Nicotinamide → Nicotinamide

HRID 2386107

反応式

HRID 2386107 の反応式
C(O)C(N)(CO)CO.Cl.[Mg+2].[Cl-].[Cl-].[C:13]([NH2:21])(=[O:20])[C:14]1[CH:19]=[CH:18][CH:17]=[N:16][CH:15]=1.[CH2:22]([O:39][P:40]([OH:43])([OH:42])=[O:41])[C@H:23]1[O:27][C@H:26]([O:28][P:29]([O:32][P:33]([OH:36])([OH:35])=[O:34])([OH:31])=[O:30])[C@H:25]([OH:37])[C@@H:24]1[OH:38]>>[CH:18]1[CH:17]=[N+:16]([CH:26]2[O:27][C@H:23]([CH2:22][O:39][P:40]([O-:43])([OH:42])=[O:41])[C@@H:24]([OH:38])[C@H:25]2[OH:37])[CH:15]=[C:14]([C:13]([NH2:21])=[O:20])[CH:19]=1.[C:13]([NH2:21])(=[O:20])[C:14]1[CH:19]=[CH:18][CH:17]=[N:16][CH:15]=1.[CH2:22]([O:39][P:40]([OH:43])([OH:42])=[O:41])[C@H:23]1[O:27][C@H:26]([O:28][P:29]([O:32][P:33]([OH:36])([OH:35])=[O:34])([OH:31])=[O:30])[C@H:25]([OH:37])[C@@H:24]1[OH:38]

参加物

7

反応物 4

HCID 936NicotinamideHCID 7339Phosphoribosyl PyrophosphateHCID 93573Tromethamine hydrochlorideHCID 5360315Magnesium Chloride

生成物 3

HCID 936NicotinamideHCID 7339Phosphoribosyl PyrophosphateHCID 46874983[(2R,3S,4R)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate

実験手順

High performance liquid chromatography was used to detect Nampt reaction products. HPLC was performed with Waters 515 pumps and a 2487 detector (Waters, Mass.) with a Supelco LC-18-T column (15 cm×4.6 cm; Supelco, Pa.). The Nampt reaction was conducted at 37° C. for 15 min in 500 μl of reaction buffer (50 mM Tris-HCl [pH 7.5], 10 mM MgCl2, 50 mM nicotinamide, 0.2 mM PRPP) with 50 μg of the recombinant Nampt protein. The reaction was terminated by adding 125 μl of 1 M HClO4. Protein was then precipitated at 18,000 g, and 500 μL of the supernatant was neutralized with 40 μl of 3 M K2CO3. After centrifugation, 100 μl of sample was mixed with 400 μl of Buffer A (50 mM K2PO4/KHPO4, pH 7.0) and loaded into the HPLC system. The products from Nampt reaction were monitored by absorbance at 261 nm. Results of HPLC detection of Nampt reaction products showed that the mouse Nampt produced nicotinamide mononucleotide (NMN) from nicotinamide and PRPP (see e.g. FIG. 2D). Nampt failed to catalyze the synthesis of nicotinic acid mononucleotide (NaMN) from nicotinic acid and PRPP (see e.g. FIG. 8), confirming the substrate specificity of this enzyme. In isolated reactions, it was also confirmed that Nmnat catalyzed the synthesis of NAD from NMN and ATP.

後処理

  1. customNampt reaction products
  2. customThe Nampt reaction
  3. customThe reaction was terminated
  4. additionby adding 125 μl of 1 M HClO4
  5. customProtein was then precipitated at 18,000 g, and 500 μL of the supernatant
  6. additionAfter centrifugation, 100 μl of sample was mixed with 400 μl of Buffer A (50 mM K2PO4/KHPO4, pH 7.0)
  7. customThe products from Nampt reaction
  8. customResults of HPLC detection of Nampt reaction products

出典と根拠

ORDレコード
ord-b58121b607694b57b1ceddc8558f8985
出典データセット
uspto-grants-2012_09
特許番号
US08268575B2

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