Pregnenolone Formation Research Articles

Mechanism of ACTH action : Intracellular flux of cholesterol during ACTH-induced corticoidogenesis

Corticoidogenic response to ACTH in the adrenal cortex is strictly controlled by the rate of cholesterol bound to cyt.P450scc which is located in the inner mitochondrial membrane. Thus, the rate of the intracellular movement of cholesterol induced by ACTH was investigated in bovine adrenocortical cells. Cholesterol and pregnenolone were measured by enzymatic assay and Cortisol by the fluorometrical assay. (1) In the presence of 1 nM ACTH, the amount of the intracellular free cholesterol was not affected, whereas about 0.3 nmoles of the cholesterol esters per 10^ cells decreased in 60 min incubation. Most of the diminished cholesterol esters in the cells were thought to be utilized as a substrate of steroidogenesis. (2) The rate_of pregnenolone formation induced by 1 nM ACTH was about 0.3 nmoles/105 cells/hr, which was just the same as that of Cortisol production for 60 min incubation. There was a stoichiometrical correlation between the decrement of the intracellular cholesterol esters and the increment of either pregnenolone or Cortisol formation in the cells. It is therefore concluded that the intracellular cholesterol esters might be the sole substrate in the corticoidogenic response to ACTH in the adrenal cortex. Thus, ACTH could activate cholesterol ester hydrolase in adrenocortical cells to facilitate the transport of the free cholesterol to the inner mitochondrial membrane, and the translocated cholesterol in the inner membrane rapidly interacts with cyt.P450scc to convert to pregnenolone and other corticoids.

Proximity of the substrate binding site and the heme-iron catalytic site in cytochrome P-450scc.

As an approach to "mapping" the active site of the cytochrome P-450 that catalyzes cholesterol side-chain cleavage, designated cytochrome P-450scc, we have synthesized steroid derivatives with the potential to interact with both the substrate binding site and the heme-iron catalytic site of the enzyme. The effects of these substrate analogs were studied with cytochrome P-450scc purified from bovine adrenal cortex. One derivative, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, was found to be a potent inhibitor of pregnenolone formation in a reconstituted enzyme system, and a kinetic analysis of the inhibition showed that binding of the derivative is competitive with respect to cholesterol. The spectral properties of a stable complex formed between the steroidal amine and the purified cytochrome suggest that the 22-amine group coordinates directly to the heme-iron. A model for the structure of this inhibitor-enzyme complex is proposed in which the 5-androstene ring system of the steroid occupies the substrate binding site, and the amine group of the side chain occupies an axial coordination position of the Fe(III) center. This places limits on the distance between these two domains in the enzyme and offers support for proposed mechanisms of cytochrome P-450-catalyzed oxygen-insertion reactions in which an iron-bound oxidant directly attacks the substrate.

Increased steroidogenesis by rat zona glomerulosa cells with increased cell concentration in vitro: evidence for a novel aldosterone-stimulating factor and implications regarding aldosterone biosynthesis.

A fourfold increase in the concentration of rat zona glomerulosa cells incubated in Krebs-Ringer bicarbonate buffer (KRBGA) increased aldosterone production per cell twofold. This cell concentration associated (CCA) increase in aldosterone production showed a dose-response relationship with the number of cells per incubation and was further increased in modified Medium 199, compared with KRBGA. The combination of a fourfold increase in cell concentration with each of maximally steroidogenic concentrations of K+, angiotensin II, ACTH, 5-hydroxytryptamine or cyclic AMP produced an increase in aldosterone production greater than that predicted from the algebraic sum of the steroidogenic effects of the factors acting separately. The CCA increase in basal aldosterone production maximal during the first 30 min of incubation, could not be accounted for by accumulation of corticosterone in the incubation medium and was largely or completely abolished by the addition of pregnenolone, progesterone, deoxycorticosterone or corticosterone to the incubation medium. Together, these data support the proposal that the CCA effect was due to a novel aldosterone-stimulating factor which increased aldosterone production by stimulating pregnenolone formation. The aldosterone and corticosterone responses to exogenous pregnenolone, progesterone, deoxycorticosterone and corticosterone demonstrated that aldosterone and corticosterone production by zona glomerulosa cells in vitro may represent separate biosynthetic pathways. Furthermore, these studies provided evidence for a mechanism by which increased levels of exogenous pregnenolone, progesterone and deoxycorticosterone were able to increase their per cent conversion to aldosterone, but not to corticosterone.

