Boyer C S, Moore G A, Moldéus P
Department of Toxicology, Karolinska Institute, Stockholm, Sweden.
J Biol Chem. 1993 Feb 25;268(6):4016-20.
The submitochondrial location of the NAD+ glycohydrolase (NADase) and its role in mitochondrial ADP-ribosyl transfer reactions were investigated in isolated rat liver mitochondria. The NADase catalyzes the hydrolysis of NAD+ to ADP-ribose and nicotinamide. Hydrolysis of intramitochondrial NAD+ has been suggested to be the first step in a nonenzymatic mono(ADP-ribosylation) by free ADP-ribose of an inner membrane-associated acceptor peptide and that this protein is involved in regulating calcium efflux from mitochondria during exposure to oxidants. The results of the present study indicate that mitochondrial NADase activity lies outside the matrix space, however. This was determined by assessing the rates of hydrolysis of externally added NAD+ by intact mitochondria and comparing these rates with those obtained when the mitochondria were permeabilized with detergent. No significant difference was observed in the rate of NAD+ hydrolysis when detergent was added indicating that NAD+ hydrolysis by mitochondria does not necessitate its access to the matrix space. The submitochondrial location of the NADase was investigated further by digitonin titration of isolated mitochondria. Digitonin titration of the mitochondria released NADase activity with the outer membrane marker, monoamine oxidase. The digitonin titration data also suggest that the outer membrane is the exclusive location of the NADase. The incorporation of radioactive label derived from [3H]NAD+ into submitochondrial particles proceeds at comparable rates in the absence or presence of NADase activity, indicating that the production of free ADP-ribose is not necessary for intramitochondrial ADP-ribosyl transfer reactions. Thus the conclusions of this study suggest that due to the submitochondrial location of the NADase, it does not participate in intramitochondrial pyridine nucleotide hydrolysis or nonenzymatic mono(ADP-ribosylation) during oxidative stress in mitochondria.
在分离的大鼠肝线粒体中，研究了NAD⁺糖水解酶（NADase）的亚线粒体定位及其在线粒体ADP-核糖基转移反应中的作用。NADase催化NAD⁺水解为ADP-核糖和烟酰胺。线粒体内NAD⁺的水解被认为是内膜相关受体肽被游离ADP-核糖进行非酶单（ADP-核糖基化）的第一步，并且该蛋白质参与在暴露于氧化剂期间调节线粒体的钙外流。然而，本研究结果表明线粒体NADase活性位于基质空间之外。这是通过评估完整线粒体对外源添加的NAD⁺的水解速率，并将这些速率与用去污剂使线粒体通透时获得的速率进行比较来确定的。添加去污剂时，NAD⁺水解速率没有观察到显著差异，表明线粒体对NAD⁺的水解并不需要其进入基质空间。通过对分离的线粒体进行洋地黄皂苷滴定，进一步研究了NADase的亚线粒体定位。线粒体的洋地黄皂苷滴定释放出与外膜标记物单胺氧化酶相关的NADase活性。洋地黄皂苷滴定数据还表明外膜是NADase的唯一位置。在存在或不存在NADase活性的情况下，源自[³H]NAD⁺的放射性标记掺入亚线粒体颗粒的速率相当，表明游离ADP-核糖的产生对于线粒体内ADP-核糖基转移反应不是必需的。因此，本研究的结论表明，由于NADase的亚线粒体定位，它不参与线粒体氧化应激期间的线粒体内吡啶核苷酸水解或非酶单（ADP-核糖基化）。
