반응 #44519

반응 방정식

Tris-HCl

MgCl2

nicotinamide

PRPP

→

nicotinamide mononucleotide

nicotinamide

PRPP

반응 조건

후처리

1. 1
   기타Nampt reaction products
2. 2
   기타The Nampt reaction
3. 3
   기타The reaction was terminated
4. 4
   workup.ADDITIONby adding 125 μl of 1 M HClO4
5. 5
   기타Protein was then precipitated at 18,000 g, and 500 μl of the supernatant
6. 6
   workup.ADDITIONAfter centrifugation, 100 μl of sample was mixed with 400 μl of Buffer A (50 mM K2PO4/KHPO4, pH 7.0)
7. 7
   기타The products from Nampt reaction
8. 8
   기타Results of HPLC detection of Nampt reaction products

실험 절차

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. 4D). Nampt failed to catalyze the synthesis of nicotinic acid mononucleotide (NaMN) from nicotinic acid and PRPP (see, e.g., FIGS. 13A and 13B), 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.

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