반응 #462850

반응 방정식

carbodiimide

β-NMN

MgCl2

HEPES NaOH

NAD+

→

cADPR

용매

water

반응 조건

후처리

1. 1
   기타evaporated to dryness
2. 2
   기타The coupling reaction
3. 3
   세척was eluted at a flow rate of 1 ml/min
4. 4
   기타over 30 minutes
5. 5
   기타The AG MP-1 purification system
6. 6
   기타The 8-amino-NAD+ peak was collected
7. 7
   기타evaporated to dryness on a SpeedVac concentrator
8. 8
   기타purified from Aplysia ovotestis
9. 9
   workup.WAITincubated for 2 to 4 hours at room temperature with 45.4 units of ADP-ribosyl cyclase

실험 절차

The chemical coupling of 8-substituted AMP's to β-NMN to form 8-substituted NAD+ 's was performed by carbodiimide coupling essentially as described by Prescott and McLennan (Prescott, M. and McLennan, A. G. (1990) Anal. Biochem. 184, 330-337). 8-amino-AMP (0.1 μmol), β-NMN (1μmol) and MgCl2 (2 μmol) were combined in a microfuge tube and evaporated to dryness using a SpeedVac concentrator. The coupling reaction was initiated by adding 20 μl of 1.5M HEPES-NaOH, pH 6.8 and 20 μl of 1.5M 1-ethyl-3(3-dimethyl-amino-propyl)-carbodiimide-HCl (EDC) and incubated at 37° C. for 12 to 18 hours. The yield of 8-amino-NAD+ was between 40 and 50%. The reaction products were diluted to 1 ml with water and injected onto an AG MP 1 column (0.6×15 cm) which was eluted at a flow rate of 1 ml/min using a trifluoroacetic acid gradient from 1.5 to 150 mM over 30 minutes. The AG MP-1 purification system has been previously described (Lee, H. C. and Aarhus, R. (1991) Cell Regulation 2, 203-209, Axelson, J. T., Bodley, J. W., and Walseth, T. F. (1981) Anal. Biochem. 18, 333-364). The 8-amino-NAD+ peak was collected and evaporated to dryness on a SpeedVac concentrator. The coupling reactions using 8-Br-AMP and 8-azido-AMP were done identically, except that all manipulations with 8-azido-compounds were done in the dark or reduced light. 8-amino-NAD+ was converted to 8-amino cADPR using ADP-ribosyl cyclase purified from Aplysia ovotestis as previously described (Lee, H. C. and Aarhus, R. (1991) Cell Regulation 2, 203-209). The 8-amino-NAD+ was reconstituted with 1 ml of 25 mM HEPES-NaOH, pH 6.8 and incubated for 2 to 4 hours at room temperature with 45.4 units of ADP-ribosyl cyclase (1 unit is defined as the amount of enzyme that produces 1 nmol of cADPR from 1 mM NAD+ at room temperature and pH 6.8). The resulting 8-amino-cADPR was purified by AG MP-1 chromatography as described above. The purified 8-amino-cADPR was evaporated to dryness on a SpeedVac concentrator and stored at -20° C. The conversion of the 8-Br and 8-azido derivatives was identical to that described for 8-amino-NAD+. The ADP-ribosyl cyclase recognized each of the 8-substituted NAD+ derivatives and converted virtually all of the NAD+ derivatives to the corresponding cADPR derivatives.

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