Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in mitochondria

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Abstract
Mitochondrial ADP‐ribosylation leads to modification of two proteins of ∼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.