Cytochrome b5 augments 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase activity

During adrenal steroidogenesis the competition between 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3βHSD) and cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1) for Δ5 steroid intermediates greatly influences steroidogenic output. Cytochrome-b5 (Cyt-b5), a small electron transfer hemoprotein, known to augment the lyase activity of CYP17A1, has been shown to alter the steroidogenic outcome of this competition. In this study, the influence of Cyt-b5 on 3βHSD activity was investigated. In COS-1 cells, Cyt-b5 was shown to significantly increase the activity of both caprine and ovine 3βHSD towards pregnenolone, 17-OH pregnenolone and dehydroepiandrosterone in a substrate and species specific manner. Furthermore, kinetic studies revealed Cyt-b5 to have no influence on the Km values while significantly increasing the Vmax values of ovine 3βHSD for all its respective substrates. In addition, the activity of ovine 3βHSD in microsomal preparations was significantly influenced by the addition of either purified Cyt-b5 or anti-Cyt-b5 IgG. The results presented in this study indicate that Cyt-b5 augments 3βHSD activity and represents the first documentation of such augmentation in any species.

► Does Cyt-b5 augment 3βHSD activity?
► Cyt-b5 significantly increases 3βHSD activity in COS-1 cells.
► 3βHSD stimulation by Cyt-b5 differs between species and between steroid substrates.
► Apo-b5, Cyt-b5 lacking the ability to bind heme, stimulates 3βHSD activity.
► Cyt-b5 significantly increases Vmax values without influencing Km values.