期刊
BIOCHEMICAL PHARMACOLOGY
卷 77, 期 10, 页码 1612-1620出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.bcp.2009.02.017
关键词
NAD; PARP; Mitochondria; FK866; NMNAT; NaPRT
资金
- University of Florence
- Italian Ministry of University and Scientific and Technological Research
- Associazione Italiana Sclerosi Multipla
- Ente Cassa di Risparmio di Firenze
The emerging key role of NAD-consuming enzymes in cell biology has renewed the interest in NAD resynthesis through the rescue pathways. The first step of the nicotinamide-dependent NAD-rescue pathway is operated by nicotinamide phosphoribosyl transferase (NaPRT) forming nicotinamide mononucleotide (NMN). Because of the difficulties in measuring NMN, numerous open questions exist about the pathophysiological relevance of NaPRT and NMN itself Here, we describe a new method of fluorimetric NMN detection upon derivatization of its alkylpyridinium group with acetophenone. By adopting this method, we analyzed the kinetics of nicotinamide-dependent NAD recycling in HeLa and U937 cells. Measurement of NMN contents in subcellular fractions revealed that the nucleotide is highly enriched in mitochondria, suggesting intramitochondrial NAD synthesis. NMN increases in cells undergoing hyperactivation of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1, or exposed to gallotannin, a putative inhibitor of NMN-adenylyl transferases. Evidence that the inhibitor of NAD resynthesis FK866 selectively inhibits NaPRT having no effect on NMNAT activity is also provided. Importantly, NMN reduces NAD and ATP depletion in cells undergoing PARP-1 hyperactivation, significantly delaying cell death. Finally, we show that a single injection of FK866 in the mouse induces long-lasting (up to 16 h) but mild (similar to 20%) reduction of NMN contents in different organs, suggesting slow rate of basal NAD consumption in vivo. Data provide new information on the biochemistry and pharmacology of NAD biosynthesis, allowing a better understanding of pyridine nucleotide metabolism. (c) 2009 Elsevier Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据