Purification and comparative characterization of cytochrome P-450scc from porcine adrenocortical mitochondria

Inl. J. Biochem.Vol. 23, No. 9, pp. 901-909, 1991

0020-711X/91$3.00+ 0.00 Copyright 0 1991Pergamon Press plc

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PURIFICATION AND COMPARATIVE CHARACTERIZATION OF CYTOCHROME P-450s~~ FROM PORCINE ADRENOCORTICAL MITOCHONDRIA KAZUHIKO IWAHASHI,’ MOTONARI TSUBAKI,‘* AKIRA MIYATAKE’ and YOSHIYUKIICHIKAWA’~ ‘Department

of Biochemistry and 2Research Center of Medical Science, Kagawa Medical School, Kita-gun, Kagawa 761-07, Japan (Recemed 25 September 1990)

Abstract-Cytochrome P-450s~~ (cholesterol side-chain cleavage enzyme) was purified from porcine adrenocortical mitochondria. 2. The purified cytochrome P-450s~~ was found to be homogeneous on SDS-polyacrylamide gel electrophoresis. 3. The heme content of the purified enzyme was 20.6 nmol/mg protein. 4. The enzymatic activity of the reconstituted cytochrome P-450scc-linked monooxygenase system amounted to 7.8 nmol of pregnenolone formed per nmole of P-450 per minute, with cholesterol as a substrate. 5. The ammo acid sequence of the amino-terminal region of the cytochrome P-450s~~ and the amino acid residue at the carboxyl terminal were determined and compared with those of other mammalian cytochromes P-450s~~.

MATERIALS AND

INTRODUCTION Cytochrome P-45Oscc, also termed as P-450XIA1, the cholesterol side chain cleavage enzyme (EC 1.14, 1.9), is a component of the mitochondrial cytochrome P-450-linked monooxygenase system (consisted from NADH-ferredoxin reductase and ferredoxin) and catalyzes the first step reaction during the synthesis of steroid hormones from cholesterol and exists in mitochondrial inner membranes of the adrenocortex (Shimizu et al., 1961, 1962; Chaudhari et al., 1962; Constantopoulos and Tchen, 1961a,b; Constantopoulos et al., 1962; Simpson and Boyd, 1966, 1967; Geuze et al., 1987), ovary (Kashiwagi et al., 1980; Matocha and Waterman, 1986), testis (Bakker et al., 1979), placenta (Rabe et al., 1983) and brain (Goascogne et al., 1987). We have purified and crystallized cytochrome P450s~~ from bovine adrenocortical mitochondria. The enzymatic properties of the bovine enzyme was reported previously (Tsubaki et al., 1986; Iwamoto et al., 1988). In this paper, we describe the purflcation of cytochrome P-450s~~ from porcine adrenocortical mitochondria, and its enzymatic and physicochemical properties in comparison with these of other mammalian cytochromes P-45Oscq to clarify the mechanism underlying the enzymatic reaction of cytochrome P-45oscc.

*Present address: Department of Life Science, Faculty of Science, Himeji Institute of Technology, Himeji, Hyogo 671-22, Japan. tTo whom all correspondence should be addressed. Abbreviations: SDS, sodium dodecyl sulfate; BSA, bovine serum albumin; DMF, N,N’-dimethyl formamide.

METHODS

Materials Porcine adrenal glands (Lard race, l-year-old) were obtained from a local slaughterhouse. The adrenal glands were put in cracked ice immediately after killing and were transported to this laboratory as soon as possible. Purification of cytochrome P-450s~~ Fat and connective tissues were removed from the porcine adrenal glands with scissors. Unless otherwise indicated, all purification procedures were performed below 4°C. The adrenal glands (about 1.0 kg total) were ground into l-2 mm pieces with a Mitsubishi electric mill. The ground adrenal glands were then homogenized with 4 vol of ice-cold 0.25 M sucrose solution (adjusted to pH 7.4 with 0.1 M Tris solution) with a Matsushita homogenizer, model MX-1405, at the speed of lO,OOOMeV/min. The homogenate was passed through two layers of gauze to remove fat and cell debris. The mitochondrial fraction was prepared from the homogenate by the method of Rosenthal and Narasimhulu (1957). Cytochrome P-450s~~ was purified from the mitochondrial fraction by the method of Tsubaki et al. (1986). Purification of NADPH-adreno-ferredoxin reductase and adreno-ferredoxin NADPH-adreno-ferredoxin reductase was purified from the mitochondrial fraction of porcine adrenal glands by the method of Hiwatashi et al. (1976). Adreno-ferredoxin was purified from the mitochondrial fraction of porcine adrenal glands by the method of Sakihama et al. (1988). The molar extinction coefficients of NADPH-adreno-ferrodoxin reductase and adreno-ferredoxin used were 11.3 x 10) M-’ cm-’ at 450 nm and 9.8 x 10) M-l cn-’ at 414 nm, respectively in 50 mM potassium phosphate buffer at pH 7.4 and 25°C (Hiwatashi et al., 1976). Reconstitution of the cytochrome P-45&c-linked monooxy genase system The enzymatic activity of the reconstituted cytochrome P-450scc-linked monooxygenase system was measured as 901

