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产品中心 药物靶点和Fc受体 小分子药物靶点蛋白

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NNMT His Tag Protein, Rhesus macaque

Nicotinamide N-methyltransferase

价格 ￥2,800.00 （ 供应商现货 ： 3-5个工作日）
货号 UA085036
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产品介绍

产品介绍 评论(0)

产品规格

  • 物种

    Rhesus macaque

  • 分子别名

    Nicotinamide N-methyltransferase

  • Accession

    F7ERX8

  • 表达序列

    Met1-Leu264 with His Tag at the C-Terminus

  • 表达宿主

    E.coli

  • 分子量

    25-35kDa (Reducing)

  • 纯度

    ＞95% by SDS-PAGE,＞90% by HPLC

  • 活性

    The specific activity of NNMT was determined to be >230 nmol /min/mg as per activity assay protocol.

  • 标记

    Unconjugated

  • 标签

    His Tag

  • 性状

    Liquid

  • 缓冲体系

    50mM Tris, 200mM NaCl, 20% Glycerol, pH7.5

  • 储存条件

    Stable for 12 months upon stored at -80℃ from the date of receipt. And avoid repeated freeze-thaws cycles.

  • 文献引用

    1.Swaminathan S, Birudukota S, Thakur MK, Parveen R, Kandan S, Juluri S, Shaik S, Anand NN, Burri RR, Kristam R, Hallur MS, Rajagopal S, Schreuder H, Langer T, Rudolph C, Ruf S, Dhakshinamoorthy S, Gosu R, Kannt A. Crystal structures of monkey and mouse nicotinamide N-methyltransferase (NNMT) bound with end product, 1-methyl nicotinamide. Biochem Biophys Res Commun. 2017 Sep 16;491(2):416-422.
    2.Parsons RB, Smith ML, Williams AC, Waring RH, Ramsden DB. Expression of nicotinamide N-methyltransferase (E.C. 2.1.1.1) in the Parkinsonian brain. J Neuropathol Exp Neurol. 2002 Feb;61(2):111-24.
    3.Kocinaj A, Chaudhury T, Uddin MS, Junaid RR, Ramsden DB, Hondhamuni G, Klamt F, Parsons L, Parsons RB. High Expression of Nicotinamide N-Methyltransferase in Patients with Sporadic Alzheimer's Disease. Mol Neurobiol. 2021 Apr;58(4):1769-1781.

背景介绍

  • NNMT (Nicotinamide N-methyltransferase) is a cytosolic methyltransferase whose crystal structure has been resolved in rhesus macaques and mice at a resolution of 1.88 Å, belonging to the methyltransferase superfamily. This enzyme utilizes S-adenosylmethionine as a methyl donor to catalyze the methylation of nicotinamide into 1-methylnicotinamide, thereby regulating NAD⁺ metabolic homeostasis; it is highly expressed in the liver, adipose tissue, and brain, and participates in energy expenditure, lipid metabolism, and insulin sensitivity modulation through the NAD⁺/Sirt1 pathway. Clinical studies have demonstrated that upregulated NNMT expression is closely associated with obesity, type 2 diabetes, and metabolic syndrome, while its levels are also significantly elevated in specific brain regions of patients with Parkinson's disease and Alzheimer's disease, suggesting that NNMT serves not only as a biomarker for metabolic disorders but also as a promising therapeutic target for drug intervention.

操作步骤

  • 实验方法

    实验原理：MTase-Glo™甲基转移酶检测法基于其反应产物S-腺苷同型半胱氨酸（SAH）来测定NNMT的活性。甲基转移酶反应完成后，只需加入MTase-Glo™ 试剂和MTase-Glo™检测溶液，即可将SAH转化为ATP，并通过荧光素酶反应测定发光信号。

    实验材料

    1.激酶检测缓冲液：20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 0.1 mg/ml BSA. 使用前加入1mM DTT。

    2.NNMT His tag Protein, Rhesus macaque

    3.MTase-GloTM Methyltransferase Assay (Promega, Catalog #: V7601)

    4.底物: Nicotinamide (Sigma, Catalog #: N481907-100GM)

    5.Solid white multi-well plate (384-well plate) (Corning, Catalog #3572)

    6.读板器（PerkinElmer）

    实验步骤

    1.将NNMT蛋白及Methyltransferase-Glo™检测试剂盒中的所有试剂于冰上解冻。

    2.配制工作液：在冰上使用激酶检测缓冲液配制以下工作液：

    2X NNMT工作液：浓度0.4 μg/mL, 0.2 μg/mL, 0.1 μg/mL, 0.05 μg/mL，0 μg/mL

    2X 底物混合液：40 μM SAM和80 μM Nicotinamide。

    3.在384孔板中加入以下组分，使初始反应总体积达到20 μL：

    组分1：10 μL 2X底物混合液

    组分2：10 μL 2X NNMT工作液

    4.置于摇床混匀2分钟，用封板膜密封，于37°C孵育60分钟。

    5.将10X Methyltransferase-Glo™ Reagent用等体积超纯水稀释，向所有反应孔中加入5 μL 5X Methyltransferase-Glo™试剂。

    6.置于摇床混匀2分钟，室温孵育30分钟。

    7.向所有反应孔中加入25 μL MTase-Glo™ Detection Solution。震荡混匀2分钟，室温孵育30分钟。

    8.分别使用终点模式读取化学发光信号。

    9.计算：根据SAH标准曲线，确定生成的 SAH 浓度（pmol），并按下述公式（或试剂盒说明）计算甲基转移酶的特异性活性：

    Specific Activity (pmol/min/µg) =

    SAH (pmol)-Blank

    Incubation time(min) ×amount of enzyme (μg)


  • 生物活性

    • The specific activity of NNMT was determined to be >230 nmol /min/mg as per activity assay protocol.

  • 电泳

    • 1μg (R: reducing condition, N:non-reducing condition).

  • 体积排阻色谱(SEC-HPLC)

    • The purity of NNMT His Tag Protein, Rhesus macaque is more than 90% determined by SEC-HPLC.

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