Sterol carrier protein2. Delivery of cholesterol from adrenal lipid droplets to mitochondria for pregnenolone synthesis.

The ability of sterol carrier protein2 (SCP2) to mediate transfer of unesterified cholesterol from adrenal lipid inclusion droplets to mitochondria has been tested in an in vitro model system. Unlike mitochondrial utilization of cholesterol added in acetone or dimethyl sulfoxide, the unesterified cholesterol of lipid droplets did not provide a readily available source of substrate for mitochondrial pregnenolone production, without the addition of a transport mediator. Addition of SCP2, but not albumin, stimulated mitochondrial utilization of droplet cholesterol in a concentration-dependent manner. In the absence of mitochondria, SCP2 sequestered lipid droplet cholesterol, and in the presence of mitochondria, which were unable to convert cholesterol to pregnenolone, this cholesterol was quantitatively accumulated by mitochondria. Both processes were concentration-dependent and demonstrated a molar ratio of SCP2 and cholesterol for both binding and transport of 1. SCP2 also enhanced pregnenolone formation by mitochondria which were incubated in the absence of an extramitochondrial source of cholesterol. However, SCP2 had no effect on steroid release from a crude particulate fraction. These studies suggest that the effects of SCP2 are related to delivery of cholesterol from preformed stores to and into mitochondria for initiation of steroid hormone synthesis, and may represent an important modulator of sterol metabolism in adrenal cortical cells.

In Vitro Effects of Cannabinoids on Follicular Function in the Rat1

delta 1-Tetrahydrocannabinol (delta 1-TCH), the major psychoactive constituent of marihuana, was found to suppress the preovulatory surge of gonadotropins and thereby to prevent ovulation in rats, rabbits and rhesus monkeys. These studies suggested that the drug acts primarily on the hypothalamus to suppress luteinizing hormone releasing hormone (LHRH) secretion. The aim of the present study was to examine the direct effect of delta 1-THC, the psychoactive constituent of marihuana and cannabidiol (CBD), one of its nonpsychoactive constituents, on preovulatory rat follicles in vitro. Both cannabinoids inhibited follicular steroidogenesis in a dose-dependent manner. Basal accumulation of progesterone (P), testosterone (T) and estradiol-17 beta (E2) was reduced up to 60% by the highest doses examined (100-200 microM). The luteinizing hormone (LH)-stimulated increase in P and T was inhibited by 75-88% by the highest doses of both cannabinoids (50-200 microM), while E2, accumulation was inhibited by only 40%. It appears that the inhibitory action of cannabinoids is exerted beyond LH binding and activation of adenylate cyclase and prior to pregnenolone formation in the gonadal steroidogenic pathway. In addition to this anti-steroidogenic effect, both cannabinoids induced resumption of meiosis in follicle-enclosed oocytes cultured in hormone-free medium; 200 microM delta 1-THC resulted in 80% maturation and CBD in 75%. It seems that the action of cannabinoids on rat follicles in vitro is unrelated to their psychotropic activity.

Formation of 22-keto-cholesterol from 22S-hydroxy-cholesterol by a NADPH and O 2 dependent, co-insensitive enzyme in bovine adrenal cortex mitochondria

(22S)-5-Cholestene-3β,22-diol is a good precursor for pregnenolone formation by the cholesterol sidechain cleavage enzyme system of adrenal cortex mitochondria. We demonstrated the presence in adrenal cortex mitochondria of another enzyme system, capable to convert (22S)-5-cholestene-3β,22-diol into 3β-hydroxy-5-cholestene-22-one. The enzyme is NADPH and O 2 dependent, but its activity is not significantly affected by carbon monoxide. The enzyme therefore should be classified as a CO-insensitive oxygenase.

The effect of ether stress and cycloheximide treatment on cholesterol binding and enzyme turnover of adrenal cortical cytochrome P450 scc

Relative rate constants for the formation of pregnenolone from cytochrome P450 scc bound cholesterol in adrenal cortical mitochondria of stressed, stressed plus cycloheximide treated and dexamethasone treated rats were calculated from the ratios of initial rates of pregnenolone formation and the pregnenolone induced difference spectrum. In mitochondria from adrenals removed under aerobic conditions in vivo, the rate constant for the enzyme in stressed rats is twice as high as the rate constant for the enzyme from the stressed plus cycloheximide group, and four times as high as that for the enzyme from dexamethasone treated rats. Anoxia for 5 min in the intact gland increases the rate constant in all groups. Pregnenolone difference spectra are higher in mitochondria from stressed plus cycloheximide treated rats than in mitochondria from stressed rats, when adrenals are removed aerobically. It is concluded that ACTH increases cholesterol binding to cytochrome P450 scc, by increasing either the enzymes affinity for its substrate or the availability of cholesterol and in addition promotes turnover of the enzyme. Both of these effects of ACTH are inhibited by cycloheximide.

