Phosphate → NADPH
Reaction equation
P([O-])([O-])([O-])=O.[Mg+2].[Cl-].[Cl-].[CH:9]1[CH:14]=[N+:13]([C@@H:15]2[O:19][C@H:18]([CH2:20][O:21][P:22]([O:25][P:26]([O:29][CH2:30][C@H:31]3[O:35][C@@H:34]([N:36]4[C:40]5[N:41]=[CH:42][N:43]=[C:44]([NH2:45])[C:39]=5[N:38]=[CH:37]4)[C@H:33]([O:46][P:47]([OH:50])([OH:49])=[O:48])[C@@H:32]3[OH:51])([OH:28])=[O:27])([OH:24])=[O:23])[C@@H:17]([OH:52])[C@H:16]2[OH:53])[CH:12]=[C:11]([C:54]([NH2:56])=[O:55])[CH:10]=1>>[CH:42]1[N:43]=[C:44]([NH2:45])[C:39]2[N:38]=[CH:37][N:36]([C@@H:34]3[O:35][C@H:31]([CH2:30][O:29][P:26]([O:25][P:22]([O:21][CH2:20][C@H:18]4[O:19][C@@H:15]([N:13]5[CH:12]=[C:11]([C:54]([NH2:56])=[O:55])[CH2:10][CH:9]=[CH:14]5)[C@H:16]([OH:53])[C@@H:17]4[OH:52])([OH:24])=[O:23])([OH:28])=[O:27])[C@@H:32]([OH:51])[C@H:33]3[O:46][P:47]([OH:50])([OH:49])=[O:48])[C:40]=2[N:41]=1Participants
Procedure
Cell membrane fractions derived from insect cells transfected with recombinant baculoviruses expressing 11-hydroxylase and complementary electron transport proteins were analyzed for 11-hydroxylase activity in a reaction mixture containing 80 mM phosphate buffer, pH 7.4, 8 mM MgCl2, and 0.9 mM NADP+ in a final volume of 200 ul. In order to insure an adequate source of reducing equivalents, an NADPH regenerating system was provided by adding glucose-6-phosphate dehydrogenase (1.5 U/ml) and 8 mM glucose-6-phosphate. Steroid substrate (e.g., androstenedione) was provided at a final concentration of 0.3 mM. Reaction mixtures were incubated at 37° C. for 30 min. The reactions were terminated by the addition of 200 ul methanol and then placed on ice. Samples were pelleted by centrifugation to remove precipitated protein.
Sources & evidence
- ORD record
- ord-31c9fcb2e22244bfb479e072a929945d
- Source dataset
- uspto-grants-2006_04
- Patent number
- US07033807B2
Reaction notes
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