KAZUHIKO IWAHASHI ef al.

902

the production of pregnenolone from cholesterol or its derivatives (Morisaki et al., 1976, 1980, 1985). The reconstituted system consisted of 0.5 pM cytochrome P-45Oscc, lO@M adreno-ferredoxin, 0.15 ,uM NADPH-adrenoferredoxin reductase and 0.1% (v/v) Tween 20 in a 2.0 ml reaction mixture containing a 20 mM potassium phosphate buffer, pH 7.4. The reaction mixture also contained 5Opg cholesterol or one of its hydroxylated derivatives as a substrate. The reaction was started by adding 0.35 ml of NADPH generating system. The NADPH-generating system consisted of 0.15 ml of 0.1 M MgCl,, 0.05 ml of 0.1 M mzsodium isocitrate, 0.15 ml of 20 mM NADPH and 0.02 ml of isocitrate dehydrogenase (NADP+ ; 3 IU~ml), in a final volume of 2.87ml. The reaction was performed at 37°C with shaking (120 times/min) under aerobic conditions. One milliliter of the reaction solution was withdrawn and the reaction was stopped by the addition of 10 ml of dichloromethane containing 2.0 ng of [I7,21,21,212H,lpregnenolone as an internal standard (Tsubaki et al., 1987). Mass spectral analysis of pregnenolone formed through the side-chain cleavage reaction was performed with a Hitachi gas chromatograph-mass spectrograph, Model M-80.

Molar extinction coe$icients The molar extinction coefficients at 340 nm of NADPH (adjusted with a sodium hydroxide solution to pH 7.4) and at 260 nm of NADP+, & iOmM potassium* phosphate buffer at DH 7.4.were taken to be 6.30 x 10”M-t cm-’ at 340 nm and 18.5 x 10’ M-’ cm-’ at 26Onm, respectively (McComb et al., 1976; Ziegenhom and Bucher, 1976; Okunuki and Nozaki, 1955). Optical spectra

Optical absorption spectra were recorded with a Shimazu s~~ophotometer, Model UV-240, and a Cary spectrophotometer, Model 17D, equipped with a thermostatically controlled cell holder. Chemicals

NADPH, NADP+ and bovine serum albumin were obtamed from Nakarai Chemical Co. or_.-Isocitrate dehydrogenase (NADP+) was obtained from Sigma Chemical Co. Emulgen 913 was a kind gift from Kao-Atlas Co. All other reagents were of the best grade available from commercial sources.

Sequencing of amino acid residues

Amino acid sequence were determined with an Applied Biosystems protein sequencer, Model 470A, equipped with a S~tra-Physics HPLC, Model SP8100 XR extended range LC. The amino acid residue at the carboxyl terminal was identified by the method of Nakai et al. (1983) using carboxypeptidase A. Amino acid composition

The amino acid composition was determined with a Hitachi amino acid analyzer, Model 835, as described previously (Tsubaki et af., 1987). Molecular weight and electrophoresis The minimum molecular weight of the protein was estimated by SDS-polyacrylamide gel electrophoresis, with 7.5% (w/v) acrylamide concentration by the method of Laemmli ( 1970). Protein contenf

Protein concentrations were measured by the method of Lowry et al. (1951) or by means of the biuret reaction (Gomall et al., 1979) using bovine serum albumin as a standard. The molar extinction coefficient of bovine serum albumin was reported to be 6.68 x IO3M-r cm-’ (Rayman and Chopra, f976) and 6.17 x lo3 M-r cm-’ (King and Spencer, 1970) at 280 nm in 50 mM potassium phosphate buffer at pH 7.4 and 25°C. Heme content The heme content of the cytochrome P-450s~~ was measured as pyridine hemochromes. Pyridine hemochrome spectra were measured in aqueous alkaline pyridine solution (final con~ntrations, 75 mM NaOH and 2.1 M py~dine) after reduction with sodium dithionite. The extinction coefficient value used was 34.4mM-‘cm-’ at 557nm and 191.5 mM-‘cm-’

at 418.5 nm (Fuhrhop

and Smith,

1975).

