X 100 → 4-Methylumbelliferone
Reaction equation
[CH3:1][C:2]1[C:8]2[CH:9]=[CH:10][C:11]([O:13][C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)=[CH:12][C:7]=2[O:6][C:4](=[O:5])[CH:3]=1.P(O)(O)(O)=O.C(O)(=O)CC(CC(O)=O)(C(O)=O)O.CCC(COC(C(N(CC[NH+](C)C)C)=O)(C1C=CC=CC=1)C1C=CC=CC=1)CC.[Cl-].C[C@@H]([C@@H]1[C@@]2(C)[C@@H](O)C[C@@H]3[C@@]4(C)CC[C@@H](O)C[C@H]4C[C@@H](O)[C@H]3[C@@H]2CC1)CCC(NCCS([O-])(=O)=O)=O.[Na+]>>[CH3:1][C:2]1[C:8]2[CH:9]=[CH:10][C:11]([OH:13])=[CH:12][C:7]=2[O:6][C:4](=[O:5])[CH:3]=1Participants
Procedure
GC enzymatic activity was measured by the method of Ohashi, T., et al., J. Biol Chem. 266:3661 (1991), the disclosure of which is incorporated herein by reference, with the synthetic fluorogenic substrate 4-methylumbelliferyl-β-D-glucopyranoside (4MU-glc) (Sigma, St. Louis, Mo.). The reaction mixture (200 μl) contained 5 mM 4MU-glc, 0.1M citrate phosphate buffer (pH 5.4), 2.1 mM Triton X-100, 0.1% bovine serum albumin, and 3.5 mM sodium taurocholate. After incubation of each cell lysate with the substrate for 30 minutes at 37° C., reactions were terminated by the addition of 3.8 ml of 0.17M glycine carbonate buffer (pH 10.4). The 4-methylumbelliferone (4MU) that had formed was measured fluorometrically. Units of GC activity were expressed as nanomoles of 4MU substrate produced/hour/mg protein. All values represent the average of duplicate measurements.
Workup
- customreactions were terminated by the addition of 3.8 ml of 0.17M glycine carbonate buffer (pH 10.4)
Sources & evidence
- ORD record
- ord-06d8481bdf45426ab4154cdc5184644f
- Source dataset
- uspto-grants-1999_06
- Patent number
- US05911983
Reaction notes
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