Cytochrome P-450 from bovine adrenocortical mitochondria: an enzyme for the side chain cleavage of cholesterol. I. Purification and properties.

Abstract One cytochrome P-450 has been purified from bovine adrenocortical mitochondria to near homogeneity. The enzyme catalyzes the conversion of cholesterol and 20α-hydroxycholesterol to pregnenolone (side chain cleavage) and shows traces of 11β-hydroxylase activity. The conversion of cholesterol to pregnenolone occurs without demonstrable accumulation of biosynthetic intermediates, i.e. hydroxylated cholesterols. The kinetic constants for side chain cleavage are as follows: cholesterol: Km, 0.19 mm; and 20α-hydroxycholesterol: Km, 0.012 mm. The P-450 described here shows the following properties: molecular weight, 850,000; s20, w0, 22 x 10-13; partial specific volume, 0.765. The enzyme is isolated in a form which is approximately 50 % high spin and 50 % low spin; it contains eight heme groups per molecule and is stable in 50 % glycerol.


SUMMARY
One cytochrome P-450 has been purified from bovine adrenocortical mitochondria to near homogeneity. The enzyme catalyzes the conversion of cholesterol and 20~ hydroxycholesterol to pregnenolone (side chain cleavage) and shows traces of 11/3-hydroxylase activity.
Ch,romatography on DEAE-cellulose-Following dialysis the sample was applied to a column of DEAE-cellulose (4 x 20 cm) and eluted with buffer of the same composition as that used for dialysis, until a light brown opaque material, appeared in the &ate.
The concentration of phosphate buffer was then increased to 100 rnM (pH 7.6) containing dithioerythritol (0.1 mM) and EDTX (0.1 mM). Cytochrome P-450 (100 to 150 ml, 650 to 1200 mg of protein) was eluted by the rnore concentrated buffer and was then dialyzed overnight against potassium phosphate buffer (10 m~\r, $1 7.0) containing dithioerythritol and EDTA (0.1 mM each). Chromatography on Bio-Gel-The concentrated sample (5 ml) was further purified by ascending chromatography on a column of Bio-Gel A-15m (2 x 90 cm). The column was elutcd at a constant flow rate of 34 ml per hour with potassium phosphate buffer (100 rnl\f; pH 7.6) containing dithioerythritol and EDTA (0.1 11131 each); hereafter this buffer mixture will be referred to as stalldart buffer. The cytochrome 1'.450 eluted between 1.4 and 2.0 void volumes n-as cycled a second time through the same column.
Unless otherwise stated, it is this material upon which all subsequent studies reported in this paper were performed. The enzyme was taken t,o a final concentration of 2 to 3 mg of protein per ml by membrane ultrafiltration.
The concentrated solution was mixed with equal parts of glycerol and stored at -20".
The reaction was started by addition of TPNH and continued for 20 min at 37".
FolloIving incubation the medium was extracted with methylene dichloride three times.
In one series of experiments, [4-14C]cholcsterol was used in place of cholesterol.
The methylene chloride extract was applied to thin layer chromatography in the system petroleum etherdiethyl ether-glacial acetic acid (64 Following purification by thin layer chromatography, cholesterol (0.1 to 1.0 pg) was meastirctl by means of a gas chromatograph (Hewlett-Packard model 402) as described elsewhere (18). The column (SE-30) was 3 x 1200 mm, and under the conditions used, the retention time of cholesterol was 38 min.

Measurement
of Phospholipid-Extraction of phospholipid from P-450 (5 to 10 mg of protein) was pcrformcd as described by Folch (19). Extracts were digested by convrntional acid hydrolysis.
The cytochrome was prepared for sedimentation equilibrium by dialyzing an aliquot against potassium phosphate in I),0 (50 rnM, pII 7.6) for 60 hours at 4". Following dialysis, D20 phosphate buffer n-as dilutctl to IO n1M by addition of further l)nO. Sedimentation was czaminccl simultaneously in II,0 and I&O (0.5 mg of protein per ml). The fluorocarbon FC-43 was used as base fluid.
Ccntrifugation was continued for 3 days at 20". Sedimentation velocity in phosphate buffer (75 m&I; pI1 7.6) was examined as a function of protein concentration.

