Nicotinamide phosphoribosyltransferase/visfatin does not catalyze nicotinamide mononucleotide formation in blood plasma.
Nicotinamide phosphoribosyltransferase/visfatin does not catalyze nicotinamide mononucleotide formation in blood plasma.
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DOI：
10.1371/journal.pone.0022781
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Tsuchiya M
中科院分区：
文献类型：
--
作者：
Hara N;Yamada K;Shibata T;Osago H;Tsuchiya M
烟酰胺（Nam）磷酸核糖转移酶（NAMPT）是哺乳动物NAD合成中的限速酶，催化Nam和5-磷酸核糖1-焦磷酸（PRPP）形成烟酰胺单核苷酸肽（NMN）。NAMPT也被描述为具有多种作用的脂肪细胞因子内脂素，尽管该蛋白的生理意义尚不清楚。已经提出内脂素的可能作用是通过NMN的细胞外形成介导的。然而，我们没有检测到小鼠血浆中的NMN，即使用高度特异性和灵敏度的液相色谱/串联质谱。此外，在细胞外间隙中没有或很少有ATP，NAMPT的激活剂。因此，我们质疑内脂素是否催化在这种细胞外环境下原位形成NMN。为了解决这个问题，我们在此使用重组人酶测定了NAMPT反应中底物Nam和PRPP在不含或含ATP的情况下的Km值，并发现1 mM ATP显著降低底物的Km值，特别是PRPP的细胞内浓度。与动力学数据一致，仅当ATP以毫摩尔水平存在时，NAMPT在底物的细胞内浓度下有效地催化NMN形成。低得多的Nam浓度和血浆中几乎不存在PRPP和ATP表明NAMPT不应有效地催化其在细胞外环境下的反应。事实上，NAMPT在血浆中不形成NMN。从这些动力学分析的酶和定量测定其底物，激活剂，和产品，我们得出结论，内脂素不参与NMN形成下的细胞外环境。连同血浆中NMN的缺乏，我们的结论不支持“NAMPT介导的系统NAD生物合成”的概念。我们的研究将促进目前的内脂素生理学的理解。
Nicotinamide (Nam) phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in mammalian NAD synthesis, catalyzing nicotinamide mononucleotide (NMN) formation from Nam and 5-phosphoribosyl 1-pyrophosphate (PRPP). NAMPT has also been described as an adipocytokine visfatin with a variety of actions, although physiological significance of this protein remains unclear. It has been proposed that possible actions of visfatin are mediated through the extracellular formation of NMN. However, we did not detect NMN in mouse blood plasma, even with a highly specific and sensitive liquid chromatography/tandem mass spectrometry. Furthermore, there is no or little ATP, the activator of NAMPT, in extracellular spaces. We thus questioned whether visfatin catalyzes the in situ formation of NMN under such extracellular milieus. To address this question, we here determined Km values for the substrates Nam and PRPP in the NAMPT reaction without or with ATP using a recombinant human enzyme and found that 1 mM ATP dramatically decreases Km values for the substrates, in particular PRPP to its intracellular concentration. Consistent with the kinetic data, only when ATP is present at millimolar levels, NAMPT efficiently catalyzed the NMN formation at the intracellular concentrations of the substrates. Much lower concentrations of Nam and almost the absence of PRPP and ATP in the blood plasma suggest that NAMPT should not efficiently catalyze its reaction under the extracellular milieu. Indeed, NAMPT did not form NMN in the blood plasma. From these kinetic analyses of the enzyme and quantitative determination of its substrates, activator, and product, we conclude that visfatin does not participate in NMN formation under the extracellular milieus. Together with the absence of NMN in the blood plasma, our conclusion does not support the concept of “NAMPT-mediated systemic NAD biosynthesis.” Our study would advance current understanding of visfatin physiology.