Antibodies to bovine cytochrome P-450~~

RESULTS

Table 1 shows the pu~fication steps for porcine cytochrome P-450s~~ from the adrenal mitochondria. The cytochrome P-45Osc.cwas purified from porcine adrenal mitochondria solubilized in 10 mM potassium phosphate buffer @H 7.8) containing 20% (v/v) glycerol, 0.2% (w/v) sodium cholate, 0.1% (v/v) Emulgen 913 and 0.1 mM EDTA (buffer A), by DEAE-cellulose column, hydroxyapatite column and adreno-ferredoxin Sepharose 4B affinity column chromatographies. The final yield was 1.4% and the heme content of the cytochrome P-450s~~ purified to 20.6 nmol/mg protein. The purity of the purified cytochrome P-450s~~ was examined by SD~polyacrylamide gel electrophoresis. The results are shown in Fig. 1. The cytochrome P-450~ gave a single stained protein band on the SDS-polyacrylamide gel. From its electrophoretic mobility relative to standard molecular markers, the minimum molecular mass of the cytochrome P-450s~~ was estimated to be 53 kDa (Fig. 2). Figure 3 shows the optical absorption spectra of the purified porcine cytochrome P-450s~ in the oxidized, dithionite-reduced, and CO-complexed states. The absorption peaks of the cytochrome P450s~ was at 278, 360, 417, 534 and 567nm in the oxidized state (cholesterol-free low spin), 413, 543 and 625 run in the dithionite-reduced state, and 448 and 548 nm in the CO-complexed state, in buffer A at 25°C. Table 1. Purification of cytochrome P-450s~~ of porcine adreno-

cortrcal mitoehondria

Antibodies against bovine cytochrome P-450s~~ were prepared from the serum of a rabbit given injections of the cytochrome P-45Oscc, as described previously (Hiwatashi and Ichikawa, 1981).

Purification steps

Ouchterlony double-diffusion agar test

Solubdization of mitochondria

The double-diffusion immunochemical precipitation reaction on an agar plate was performed by the method of Ouchterlony with some modifications, as described previousfy (Hiwatashi and Ichikawa, 1981).

DEAE-cellulose Hydroxyapatite

Adreno-ferredoxin affinitySepharose

Total protein (mgf

3001 240 51

25

Specific content (nmoi/mg protein)

Total content (nmoi)

Yteld (%I

1.o

3001

4.5 10.0

1080 510

36.0 17.0

16.4

41

1.4

100

A

KDa

67

-

43

-

B

-

Front

Fig. 1. Electrophoretogram of the purified porcine and bovine cytochromes P-450s~ on an SDSpolyacrylamide gel. 2.5 or 5 fig of protein was electrophoresed. A-Standard proteins; B-porcine cytochrome P-45Oscc (5 pg protein); C-bovine cytochrome P-450s~~ (2.5 pg protein).

903

Fig. 6. Ouchterlony double-diffusion analysis of porcine, bovine and ovine cytochromes P-450s~~ against antibodies to porcine cytochrome P-450s~~. Well P, porcine cytochrome P-450s~~; well B, bovine cytochrome P-450s~~; well S, ovine cytochrome P-450s~~; center well, antibodies to porcine cytochrome P-45oscc.

904

Porcine cytochrome P-450~

905

loo-

76.

A

Rf

Fig. 2. Estimation of the minimum molecular weight of the purified porcine cytochrome P-450s~~ by SDSpolyacrylamide gel electrophoresis, with 7.5% acrylamide concentration. Solid circles, standard marker proteins; open circle, purified porcine cytochrome P-450s~~; open triangle, purified bovine cytochrome P-450s~~.

g

0.10

2 k B az

0.08

0.06

0.04

0.02

The effect of substrate (cholesterol) binding on the spectrum of the oxidized cytochrome P-450s~~ (substrate-free low-spin) is shown in Fig. 4. The spin state

of the oxidized cytochrome P-450s~~ changed slowly from the low-spin to the high-spin upon the addition of the substrate (cholesterol) during incubation on ice. In the cholesterol-bound state, absorption peaks were observed at 278,392, 519 and 647 nm. The peak height ratio at 278 and 392 nm was 1.2 for the cytochrome P-450s~~ in the high-spin state, and the peak height ratio at 278 and 417 nm was 1.0 for the low-spin state. Table 2 shows the molar extinction coefficients for the porcine cytochrome P-450s~~ in various states determined from spectra measured on a protoheme basis in 10 mM K-phosphate buffer, pH 7.4, containing 20% (v/v) glycerol, 0.1% (v/v) Emulgen 913 and 0.1 mM EDTA at 25°C. The value of 98.2 mM_’ cm-r obtained for the difference in molar extinction between 450 and 490 nm in the CO-re-

