IDENTIFICATION AND PURIFICATION OF A BOVINE LIVER MITOCHONDRIAL NAD(-GLYCOHYDROLASE())

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 speci fic NAD(+)glycohydrolase, which catalizes hydrolysis of NAD(+) to ADP- ribose and nicotinamide, We purified this enzyme from bovine liver mit ochondrial membranes. The final preparation had a 1660-fold purified e nzyme activity and contained a main protein band with an apparent mola r mass of 32,000 in a SDS-polyacrylamide gel. The identity of this pro tein band with NAD(+)-glycohydrolase was verified by renaturation of i ts enzymatic activity, Partial amino acid sequence information was obt ained from two enzyme fragments after proteolytic cleavage of the prot ein 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(+)-glycohyd rolase sequences.