The general description for the construction of vectors used in the COS-1 expression studies of mutated CYP21A2 has been previously described [11 (link), 22 (link)].
Expression of wild-type and mutant CYP21A2 enzymes and assays of 21OH activity were performed as previously described [23 (link), 24 (link)]. Enzyme activities were expressed as a percentage of conversion, taking the apparent specific activity of the CYP21A2 wild type as 100%. Assays were performed after 40 min of incubation time.
Apparent kinetic constants were determined 24 h after transfection. Intact cells were incubated as previously described [23 (link)] together with 0.5, 1.0, 2.0, 3.0, or 6.0 μM of unlabeled 17-hydroxyprogesterone (Sigma-Aldrich, Saint Louis, USA) as substrate. After incubation at 37°C for 20 min, steroids were extracted and analyzed as previously described [23 (link)]. Apparent kinetic constants were calculated after linear regression of the data derived from determinations of enzymatic activity at each of the five different substrate concentrations.
Expression of wild-type and mutant CYP21A2 enzymes and assays of 21OH activity were performed as previously described [23 (link), 24 (link)]. Enzyme activities were expressed as a percentage of conversion, taking the apparent specific activity of the CYP21A2 wild type as 100%. Assays were performed after 40 min of incubation time.
Apparent kinetic constants were determined 24 h after transfection. Intact cells were incubated as previously described [23 (link)] together with 0.5, 1.0, 2.0, 3.0, or 6.0 μM of unlabeled 17-hydroxyprogesterone (Sigma-Aldrich, Saint Louis, USA) as substrate. After incubation at 37°C for 20 min, steroids were extracted and analyzed as previously described [23 (link)]. Apparent kinetic constants were calculated after linear regression of the data derived from determinations of enzymatic activity at each of the five different substrate concentrations.