High-affinity steroid binding to rat testis 17α-hydroxylase and human placental aromatase

Abstract

The high affinity binding of progesterone to rat testis 17α-hydroxylase and of androstenedione to human placental aromatase was analyzed using spectral, enzymatic and radioactive binding methods. For progesterone interaction with rat testis microsomal cytochrome P-450 the apparent spectral dissociation constant ( K s ) varied between 10 and 33 nM and was dependent on the protein concentration. For androstenedione binding to human placental cytochrome P-450 the apparent K s was 9–10 nM. For progesterone 17α-hydroxylation the apparent Michaelis-Menton constant ( K m ) was 13 nM. For andros-tenedione aromatization the apparent K m was 10 nM. In rat testis microsomes radioactive progesterone could be displaced from a high affinity site with a dissociation constant of 30–50 nM. Methodologie problems such as substrate depletion must be avoided in order to obtain accurate K s and K m values for certain steroid-cytochrome P-450 high affinity interactions.