3-Phosphoglyceraldehyde → Ribose 5-phosphate
반응식
[P:1]([O:5][CH2:6][CH:7]([OH:10])[CH:8]=[O:9])([OH:4])([OH:3])=[O:2].C(O)[CH:12]([OH:19])[CH2:13][O:14]P(O)(O)=O.C1N=C(N)C2N=CN([C@@H]3O[C@H](COP(OP(OC[C@H]4O[C@@H](N5C=C(C(N)=O)CC=C5)[C@H](O)[C@@H]4O)(O)=O)(O)=O)[C@@H](O)[C@H]3O)C=2N=1>>[P:1]([O:5][CH2:6][C@@H:7]([OH:10])[C@@H:8]([OH:9])[C@@H:12]([OH:19])[CH:13]=[O:14])([OH:4])([OH:3])=[O:2]참가 물질
4
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- 온도
- 30 °C
실험 절차
Crude cell extracts were prepared in 100 mM Tris/HCl at pH 7.8. Determination of TKT activity required application of several coupling enzymes. RPI and RPE served as coupling enzymes to provide the substrates ribulose 5-phosphate and xylulose 5-phosphate from ribose 5-phosphate (R5P) supplied in the assay. Online monitoring at 340 nm was enabled by further conversion of glyceraldehyde 3-phosphate as product of the TKT reaction by triosephosphate isomerase and glycerophosphate dehydrogenase (TPI/GDH). In contrast to the above mentioned procedure, two distinct mastermixes were prepared. Mix A contained buffer, TPI, RPI, RPE and NADH in a total volume of 650 μl, mix B contained buffer, TPP, MgCl2 and cell extract in a total volume of 300 μl. Both were pre-warmed for 10 min at 30° C. Ribose 5-phosphate was provided in the cuvette and the reaction was started by subsequent addition of mix B and mix A. The final reaction mix contained 50 mM Tris/HCl (pH 7.8), 0.5 mM NADH, 1 U RPI, 0.5 U RPE, 1 U TPI/GDH, 10 mM MgCl2, 0.2 mM TPP, 20 mM R5P and 50 μl of crude cell extract.