Spectra2
Studies-CO-difference spectra wcrc recorded in potassium phosphate buffer (pH 7.0, 100 maf) containing cysteinc (5 mw) and EDTA (1 IrIM). 1'.450 was rctluc~l with sodium dithionitc (2.5 mg per ml) before treatment with carbon monoxide. The 1'.450 content of the enzyme has been calculated on the basis of an extinction coefficient of 91 ml1-l cm-l (21); the reason for this method of calculation is given under "Discussion." l'yridine hemochrome was prepared and measured as described by Falk (22) ; millimolar difference cstinction coefl?cients of 20.7 (Reference 23) and 32.4 (Reference 21) were used to determine the content of hcmc (see "Results").
Miscellaneous-Protein determinations were performed by the method of Lowry et al. (24), using crystalline bovine serum albumin as standard.
Phosphate was cletcrmined by the method of Allen (25). Absorbance at 280 and 415 nm was measured using a Gilford recording spectrophotometcr.
Absorbance of column effluents was monitored at 280 nm by means of an ultraviolet flow analyzer (Isco model 224). Liquid scintillation spectrometry was performed by published methods (17) using a Packard Tri-Carb model 3375. Ultrafiltration membranes (Diaflo X?&lOOA) were purchased from Amicon, Lexington, Massachusetts.

Chemicals
Hemin was purchased from Sigma Chemical Co. and was recrystallized twice as described by Falk (22 Proteins-An enzyme preparation containing 3&hydroxysteroid dehydrogenase and As-3-ketosteroid isomerase activities was prepared from Pseudomonas testosteroni by ammonium sulfate fractionation (14). Diaphorase and adrenodoxin were prepared from bovine adrenocortical mitochondria according to Kimura and Suzuki (26). It was found that both the diaphorase and the adrenodoxin preparations used in these studies each cont,ained less than 0.05 nmole of cholesterol per mg of protein.

PuriJication of Cytochrome
P-450-The cholic acid extract of sonicated adrenal mitochondria can be resolved into two overlapping protein components containing P-450 with side chain cleavage and llfl-hydroxylase activities by chromatography on hydroxylapatite (Fig. 1). The studies reported here are concerned with the second of these components to be eluted from the column.
This component was further purified by chromatography on Bio-Gel (Fig. 2). In addition to the peak shown in Fig. 2 (void volume, 1.6 to 1.9), two other protein fractions are separated from the major P-450 component during exclusion chromatography (void volumes, 1.0 to 1.2 and 2.1 to 2.5); one of these compounds contains P-450 (void volume, 1.0 to 1.2). At this stage the major P-450 component appears to be highly purified according to the following criteria: (a) the specific activity of the enzyme, through the peak fractions from chromatography on Bio-Gel, is a straight line of zero slope (Fig. 2); (b) the protein appears as a single peak on analytical ultracentrifugation (Fig.   3), although some evidence of asymmetry of the peak is seen in the direction of centrifugation; (c) sedimentation equilibrium shows a single linear relationship between log10 recorder deflection (f) and distance squared (z") in HsO, and a second such relationship in DzO (Fig. 4).  P-450 was examined at 20" in potassium phosphate buffer (75 mM, pH 7.5, 7.0 mg of protein per ml). Exposures were made at 4-min intervals.
The rotor speed was 40,000 rpm. The ~20,~ value calculated from these observations was 17.0 x 10-13.

