P450 17α Research Articles

Interaction of steroids with adrenal cytochrome [formula omitted] ( [formula omitted]) in liposome membranes

Purified cytochrome P-450 17α, lyase from guinea-pig adrenal microsomes, which catalyzes progesterone 17α-hydroxylation and sequentially C17C20 bond cleavage of the 17α-hydroxyprogesterone, was successfully incorporated into liposomal membranes composed of only phosphatidylcholine or of a phospholipid mixture of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine at a molar ratio of 5:3:1. Although the purified P-450 17α, lyase was readily converted into P-420 in the detergent-solubilized system without substrates, the P-450 embedded in the liposomal membranes was found to be quite stable without the substrates. Using the P-450 17α, lyase - proteoliposomes , the interaction of steroids with P-450 17α, lyase was studied for progesterone, 17α-hydroxyprogesterone and androstenedione in the liposomal system by optical difference spectroscopy and by equilibrium dialysis. The partition coefficients of steroids between the aqueous phase and the liposomal membranes were determined by the equilibrium dialysis. They were about 1.4–1.6-times higher in phosphatidylcholine liposomes than in the liposomes of the lipid mixture. The dissociation constants of the P-450- steroid complexes were calculated from the apparent dissociation constants using the partition coefficients for the situation where the substrate-binding site faces the lipid phase of the membranes or where it faces the aqueous phase. The dissociation constant in the former case was not affected by the lipid composition. These results suggest that P-450 17α, lyase might interact only with the substrates in the lipid phase of the liposomal membranes.

Ontogeny of adrenal steroid hydroxylases: evidence for cAMP-independent gene expression

Total RNA from normal and anencephalic human fetal adrenals was examined by blot analysis for transcripts encoding P-450 scc, P-450 11β P-450 17α P-450 c21 and adrenodoxin using bovine cDNA clones specific for these different enzymes. The specific contents of RNA encoding these components of the adrenocortical steroidogenic pathway were found to be similar in both types of adrenal tissue. Likewise, immunoblot analysis showed comparable concentrations of P-450 scc, P-450 17α and adrenodoxin protein to be present in adrenal tissues from normal and anencephalic human fetuses. Immunoblot analysis of homogenates of fetal sheep adrenals of increasing gestational age (85–145 days) showed constant levels of P-450 scc and P-450 11α, but increasing P-450 17α content, especially near term. Both sheep fetuses prior to 136 days gestational age and human anencephalic fetuses are known to have extremely low circulating levels of immunoreactive ACTH as well as very low adrenal adenylate cyclase activity. Thus, it is concluded that factors other than pituitary ACTH which operate independent of adenylate cyclase activation are required for the initial expression (imprinting) of steroid hydroxylase genes.

Regulation of the biosynthesis of steroidogenic enzymes

Recombinant DNA technology can permit study of the regulation of steroid hydroxylase gene expression at three levels. The first of these is cAMP-regulated gene expression. In the adrenal, ACTH, via cAMP, increases the expression of the genes for all of the cytochrome P-450 species involved in the steroid biosynthetic pathway, as well as the iron-sulfur protein, adrenodoxin. This action of cAMP is inhibited by cycloheximide, suggestive of the involvement of a regulatory protein factor in mediating this action of cAMP. The second level is tissue-specific regulation of steroid hydroxylase gene expression. An example of this which we have studied is the expression of cholesterol side-chain cleavage cytochrome P-450 (P-450 scc) and 17α-hydroxylase cytochrome P-450 (P-450 17α) in the bovine ovary. P-450 scc is expressed at high levels in the corpus luteum but at low levels in follicles, whereas P-450 17α is expressed in follicles, but is undetectable in the corpus luteum. The third level is fetal imprinting. A number of the cytochrome P-450 species involving in the steroidogenic pathway are expressed in the fetal adrenal at a time when exposure of the gland to ACTH is very low, suggestive that factor(s) other than pituitary ACTH mediate this expression in fetal life.

Regulation of synthesis and activity of bovine adrenocortical NADPH-cytochrome P-450 reductase by ACTH

Primary, monolayer cultures of bovine adrenocortical cells have been utilized to demonstrate that ACTH regulates the synthesis and thus the activity of NADPH-cytochrome P-450 reductase. The temporal pattern of induction correlates well with that previously reported for cytochromes P-450 17α and P-450 C21, microsomal steroid hydroxylases serviced by P-450 reductase. Results of in vitro translation studies suggest that ACTH regulates P-450 reductase synthesis at the transcriptional level and show that the adrenocortical enzyme is synthesized as the mature form. The action of ACTH to induce P-450 reductase synthesis in the adrenal cortex can be mimicked by dibutyryl cAMP, but not by phenobarbital which induces P-450 reductase synthesis in liver.

Studies on cytochrome P-450 (P-450 17α,lyase)from guinea pig adrenal microsomes. Dual function of a single enzyme and effect of cytochrome b5

We have reported (Kominami S., Shinzawa K. and Takemori S. (1982) Biochem. Biophys. Res. Commun. 109, 916–921) that a cytochrome P-450 purified from guinea pig adrenal microsomes shows 17α-hydroxylase and C-17,20-lyase activities in a reconstituted system with NADPH-cytochrome P-450 reductase. The homogeneity of the purified cytochrome P-450 was examined with the following methods: isoelectric focusing, immunoelectrophoresis and affinity chromatography on cytochrome b 5-immobilized Sepharose. It was found that progesterone competitively inhibited C-17,20-lyase reaction and that progesterone was converted into androstenedione by 17α-hydroxylation followed by the lyase reaction. These results indicate that the dual activities are carried out by a single enzyme (P-450 17α,lyase). P-450 17α,lyase had the maximum activity at pH 6.1 both for 17α-hydroxylation (6.0 nmol/min per nmol of P-450) and the lyase reaction (11.0 nmol/min per nmol of P-450). Upon addition of cytochrome b 5 to the reconstituted system, the optimal pH for 17α-hydroxylation was shifted to 7.0 and that of the lyase reaction to 6.6. The maximum activities at these optimal pH values were almost the same in the presence or absence of cytochrome b 5. With the addition of cytochrome b 5, both the activities were stimulated above pH 6.3–6.5 and were suppressed below pH 6.3–6.5. These results indicate that cytochrome b 5 plays some important role in controlling the dual activities of P-450 17α,lyase.

The role of cytochromes P-450 in adrenal steroidogenesis

Steroid hormones from adrenal glands are synthesized from cholesterol via several monooxygenase reactions catalysed by different species of cytochrome P-450. P-450 scc and P-450 11β are located in the mitochondria and P-450 c21 and P-450 17α,lyase are located in the endoplasmic reticulum. There are different electron transfer pathways from NADPH to cytochrome P-450 in the two organelles. The metabolic intermediates move back and forth between the organelles during the synthetic process. Recently, steroidogenic electron transfer systems have been reconstituted and well characterized.