Characterization of the desensitized state of Leydig tumor cells

A perifusion system has been used to study the in vitro desensitization of isolated Leydig tumor cells. It was observed that the cells become refractory, as measured by decreased rates of steroidogenesis, during continuous perifusion with saturating concentrations of either human choriogonadotropin (CG), cholera toxin, or 8-bromo-cyclic AMP. Furthermore, an initial perifusion of the cells with either human CG, cholera toxin, or 8-bromo-cyclic AMP causes subsequent desensitization towards all three stimuli. Thus, each of these stimuli is equally effective in inducing a state of desensitization in these cells that is manifested by a steroidogenic lesion(s) distal to cyclic AMP formation. It was found that the post-cyclic AMP lesion(s) in the desensitized state occurs prior to the formation of pregnenolone. However, the decreased rates of steroidogenesis do not seem to arise from a depletion of intracellular cholesterol.

Inhibition of bovine adrenocortical cytochrome P-450scc by 3,3'-dimethoxybenzidine

The effect of 3,3'-dimethoxybenzidine (o-dianisidine) on the conversion of cholesterol to pregnenolone was investigated in a reconstituted side chain cleavage system using enzymes purified from bovine adrenal cortex; d-p-aminoglutethimide was also assayed under similar conditions for comparison. 3,3'-Dimethoxybenzidine was found to be a potent inhibitor of pregnenolone formation, causing 50% inhibition at a concentration of 1.5 μM when using 70 μM cholesterol — this dose is approximately one fourth that required of 3-methoxybenzidine and one twentieth that required of benzidine for equal inhibition. In the same system, d-p-aminoglutethimide exhibited an I50 value of about 55 μM. No effects of 3,3'-dimetoxybenzidine on adrenodoxin reductase or adrenodoxin activities could be detected, and inhibition of side chain cleavage could be relieved by dilution suggesting that the inhibitor acts by reversibly binding to cytochrome P-450scc.

Phosphatidylcholine vesicle reconstituted cytochrome P-450scc. Role of the membrane in control of activity and spin state of the cytochrome.

Cytochrome P-450scc from bovine adrenal cortex mitochondria was purified and reconstituted into phosphatidylcholine vesicles which varied in both cholesterol content and in the fatty acyl composition of the phospholipid. Under conditions of optimal ionic strength, pH, and excess adrenodoxin and adrenodoxin reductase, it was found that at a constant cholesterol: phospholipid ratio, the membrane composition had large effects on the rate of hemoprotein-catalyzed side chain cleavage of cholesterol. Rate effects were due to phospholipid-induced changes in the enzyme's Km for cholesterol, and not due to Vmax effects. Binding of cholesterol to cytochrome P-450 could also be monitored optically by measuring the fraction of enzyme in the high spin form. Dissociation constants determined in this manner for cholesterol binding in phospholipid of differing fatty acyl composition showed an excellent inverse correlation with the rates of pregnenolone formation in the same lipids (at constant cholesterol concentration) (see Fig. 6); thus, phospholipid exerts its rate effects by modulating the binding of cholesterol to the cytochrome. The membrane-mediated effects on spin state and activity mimic closely the effects seen in mitochondria isolated from adrenocorticotropic hormone-treated versus control adrenal cells. This behavior suggests to us that acute steroidogenic action of adrenocorticotropic hormone may be mediated through changes in the composition of the inner mitochondrial membrane in which cytochrome P-45scc is embedded.

Regulatory actions of angiotensin II on receptors and steroidogenic enzymes in adrenal glomerulosa cells.

