modification in vivo. Purified GDH was ADP-ribosylated in a cysteine residue in the presence of the mitochondrial activity that transferred the ADP-ribose from NAD+ onto the acceptor site. ADP- ribosylation of GDH led to substantial inhibition of its catalytic activity. The stoichiometry between incorporated ADP-ribose and GDH subunits suggests that modification of one subunit per catalytically active homohexamer causes the inactivation of the enzyme. Isolated, ADP-ribosylated GDH was reactivated by an Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase. GDH, a highly regulated enzyme, is the first mitochondrial protein identified whose activity may be modulated by ADP-ribosylation.
Herrero-Yraola
A
A
Institut für Biochemie, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany.
Bakhit
S M
SM
Franke
P
P
Weise
C
C
Schweiger
M
M
Jorcke
D
D
Ziegler
M
M
D000244:Adenosine Diphosphate / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D008274:Magnesium; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism
D002413:Cations, Divalent; D009243:NAD; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D009249:NADP; D000244:Adenosine Diphosphate; D005969:Glutamate Dehydrogenase; D008274:Magnesium