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

Abstract

Nonenzymatic ADP-ribosylation of mitochondrial proteins is thought to play a role in the regulation of Ca 2÷ 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 mitocbondrial 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.

Forever young: SIRT3 a shield against mitochondrial meltdown, aging, and neurodegeneration

“…In response to ROS and increased Ca 2+ , binding of CypD to ANT initiates formation of a tunnel-like structure, which connects the mitochondrial matrix to the cytosol resulting in the rapid flow of NAD + from the mitochondria to the cytosol (Lemasters et al, 2009). Within the cytosol, NAD + is quickly hydrolyzed by multiple NADases to yield ADP-ribose and NAM (Zhang et al, 1995; Bernardi, 1999). The frequency of mtPTP formation may result in the destruction of defective mitochondria by autophagy or possibly even cell death via apoptosis (Kim et al, 2007).…”