Identification and purification of a bovine liver mitochondrial NAD+-glycohydrolase

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Abstract
Nonenzymatic ADP‐ribosylation of mitochondrial proteins is thought to play a role in the regulation of Ca2+ efflux from mitochondria. It has been shown that intramitochondrial ADP‐ribose is generated by a specific NAD+ glycohydrolase, which catalizes hydrolysis of NAD+ to ADP‐ribose and nicotinamide. We purified this enzyme from bovine liver mitochondrial membranes. The final preparation had a 1660‐fold purified enzyme activity and contained a main protein band with an apparent molar mass of 32,000 in a SDS‐polyacrylamide gel. The identity of this protein band with NAD+‐glycohydrolase was verified by renaturation of its enzymatic activity. Partial amino acid sequence information was obtained from two enzyme fragments after proteolytic cleavage of the protein band in the SDS‐polyacrylamide gel. Searches in protein databases revealed that an arginine ADP‐ribosyl hydrolase harbours two stretches of amino acids that are highly similar to the partial NAD+‐glycohydrolase sequences.