Fig. 3.

Human placental cytosolic COMT-mediated O-methylation of 2-OH-E₂ and 4-OH-E₂ (top panel) and their sensitivity to heat inactivation at 42°C (middle panel) and 46°C (bottom panel). The incubation mixture consisted of 10 μM 2-OH-E₂ or 4-OH-E₂, 250 μM [methyl-³H]AdoMet (containing 0.2 μCi), 0.5 mg/ml human placental cytosolic protein, 1 mM dithiothreitol, and 1.2 mM MgCl₂ in a final volume of 0.25 ml of Tris-HCl buffer (10 mM, pH 7.4). Incubations were performed at 37°C for 10 min. For heat inactivation, the cytosolic proteins were first incubated at 42 or 46°C for 15 min before analysis of their catalytic activity. Each point is the mean of duplicate determinations (with variations <5%). H/C refers to the ratio of the catalytic activity (H) of COMT that was preincubated at 42°C or 46°C for 15 min over the control catalytic activity that was preincubated at 30°C for 15 min.