Shigeki Takemori

Purification and some properties of cytochrome P-450 specific for steroid 17α-hydroxylation and C17C20 bond cleavage from guinea pig adrenal microsomes

Abstract A novel species of cytochrome P-450 was purified to a specific content of 15 nmol/mg of protein from guinea pig adrenal microsomes. The purified preparation exhibited a single polypeptide band (Mr = 52,000 ± 1,000) when submitted to a sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both progesterone-17α-hydroxylase (1.67 nmol 17α-hydroxyprogesterone/min/nmol cytochrome P-450) and 17α-hydroxyprogesterone C17C20-lyase (0.68 nmol androstenedione/min/nmol cytochrome P-450) activities could be reconstituted upon mixing the purified cytochrome P-450 with NADPH-cytochrome P-450 reductase. The absorption spectrum of the oxidized cytochrome P-450 in the presence of progesterone had maxima at 394 and 650 nm, characteristic of a high spin form. The reduced CO complex of the cytochrome P-450 showed an absorption maximum at 448 nm.

Studies on cytochrome a VII. Physico-chemical properties of purified cytochrome a

This paper gives results of an investigation on the homogeneity and molecular state of preparations of cytochrome a solubilized with surface-active agents and studied by sedimentation experiments and electrophoresis. The sedimentation pattern of cytochrome a in the presence of the non-ionic detergent. Emasol 1130, shows a single homogeneous and symmetric monodispersed component, whereas in the presence of the ionic detergent, sodium cholate, the protein behaves as a polydispersed, aggregated system. Based upon experimental results indicating a sedimentation coefficient of 21.9 S, a diffusion constant of 3.58·10−7 cm2/sec and a partial specific volume of 0.72 ml/g, it si calculated that cytochrome a solubilized with Emasol 1130 has a molecular weight of approx. 530 000, whereas its minimum molecular weight calculated from its iron content is 100 000. Thus the existence of the rigid structure of a pentamer is indicated. It was, furthermore, found that by treatment with reagents such as sodium docecyl sulfate or urea the protein is split into smaller subunits. Based on these results, the molecular state of cytochrome a is discussed.

The role of cytochromes P-450 in adrenal steroidogenesis

Abstract 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.

Steroidogenesis in liposomal system containing adrenal microsomal cytochrome P-450 electron transfer components

Purified adrenal microsomal P-450C21 and/or P-45017 alpha,lyase were incorporated with purified NADPH-cytochrome-P-450 reductase into liposome membranes composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine at a molar ratio of 5:3:1. The rate dependences of reduction of liposomal P-450C21 in the fast phase as well as progesterone hydroxylation activities of P-450C21 and P-45017 alpha,lyase on the reductase concentration in the liposome membranes suggested that electrons were delivered through random collisions between the reductase and cytochrome P-450s in the liposome membranes. A rapid exchange of the steroid metabolic intermediate between vesicles was observed in a reaction system consisting of P-450C21-proteoliposomes and P-45017 alpha,lyase-proteoliposomes. Using the combined liposomal system, it was definitely proved that androstenedione was formed from progesterone mainly by a successive hydroxylation reaction without the intermediate 17 alpha-hydroxyprogesterone leaving from P-45017 alpha,lyase. It was also found that 21-hydroxylation of 17 alpha-hydroxyprogesterone into 11-deoxycortisol was inhibited by a physiological concentration of progesterone.

Immunochemical studies on cytochrome P-450 in adrenal microsomes

An antibody was prepared against electrophoretically homogeneous cytochrome P-450C21 purified from bovine adrenal microsomes. This antibody was used to compare various cytochromes P-450 in bovine and guinea pig adrenal microsomes. In an Ouchterlony double diffusion test, a spur formation was observed between the precipitin lines of the purified bovine cytochrome P-450C21 and guinea pig adrenal microsomes against anti-cytochrome P-450C21 IgG. Anti-cytochrome P-450C21 IgG inhibited 21-hydroxylation both of bovine and guinea pig adrenal microsomes but the inhibition was much more effective in the bovine microsomes than in the guinea pig microsomes. These results suggest that the 21-hydroxylase in the guinea pig microsomes has some molecular similarities to the bovine cytochrome P-450C21 and a part of the antibodies cross-reacts with the 21-hydroxylase in the guinea pig microsomes. Anti-cytochrome P-450C21 IgG did not inhibit the activities of 17 alpha-hydroxylase and C17,20-lyase in the bovine and guinea pig microsomes but stimulated these activities. This result shows that different species of cytochrome P-450 other than cytochrome P-450C21 catalyzes the 17 alpha-hydroxylation and C17,20 bond cleavage. The stimulation of 17 alpha-hydroxylation and C17,20 bond cleavage by blocking 21-hydroxylation indicates that the electron transfer systems for various cytochromes P-450 are intimately linked in adrenal microsomes.

Adrenal cytochrome P-45011β-proteoliposomes catalyzing aldosterone synthesis: preparation and characterization

Purified cytochrome P-45011 beta from bovine adrenocortical mitochondria was successfully incorporated into the liposome membranes composed of phosphatidylcholine, phosphatidylethanolamine and cardiolipin at a molar ratio of 2:2:1. The incorporation of P-45011 beta into the liposome membranes was ascertained by the Ficoll density gradient centrifugation and the protein refractoriness to trypsin digestion. The prepared proteoliposomes containing P-45011 beta and phospholipid at a molar ratio of 1:3000 were unilamellar vesicles of about 40 nm in average diameter. The P-45011 beta embedded in the liposome membranes was found to be more stable than the detergent-solubilized form. The reconstituted system containing the P-45011 beta-proteoliposomes, adrenodoxin and NADPH-adrenodoxin reductase showed catalytic activities not only for the hydroxylation of 11-deoxycorticosterone at 11 beta- and 18-positions but also for its conversion into aldosterone with a turnover number of 2.3 nmol/min per nmol of P-45011 beta. A successive reaction without the intermediates leaving from the enzyme was suggested for the P-45011 beta-mediated conversion of 11-deoxycorticosterone to aldosterone following the result that the formation of aldosterone was linear with respect to time without the lag phase; this was confirmed by the result that radioactivity in aldosterone from 3H-labeled 11-deoxycorticosterone was scarcely decreased by the addition of unlabeled intermediates to the reactions system.