The trophic effects of angiotensin II (AII) on adrenal glomerulosa function were studied by prolonged infusion of the Ile5-octapeptide in intact and 14-day hypophysectomized rats. The administration of a nonnatriuretic dose of AII (50 ng/ min) for 6 days elevated blood levels of AII 3-fold and increased the plasma aldosterone concentration from 9.6 ± 1.2 to 29.7 ± 5.7 ng/dl in intact rats and from 7.5 ± 1.5 to 31.6 ± 7.4 ng/dl in hypophysectomized rats. AII receptors measured in adrenal glomerulosa fractions from hypophysectomized rats were increased 2-fold, from 1408 ± 138 to 2270 ± 324 fmol/mg. Significant increases in enzymes of the aldosterone biosynthetic pathway, more marked in hypophysectomized animals, were observed after AII infusion. Mitochondrial cholesterol content and pregnenolone formation were increased by 30% in intact rats and by 100% and 240%, respectively, in hypophysectomized rats. The conversion of corticosterone to aldosterone was also increased, by 100% in intact rats and by 180% in...

The subzero temperature stabilized oxyferro complex of purified cytochrome P450 SCC

Purified preparations of bovine adrenocortical mitochondrial cytochrome P450, capable of catalyzing the side-chain cleavage of cholesterol have been obtained [ 1,2]. The spectral, biochemical and immunological characteristics of this hemoprotein are now known [3,4], and the multistep nature of this bioconversion leading through enzyme-bound intermediates to pregnenolone formation, established [5-71. The first step in the activation of molecular oxygen by cytochrome P450 involves the formation of an oxyferro complex [a]. Although oxyferro intermediates of a number of cytochrome P45Os have been described [9] no information is available about this form of cytochrome P450,. The development of subzero temperature techniques [lo-l 31 together with our studies on proton activity, dielectric constants and viscosity in fluid hydroorganic media, enabled the stabilization and detection of intermediates in various biological reactions [ 14-161. Among these are the oxyferro species of cytochrome P450, from Pseudomonas putida [ 171 and the highly purified microsomal cytochrome P450 (LM) from rabbit liver [ 181. Here we describe the formation and stabilization at low temperature of the oxyferro intermediate of cytochrome P450, isolated from bovine adrenocortical mitochondria.

Steroid biosynthetic lesions in gonadotropin-desensitized Leydig cells

Administration of exogenous gonadotropins to rats causes loss of LH receptors and a decrease in the maximum steroidogenic response when Leydig cells are subsequently stimulated by LH or hCG. The impaired testosterone response of desensitized Leydig cells is more evident after intravenous than after subcutaneous treatment with hCG, and is due to a partial defect in testicular 17,20 lyase activity. The enzyme defect is present in Leydig cells from animals given intravenous hCG to produce about 70% loss of LH receptors, yet is relatively small after treatment with subcutaneous hCG to produce the same degree of receptor loss, suggesting that the steroidogenic block is not only due to receptor depletion. Acute elevations of endogenous LH by single doses of LHRH caused the same changes as exogenous hCG, with marked receptor loss and reduction in testosterone responses to hCG, dibutyryl cyclic AMP and choleragen. However, pregnenolone formation (measured in the presence of cyanoketone and spironolactone to inhibit pregnenolone metabolism) was normal or increased in desensitized Leydig cells from LHRH-treated animals. Accumulation of progesterone and 170H-progesterone occurred in serum and incubated Leydig cells of desensitized animals, localizing the major steroidogenic block to the conversion of 170H-steroids to androgens. In cultures of normal rat and murine tumor Leydig cells, 17–20 lyase and 17α-hydroxylase were markedly decreased when LH receptors were lost, suggesting coordinate regulation of receptors and steroidogenic enzymes in vitro. However, the in vivo development of the steroid block was not coincident with the loss and return of LH receptors. A role of oestradiol in the gonadotropin-induced enzyme defect was suggested by acute elevations of testicular oestradiol 30 min after intravenous hCG (whereas more gradual increases occurred 4–8 h after subcutaneous hCG), and by the ability of an oestrogen antagonist (Tamoxifen) to prevent the reduction of testosterone responses caused by intravenous hCG. These results indicate that acute elevation of oestradiol production is a contributory factor in the steroidogenic lesions of LHRH- and hCG-desensitized Leydig cells in the rat testis.

