TABLE 3

Effect of assay conditions on estradiol-17-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
		V_max	K_m	CL_int	V_max	K_m	CL_int	V_max	K_m	CL_int
		pmol/min/mg	μM	μl/min/mg	pmol/min/mg	μM	μl/min/mg	pmol/min/mg	μM	μl/min/mg
Phosphate	A	110, 110	11, 8	10, 13	100, 70	12, 8	8.2, 9.1	120, 120	12, 9	10, 13
Phosphate	B	230, 200	11, 11	21, 18	100, 100	14, 11	6.9, 8.9	190, 200	13, 15	15, 13
Sucrose	A	80, 120	13, 15	6.4, 7.9	60, 60	9, 13	6.2, 4.5	90, 110	13, 13	7.2, 8.4
Sucrose	B	190, 180	16, 18	12, 9.8	80, 70	19, 16	4.0, 4.3	120, 150	26, 18	4.5, 8.4