Kinetic control of steroidogenesis by steroid concentration in guinea pig adrenal microsomes

For clarification of the effects of steroid concentration on steroidogenesis of adrenal microsomes, the kinetic parameters, Km and kcat, were determined in the steady-state for progesterone and 17 alpha-hydroxyprogesterone metabolism catalyzed by P-450C21 and P-450(17 alpha lyase) in guinea pig adrenal microsomes. At a high concentration of progesterone, it was equally metabolized by P-450C21 and P-450(17 alpha lyase), while at a low concentration, it was hydroxylated at 17 alpha-position with twice higher rate than at 21-position. 17 alpha-Hydroxyprogesterone is apparently metabolized preferentially by P-450C21 at any concentration. Although the productions of deoxycortisol and androstenedione from 17 alpha-hydroxyprogesterone were strongly inhibited by progesterone, androstenedione formation from progesterone was not inhibited by a high concentration of progesterone. The addition of liposomal P-450C21 to the reaction medium containing adrenal microsomes caused a decrease in the concentration of 17 alpha-hydroxyprogesterone released into the medium in the steady state reaction, but this had no effect on the activity of androstenedione formation from high concentrations of progesterone. It thus follows that androstenedione is produced by successive monooxygenase reactions without the release of 17 alpha-hydroxyprogesterone from P-450(17 alpha lyase) at a high concentration of progesterone, which is the condition of the adrenal microsomes in vivo.

In vitro synthesis of mitochondrial cytochromes P-450(scc) and P-450(11-β) and microsomal cytochrome P-450(C-21) by both free and bound polysomes isolated from bovine adrenal cortex

Abstract In vitro synthesis of mitochondrial cytochromes P-450(scc) and P-450(11-β), and microsomal cytochrome P-450(C-21) programmed by bovine adrenal cortex polysomes was carried out using rat liver cell sap and wheat germ lysate systems. Synthesis of P-450 proteins in the cell-free systems was determined by immunoprecipitation and immunoadsorption using mono-specific antibodies to each species of P-450, and the sizes of the in vitro products were analyzed by SDS-polyacrylamide gel electrophoresis. Both free and bound polysomes synthesized these three species of P-450 in the cell-free systems. P-450(scc) and P-450(C-21) were synthesized apparently as the mature size products, whereas P-450(11-β) was synthesized as a putative precursor approximately 5,000 daltons larger than the mature form. Mitochondrial and microsomal P-450 proteins seem to share common sites of synthesis in the cytoplasm of adrenal cortex cells.

[12] Purification of adrenal cytochrome P-450 (cholesterol desmolase and steroid 11β- and 18-hydroxylase)

Publisher Summary This chapter provides information on the purification of adrenal cytochrome P-450. Two different kinds of cytochrome P-450 play an essential role as oxygenative enzymes in steroid hormone biosynthesis of adrenocortical mitochondria. One is P-450 scc , which is responsible for the conversion of cholesterol to pregnenolone. The other is P-450 11β , which catalyzes 11β-hydroxylation of 11-deoxycorticosteroids. In the method that is described in the chapter, the activity of P-450 scc and P-450 11β is assayed conveniently at 37° with radioactive substrate by measuring the formation of pregnenolone from cholesterol and of corticosterone from DOC, respectively. The reaction catalyzed by each P-450 proceeds in the presence of molecular oxygen, NADPH, a ferredoxin (adrenodoxin), and an electron-transferring flavoprotein (adrenodoxin reductase). Studies employing an antibody to P-450 demonstrate that no immunochemical similarity exists between the two P-450s, either by the Ouchterlony technique or by inhibition study of the catalyzed reactions by the comparable antibody. The activity of P-450 can be measured by using the reaction system composed with NADPH, adrenodoxin, adrenodoxin reductase, the substrate, and molecular oxygen.

Cytochrome P-45017α,lyase-mediating pathway of androgen synthesis in bovine adrenocortical cultured cells

Abstract Cytochrome P-45017α,lyase-mediating pathway of dehydroepiandrosterone (DHA) formation from pregnenolone was investigated in primary cultures of bovine adrenocortical fasciculata-reticularis cells. To determine whether DHA formation proceeds predominantly by successive monooxygenase reactions without 17α-hydroxypregnenolone leaving P-45017α,lyase the cells were incubated with [14C]pregnenolone and 17α-[3H]hydroxypregnenolone in the presence of Trilostane. Results of the double-substrate double-label experiments indicate that in the presence of high concentration of pregnenolone most of DHA was formed directly from pregnenolone by the successive reactions. Since the concentration of pregnenolone usually exceeds that of 17α-hydroxypregnenolone in the adrenal glands, DHA is concluded to be formed predominantly by successive reactions from pregnenolone without 17α-hydroxypregnenolone leaving P-45017α,lyase in vivo. By chronic ACTH treatment, the activities of 17α-hydroxylation and DHA formation in adrenocortical cultured cells became higher concomitantly with the increase of P-45017α,lyase content. Most of DHA was found to be formed by successive reactions from pregnenolone even under such conditions.