TABLE 2

Effect of assay conditions on estradiol-3-glucuronidation by human liver microsomes

Kinetic parameters were determined using HLM under varying assay conditions as described under Materials and Methods. Results are from two separate experiments (each kinetic determination was performed using nine substrate concentrations in duplicate). Assay buffer A contained 100 mM potassium phosphate, pH 7.1, 1 mM MgCl2, 5 mM saccharic acid 1,4-lactone. Assay Buffer B contained 100 mM Tris/malate, pH 7.4, 10 mM MgCl2, 10 mM saccharic acid 1,4-lactone.

Activating Agent
Microsomal Preparation Assay Buffer Alamethecin
Sonication
Brij 58
Vmax
S50
n
Vmax
S50
n
Vmax
S50
n
pmol/min/mg μM pmol/min/mg μM pmol/min/mg μM
Phosphate A 1670, 810 50, 30 1.3, 1.3 1080, 1070 40, 39 1.4, 1.4 1170, 710 44, 49 1.5, 1.4
Phosphate B 2190, 1150 50, 38 1.3, 1.3 730, 730 50, 50 1.2, 1.2 840, 870 38, 83 1.7, 1.3
Sucrose A 580, 480 35, 30 1.3, 1.4 330, 300 33, 30 1.2, 1.4 480, 470 37, 50 1.5, 1.3
Sucrose
B
820, 690
30, 43
1.3, 1.3
360, 360
53, 61
1.2, 1.2
420, 390
50, 48
1.6, 1.5