Loci of Action of Regulators of Aldosterone Biosynthesis in Isolated Glomerulosa Cells

The effects of angiotensin II (All), ACTH, and potassium on early and late steps of aldosterone biosynthesis were studied in collagenase-dispersed cells from rat adrenal capsules and dog zona glomerulosa. Isolated cells were incubated with a cyanoketone derivative (WIN 19,578) to isolate the nearly and late portions of the biosynthetic pathway. In this way, the formation of pregnenolone and later steps of biosynthetic conversion in the absence of endogenous precursors could be measured independently during stimulation by the three regulators. During incubation for 3 h with 10 nM All, 10 nM ACTH, or 15 mM potassium, marked stimulation of pregnenolone production was observed in glomerulosa cells of both species. In rat glomerulosa cells, each of the three regulators also stimulated conversion of exogenous corticosterone to aldosterone. In dog glomerulosa cells, All and potassium, but not ACTH, stimulated the conversion of corticosterone to aldosterone. In rat glomerulosa cells, aldosterone production from e...

In Vitro biosynthesis of steroids from cholesterol by the ovaries and pyloric caeca of the starfish Asterias rubens

1. 1. In vitro biosynthesis of steroids from cholesterol in ovaries and pyloric caeca of Asterias rubens has been investigated. 2. 2. The formation of pregnenolone and progesterone was demonstrated in both tissues by using 1,2- 3H-cholesterol as a precursor. 3. 3. Pregnenolone and progesterone are produced in small quantities; the ovaries appear to be more active in the biosynthesis of these steroids than the pyloric caeca. 4. 4. The results indicate the presence of the following biosynthetic enzyme systems in ovaries and pyloric caeca of Asterias rubens: a side-chain-cleaving enzyme complex and a Δ 5–Δ 4- isomerase-3β- HSD complex. 5. 5. The importance of these enzymes and the function of cholesterol for the biosynthesis of steroids will be discussed.

Biosynthesis of pregnenolone from cholesterol by mitochondrial enzymes of bovine adrenal cortex. The question of the participation of the 20(22)-olefins and 20, 22-epoxides of cholesterol.

The two isomeric 20(22)-olefins and the four isomeric 20,22-epoxides of cholesterol have been examined as possible intermediates in the conversion of cholesterol to pregnenolone both by crude extracts of acetone-dried bovine adrenal cortex mitochondria, and by a partially purified cytochrome P-450 specific for the side chain cleavage (P-450SCC). The quantities of pregnenolone (measured by radioimmunoassay) formed from these putative intermediates and from cholesterol, (20S)-20-hydroxycholesterol, (22R)-22-hydroxycholesterol, and (20R,22R)-20,22-dihydroxycholesterol have been compared. In both crude and purified systems, the conversion of the monohydroxycholesterols is somewhat more rapid than that of cholesterol. However, the dihydroxycholesterol is a very much more efficient precursor of pregnenolone. Whereas small amounts of pregnenolone were formed from the four 20,22-epoxycholesterols, and slightly larger amounts from the two 20(22)-didehydrocholesterols in the crude enzyme systems, almost no conversion of these six compounds was observed with the purified system containing cytochrome P-450SCC, adrenoxin, adrenodoxin reductase, and an NADPH-generating system. It is concluded that the olefins and epoxides are not obligatory intermediates in the conversion of cholesterol to pregnenolone, and the conversions of cholesterol olefins and epoxides observed by us and by others may reflect the presence of extraneous enzymes such as double bond reductases and epoxide hydratases which are unrelated to pregnenolone biosynthesis. The relative effectiveness of the cholesterol olefins and epoxides, as well as 20-isocholesterol in inhibiting the formation of pregnenolone from [4-14C]cholesterol has been determined. The compounds which have the same configuration as cholesterol at C-20 are more potent inhibitors of this conversion than the olefins in which the C-21, C-20, C-22, C-23 carbon system is planar, or than those compounds which have the opposite configuration to cholesterol at C-20 (i.e. 20-isocholesterol, (20S,22S)-20,22-epoxycholesterol and (20S,22R)-20,22-epoxycholesterol). The enzyme appears to be more fastidious in its steric requirement at C-20 than at C-22. Since the olefins and epoxides are stereochemically rigidified around the C-20 to C-22 bond, these inhibitor studies suggest that the natural conformation of cholesterol on the side chain cleavage enzyme system is such that the side chain is fully extended and thus closely resembles the conformation of cholesterol in the crystal state.