Evidently
there is no loss of heme relative to protein. The content of endogenous cholesterol decreases to negligible levels (<0.002 nmole per nmole of P-450) as the enzyme is purified. On the other hand, side chain cleavage activity increases during purification (Table I), although a number of factors to be discussed make the interpretation of enzyme activity complicated (see "Discussion").
The purified enzyme contains phospholipid in an amount of 0.08 to 0.09 pmole of phosphorous per mg of protein.
Spectral Properties- Fig.  6 shows certain important spectral properties of cytochrome P-450 from bovine adrenocortical mitochondria.
The absolute spectrum shows five peaks (395, 412, 520, 565, and 650 nm), together with a small shoulder at 360 nm. The spectrum shows a trough at 312 nm. The carbon monoxide difference spectrum shows a peak at 447 nm and a small shoulder at 420 nm. The characteristics of these spectra, together with spectra of chemically reduced P-450 and the pyridine hemochrome, are summarized in Table II. When the millimolar extinction coefficient for pyridine hemochromogen AA557-580 nm was assumed to be 32.4 (Reference 21), the heme content of P-450 was calculated to be 7.4 moles of heme per mole of P-450. When the millimolar extinction coefficient of 20.7 for AA557..538 nm (Reference 23) was used, heme content was calculated to be 8.4 moles per mole of P-450. The enzyme shows lip-hydroxylase activity which decreases Since cytochrome P-450 was normally kept in glycerol (see during purification.
The amount of this enzyme activity is too "Experimental Procedure"), it was decided to examine the small for accurate determination of specific activity and kinetic influence of glycerol upon the spectral properties of P-450. Beconstants, so that the difference between the last two values tween the concentrations of 10 and 50%, glycerol increased Ab12 shown in Table I  not increased by changing either the absolute or the relative amounts of diaphorase and/or adrenodoxin through a IO-fold range of concentrations above and below those routinely used in the assay system (see "Experimental Procedure").

Molecular
Weight and Sedimentation Properties-The sedimentation coefficient of P-450, as determined by sucrose gradient (Fig. 5), was found to be 22 x 10-13. This value is consistent with that determined by sedimentation velocity (s&,, = 21 x lo-r3), as already reported from this laboratory (12). In addition, the following data were calculated from sedimentation equilibrium (27) (Fig. 4) : partial specific volume, 0.765; molecular weight, 850,000.
This estimate of molecular weight is in agreement with the value (800,000) obtained from exclusion chromatography ( Fig. 2 and Reference 12). An aliquot (0.5 ml) of the concentrated sample was layered onto a gradient (30 ml) of sucrose (5 to 20%) in phosphate buffer (0.1 M, pH 7.6). The gradient was centrifuged at 28,000 rpm for 16 hours. Fractions (0.6 ml each) were collected from the gradient and analyzed as follows: (a) enzymatic activities (side chain cleavage, ll& hydroxylation, catalase); (6) absorption at 280 nm; and (c) P-450 content.
Enzymatic Activities-The adrenal P-450 which forms the subject of this report shows side chain cleavage activity with both cholesterol and 20a-hydroxycholesterol as substrates. The following kinetic constants were calculated for cholesterol: K,, 0.19 mM; v,,,, 20.6 nmoles per min per mg of protein; and for ZOcr-hydroxycholesterol: K,, 0.012 mM; V,,,, 20.6 nmoles per min per mg of protein.
Conversion of both substrates to pregnenolone was linear with respect to time (0 to 30 min) and to concentration of P-450 (0.05 to 0.5 nmole).
The enzyme reaction is absolutely dependent on the electron transport system; removal of TPNH or adrenodoxin or adrenodoxin reductase from the reaction mixture completely abolished the production of pregnenolone from cholesterol. Addition of either hemin (1 to 4 nmoles) or MgS04 (1 to 20 nmoles) or bovine serum albumin (100 pg) to the assay solution (2 ml) had no effect on the side chain cleavage of cholesterol. On the other hand, addition of glycerol (5 to 2001,) increased the enzyme activity by 20 to 50%.
It is also of interest to notice that with [4-r4C] 6. Absorption spectroscopy of cytochrome P-450. P-450 was examined at room temperature both as the native protein and as the carbon monoxide complex (inset) as described under "Experimental Procedure." The concentrations used were as follows: absolute spectrum, 1.60 mg of protein per ml; CO-difference spectrum, 0.12 mg of protein per ml. Full scale for absorbance with the absolute spectrum is 1.0, while that for CO-difference spectrum is 0.1. of adrenal mitochondria Purified cytochrome P-450 was subjected to absorption spectroscopy as the native protein, after reduction by sodium dithionite, as carbon monoxide complex and as pyridine-hemochrome (see "Experimental Procedure"). Values for millimolar extinction coefficient are based on the protein concentration and the molecular weight (850,000) of P-450; these values represent means of determinations on three different preparations of P-450. Enzyme activity (side chaiii cleavage) of the purified cytorhrome P-459 is stable for at least 3 months, if stored at -20" in 50 mu potassium phosphate buffer (pH 7.0) containing 50y0 glycerol.
When stored at 5" without glycerol, activity derreased by 21 to 43c/, within 30 days and was completely lost in 4 months.
The optimal pH for the conversion of both cholesterol and 20a-hydroxycholesterol to pregnenolone in 75 mlnl potassium phosphate buffer was 6.8. The enzyme activity decreased by more than 50"/;, at pH >7.5 and <6.0.
The pH optimum for the lip-hgdroxylase activity was also found to be about 6.8. In addition, the magnitude of the CO-difference spectrum was greatest when the l-'-450 was dissolved in phosphate buffer at pH 6.5 to 7.0 (before addition of sodium dithionite) .

