Figure 1.
Schematic overview of the steroid hormone biosynthesis.
Figure 2.
A) SDS-Polyacrylamid gel electrophoresis.
Lane 1: protein marker IV (peqlab), lane 2: CYP11A1 after Ni-NTA purification, lane 3: CYP11A1 after Ni-NTA and Sp-sepharose purification. B) Absolute spectrum of purified CYP11A1 in the range of 260–700 nm.
Figure 3.
Determination of kinetic parameters for the conversion of cholesterol to pregnenolone catalyzed by CYP11A1 using Adx and AdR as redox partners. The product formation analyzed by HPLC is represented as mean ± standard deviation of four individual experiments.
Figure 4.
Comparison of pregnenolone formation by CYP 11A1 in the presence of 5-fold excess of DHEA or DHEAS (n = 5 for each group, asterisk: p<0.05).
Figure 5.
Determination of kinetic parameters for the conversion of cholesterol to pregnenolone catalyzed by CYP11A1 with Adx and AdR as redox partners in the presence of DHEAS. The product formation analyzed by HPLC is represented as mean ± standard deviation of four individual experiments.
Table 1.
Kinetic parameters of CYP11A1, metabolizing cholesterol with or without the addition of DHEAS.
Figure 6.
Determination of substrate affinity to CYP11A1.
Kd values of CYP11A1 for cholesterol in absence (n = 5) and in the presence of a 5-fold excess of DHEAS (n = 5).
Figure 7.
Binding affinity of Adx and CYP11A1.
a) Overlay of Biacore3000 sensograms of measurements with 7.5 nM CYP11A1 in absence of cholesterol, in the presence of cholesterol and in the presence of cholesterol and DHEAS (CYP11A1: cholesterol: DHEAS, 1∶15∶75), b) Comparison of Kd values for the interaction between CYP11A1 and Adx without cholesterol (n = 8), with cholesterol (n = 8, CYP11A1: cholesterol, 1∶15) and with cholesterol in the presence of DHEAS (n = 8, CYP11A1: cholesterol: DHEAS, 1∶15∶75).
Table 2.
Binding parameters for CYP11A1 and Adx without cholesterol (n = 8), with cholesterol (n = 8) and with cholesterol and in the presence of DHEAS (n = 8) determined from measurements with a Biacore3000 system.