The role of protein kinases in ACTH-stimulated steroidogenesis

The effect of highly purified bovine cytosolic adrenal cortical protein kinase isozyme II catalytic subunit (ATP: Protein Phosphotransferase EC 2.7.1.37) on the formation of pregnenolone from cholesterol in rat and bovine adrenal mitochondrial extracts has been investigated. No stimulation was observed although a low level incorporation of [ 32P] from [ 32P]-ATP into a component or components of the extract was detected. The mitochondrial extracts contained alkaline phosphatase activity that was inhibited by L-cysteine and dithiothreitol. It is concluded that the acute stimulation by ACTH of corticoid production in the rat adrenal does not involve protein kinase mediated phosphorylation of a component or components of the cholesterol sidechain cleavage mixed-function oxygenase system.

Cholesterol side-chain cleavage, cytochrome P450, and iron-sulfur protein in human placental mitochondria

Cytochrome P450 and the associated iron-sulfur protein have been characterized in human placental mitochondria by means of optical absorbance difference spectrophotometry and electron paramagnetic resonance spectrometry. These two proteins occur in a molar ratio of about 1:1 in human placental mitochondria, and the cytochrome P450 appears to be that form involved in cholesterol side-chain cleavage. Pregnenolone formation from endogenous mitochondrial cholesterol, as measured by radioimmunoassay, follows a biphasic time-course similar to the situation in other steroidogenic tissues. The specific activity of cholesterol side-chain cleavage, and the specific contents of cytochrome P450 and the iron-sulfur protein in the mitochondria, are 2- to 3-fold higher at term than in the 1st and 2nd trimesters. When expressed in terms of the cytochrome P450 content, the rate of pregnenolone formation is high, suggesting that cholesterol side-chain cleavage in human placenta is in an activated state.

Angiotensin stimulates both early and late steps in aldosterone biosynthesis in isolated bovine glomerulosa cells

The present study was designed to examine the effect of angiotensin on both the early and the late phases of aldosterone biosynthesis. In order to isolate the early phase (the formation of pregnelone). it was necessary to identify an agent that would inhibit the 3β-hydroxysteroid dehydrogenase, 4–5 eneisomerase enzyme system which facilitates the conversion of pregnenolone to progesterone. Such an agent was found in trilostane. Treatment of zona glomerulosa cells with trilostane resulted in accumulation of pregnenolone, and the addition of angiotensin increased the accumulation of pregnenolone still further. This observation demonstrates that angiotensin stimulates aldosterone biosynthesis at a point prior to pregnenolone formation. The late phase of aldosterone biosynthesis was isolated by using aminoglutethimide to inhibit the formation of pregnenolone by cells of the zona glomerulosa. Aldosterone was formed when aldosterone precursors occurring in the biosynthetic pathway from pregnenolone onward were added. When angiotensin was added along with deoxycorticosterone, the conversion of deoxycorticosterone to aldosterone was enhanced ( P < 0.05). This observation demonstrates that angiotensin stimulates aldosterone formation at a point distal to deoxycorticosterone formation. Thus, angiotensin stimulates aldosterone biosynthesis at at least 2 independent points, one early and one late in the biosynthetic pathway.

Purification and immunochemical characterization of the two adrenal cortex mitochondrial cytochrome P-450-proteins

A procedure for the separation and purification of two distinct P-450 cytochromes, termed P-450 scc and P-450 11 β , solubilized from bovine adrenal cortex mitochondria is reported. Important features of the purification procedure are uses of aniline-substituted Sepharose chromatography and utilization of the markedly different characteristics in stability and solubility of each cytochrome. Polyacrylamide gel electrophoresis of the two purified preparations in sodium dodecyl sulfate reveals single protein bands. The P-450 scc, which catalyzes the formation of pregnenolone from cholesterol, has a turnover number of 16 mol of pregnenolone formed per minute per mole of P-450-heme. The P-450 11 β catalyzes the hydroxylation of deoxycorticosterone at 11β- and 18-positions with turnover numbers of 110 and 18, and of 4-androstene-3,17-dione at 11β- and 19-positions with turnover numbers of 41 and 12, respectively. The recoveries of the P-450 scc and P-450 11 β , in terms of the catalytic activities, are 20% and 15%, respectively, from the crude extract which contains the two activities in a ratio of roughly 2:1. Each rabbit antibody prepared against the two purified P-450-proteins interacts with respective, but not alternative cytochrome P-450, whether in the crude mitochondrial preparation or in the purified preparation. The observed patterns of immunoprecipitation and inhibition of catalytic activity indicated that the two P-450 proteins are immunochemically different from each other. Neither antibody immunoprecipitates with a highly purified bacterial cytochrome P-450, P-450 cam.