l~IscuYsIol\j I I
The present observations deal with only one of the cytochromes I'-450 cwtractctl from bovine adrenal mitochondria.
The yield of P-450 from the initial soriicate to final purification is approximately 7%. The enzymatic activities and other properties of the 1'.459 lost during purification have not been studied in detail. The rnethod of isolating I'-450 reported here is based upon cholic acid extraction of mitochondria as described by Mitani and Horir (28).
The purified cytochrome 1'.450 appears to be almost homogeneous according to three criteria-sedimentation rclocity, sedimentation equilibrium in D20 and HsO, and chromatography on Rio-Gel.
HoJvever the extent of purification effected by the methods used here is difficult to compute bccausc certain changes, occurring in the enzyrnc during purification, rnake interpretation of enzyme assays difficult.
For example, cholic acid is removed from the enzyme during purification, and since this steroid induces a difference spectrum with 1'-450,2 it may be that cholic arid influences enzymatic activities.
Secondly, the concent,ration of adrenodosin and adrenodosin reductase (diaphorase) required t,o secure masirnal activity varies during purification. Although t,hc Mivity of the purified enzyme is measured under optimal coutlit~ious, available supplies of adrenal tissue rnake it unrealistic to perform sufficient measurcmeiits to determine opt,imal conditions at crcry step in the purification of the cytochrome. hgain 1'.450 contains bourrc~ (endogenous) cholesterol which is removed during purification.
It is not known whether cndogenous and exogenous cholcstcrol have equal access to t,he enzyme.
I\Ioreover the electron carriers and l'-450 are presumably lipidbound within tlic mit~ochondrioii. During purification, lipid is removed from the cnzymc, and it has been shown that addition of adrenal mitochondrial lipid accelerat,es the activity of 1'.450 (29). It may be then, that the activity of the enzyme is not optimal in aqueous solution and heiice the conparison of crude and purified samplrs of 1'.450 may be misleading.
Purified 1'.450 shows, in addition to side chain cleavage actirity, IQ'-hydrosylnse activity but 110 18.hydroxylase activity. The capacity of the enzyme to support some lib-hydrosylation relative to side chain cleavage activity decreases during purifica-2 A. h/I. Shikita and I'. F. Hall, unpublished observations. tion to a point at which lip-hydroxylase activity can be measured wit,h only limited accuracy.
This observation would be compatible with the presence of small amounts of ll@hydroxylase as a contaminant, although it is not possible to exclude some inherent llfi-hydroxylase activity in the side chain cleavage enzyme.
Evidently Whydrosylase activity requires a different enzyme system.
The cytochrome P450 which is not extracted by cholic acid under the conditions reported is relatively rich in llfl-hydroxylase act.ivity as compared to side chain cleavage (30). It seems probable that the P-450 described here is a major, if not the major, side chain cleavage enzyme.
The conversion of cholesterol to pregnenolone by the side chain cleavage enzyme occurs without demonstrable accumulation of the postulated intermediates 20oc-hydroxycholesterol and ZOa, 22.dihydroxycholesterol (10, 11)) in keeping with findings using a less purified enzyme (31,32). If such intermediates do occur, they must be present in very small amounts.
The spectral properties of cytochrome P-450 indicate that it fulfills the requirements for classification as a cytochrome of the b type since the pyridinc hemochromogen reveals the presence of protoheme (33). In addition the pyridine hemochromogcn reveals the presence of eight heme groups per molecule of P-450.
