Tissue was prepared at 4°C and, for experiments with cell-free preparations, thawed samples (25 mg of brain tissue and 5 mg of placental tissue for STS activity, 100 mg of brain tissue, and mg of adrenal cortex tissue for P450c17 and SULT2 activity) were homogenized in 1 mL ice-cold homogenization buffer as described previously ( Steckelbroeck et al . 2002 ). Aliquots were removed for protein determination ( Lowry et al . 1951 ). To obtain high protein concentrations for the measurement of enzyme activity in subcellular fractions, 200 mg of brain tissue were homogenized in 1 mL homogenization buffer. Whole homogenate, nuclear, membrane, and cytosolic fractions were prepared as previously described ( Steckelbroeck et al . 2002 ).
As a mitochondrial P450 system, P450c11 is dependent on two electron transfer proteins, adrenodoxin reductase and adrenodoxin that transfer 2 electrons from NADPH to the P450 for each monooxygenase reaction catalyzed by the enzyme. In most respects this process of electron transfer appears similar to that of P450scc system that catalyzes cholesterol side chain cleavage.  Similar to P450scc the process of electrons transfer is leaky leading to superoxide production. The rate of electron leakage during metabolism depends on the functional groups of the steroid substrate.