The function of NADH-semidehydroascorbate reductase and ascorbic acid in corticosteroid hydroxylation

Area of Science:

• Biochemistry
• Mitochondrial Physiology
• Steroidogenesis

Background:

• NADH-semidehydroascorbate reductase and ascorbate form an electron transport pathway (ETP) supplying reducing equivalents to cytochrome P-450scc in rat adrenal mitochondria.
• The role of this ascorbate-dependent ETP in other mitochondrial hydroxylases was previously unclear.

Purpose of the Study:

• To investigate the role of the ascorbate-dependent ETP in supplying reducing equivalents to cytochrome P-450(11 beta/18) in adrenal mitochondria.
• To compare the efficiency of the ascorbate-dependent ETP with the classical pathway for mitochondrial hydroxylation.
• To reconstitute the ascorbate-dependent 11 beta/18-hydroxylase activity.

Main Methods:

• Studied ascorbate-dependent electron transport in rat adrenal and bovine adrenal cortex mitochondria.
• Assessed 11 beta- and 18-hydroxylation rates supported by the ascorbate-dependent ETP and TCA cycle intermediates.
• Partially reconstituted 11 beta/18-hydroxylase activity using purified mitochondrial membranes and sonified mitochondrial supernatant.

Main Results:

• The ascorbate-dependent ETP supplies reducing equivalents to cytochrome P-450(11 beta/18) in both rat and bovine adrenal mitochondria.
• This pathway's activity depends on catalase or cold shock treatment of mitochondria.
• The ascorbate-dependent pathway may be essential for maximal in vivo hydroxylation by mitochondrial hydroxylases.
• Reconstituted preparations lacking catalase or cold shock treatment showed restored activity.

Conclusions:

• The ascorbate-dependent ETP is a significant pathway for supplying reducing equivalents to mitochondrial hydroxylases in the adrenal cortex.
• This pathway is likely essential for maximal steroid 11 beta- and 18-hydroxylation in vivo.
• The reconstituted system provides insights into the components required for this specific hydroxylase activity.