The molar extinction coefficient of the carbon monoxide complex (Table II) requires comment.
The table shows the coeflicient for 1 mole of P-450, so that the value per hemc group would be 23, which is considerably lower than values observed with other cytochromes P-450 (21, 34). Moreover a variety of conditions including regassing, variations in the amount and st,ate of dithionite added, etc., have all failed to increase the value. -it t'his time we are unable to resolve this parados which may result from failure of some heme groups to react with carbon monoxide or from an inherently low extinction for the hemc groups of this P-450.
In vielv of the findings with other cytochromes P-450, this last explanation seems less likely.
It should be added that examination of the absolute spectrum of the carbon monoxide complex has failed to resolve these possibilities. Foi this reason, values for the amounts of P-450 prcscnted in this paper arc based upon a millimolar extinction coefficient of 91 in accordance with current practice; until this problem can be resolved, this value might be regarded as the contribut,ion from active heme groups.
The carbon monoxide differcncc spectrum also shows that this side chain cleavage I'-450 contains no P-420 (i.e. less than 1°~o, Fig. 6). It is interesting that this feature of this I"-450 distinguishes it from most other cytochromes 1'.450 iii which large amounts of 1'.420 are usually observed.
Moreover, no more than minute amounts of P420 arc observed at any st,age of the preparation.
The amounts of 1'.420 are always too low to measure accurately in the presence of 1'.450. :1ndo and Horie (34) have also succeeded in preparing a cytochrome r-450 from adrenal mitochondria with very little P-420. The abeolut,c absorption spectrum of l-'-450 at room temperature shows peaks of approximately equal intensity at 412 and 395 nm. hbsorbance at 412 nm (low spin ferriporphyrin) inclicates the csistence of four such heme groups per molecule of P-450. The peak at 395 nrn reveals the presence of four high spin ferriporphvrin groups per molecule, suggesting that under the conditions of isolation, P-450 is present as an approsimately equal mixture of two forms in which the iron is present in high spin and low spin states, respectively.
This conclusion is supported by spectral changes resulting from addition of glycerol. It has been found that glycerol converts the high spin state by guest on March 24, 2020 http://www.jbc.org/ Downloaded from (peaks at 650, 520, and 395 nm) to the low spin state (peaks at 565, 532, 417, and 356 nm).
The observations presented here are of interest in relation to those reported by Schleyer et al. (35) for a cytochrome P-450 from the same source as that from which the P-450 reported in the present paper was prepared.
The enzyme isolated by Schleyer et al. catalyzes ll&hydroxylation, shows little side chain cleavage activity, and is isolated in low spin state (35). Further comparison between these two cytochromes must await a detailed report of the physical properties of this ll/%hydroxylase cytochrome.
The cytochrome P-450 discussed here is of high molecular weight (850,000), contains eight heme groups per molecule, and shows an unusually high partial specific volume (0.765), suggesting that the molecule is rather loosely organized or packed. It is therefore not surprising to learn that P-450 is composed of subunits.
The subunit structure of the adrenocortical side chain cleavage cytochrome P-450, forms the subject of the accompanying paper (36)