X 100 → 4-Methylumbelliferone

Participants

Reaction Steps

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.

Provenance & Evidence