,

400

460

6w

0.00 300

400 Wavelength

500

600

700

(am)

Fig. 4. Effect of cholesterol binding on the absolute optical spectrum of porcine cytochrome P-450s~~ in the oxidized state in 50 mM K-phosphate buffer, pH 7.4 and 25°C. Solid line, cholesterol-free low spin form; broken and dotted line, cholesterol-bound high spin form. The cuvette contained 2.3 PM of the cytochrome P-450~ in buffer A (Tsubakl er al., 1986) pH 7.4 and 25°C.

duced minus reduced difference spectrum. This value was clearly different from the conventional value of 91 mM_’ cm-i usually used for microsomal cytochromes P-450. The cytochrome P-450scc-linked monooxygenase system was reconstituted with NADPH-ferredoxin reductase and adreno-ferredoxin (both purified from

SW 600 650

Wavelength (rm) Fig. 3. (a) Absolute optical absorption spectra of porcine cytochrome P-450s~~ in the oxidized (low spin), dithionite-reduced and CO-bound states. The cuvette contained 0.4 PM of the cytochrome P-450s~~ in buffer A (Tsubaki et al., 1986), pH 7.4 and 25°C. Solid line, oxidized state; broken and dotted line, dithionite-reduced state; broken line, CO-complex state; dotted line, base line. (b) Difference spectrum of porcine cytochrome P-45Oscc, CO-complex minus reduced state (solid line). The cuvette contained 0.5 PM of the cytochrome P-450s~~ in buffer A, pH 7.4 and 25°C. Solid line, dithionite-reduced cytochrome P-450s~~ CO-complex minus dithionitereduced cytochrome P-450s~~; dotted line, base line.

906

KAZUHIKO IWAHAW et al. Table 2. Extinctmn coefficients of porcme cytochrome P-45Oscc in various states Oxidized state Low spl” (cholesterol-free)

High spm (cholesterol-bound)

i ma” (“m)

L (mM_’ cm-‘)

1mar (nm)

278 360 417 534 567

157.0 43.8 138.7 13 6 15.9

278 392 519 647

Dlthionite-reduced state

L (mM~‘cm~‘) 156.5 115.6 14.8 114

I Illax (nm)

f (mM_’ cm-‘)

Lx (nm)

413 543

95.9 17.1

448 548

porcine adrenal mitochondria) as described under Materials and Methods. The reconstituted enzymatic activities of porcine and bovine cytochromes P450s~~ for various substrates were measured in the presence of 0.1% (v/v) Tween 20. We chose 0.1% (v/v) Tween 20 as the optimum concentration for the side-chain cleavage reaction for cholesterol as a substrate. Cholesterol, 20(R)- or ZZ(R)-hydroxycholesterol, and 20(R), 22(R)-dihydroxycholesterol were also used as substrates. The enzymatic activities were expressed as pregnenolone formed through the sidechain cleavage reaction, as summarized in Table 3. The enzymatic activities of the porcine side chain cleavage enzyme are compared with those of bovine cytochrome P-450s~~ in this table. Both of the reconstituted systems showed remarkable similarities, each other in the activity toward each of the substrates examined. The turnover rate (at a saturating substrate concentration) was highest for 20(R), 22(R)-dihydroxycholesterol and lowest for cholesterol. When pregnenolone formation was measured using 22(R)hydroxycholesterol or 20(S)-hydroxycholesterol as a substrate, the turnover rate had an intermediate value. Table 4 shows the amino acid composition of the purified porcine cytochrome P-450s~~. The cysteinyl residues of the cytochrome P-450s~~ were measured spectrophotometrically. In addition, the amino acid compositions of bovine and human cytochromes P-450s~~ (estimated from the sequences of both cDNA and proteins) are compared in this table. Cysteinyl analysis showed 2 residues per molecule and tryptophanyl analysis showed 8 residues per molecule of porcine cytochrome P-450s~~. The values obtained for both the numbers of cysteinyl and tryptophanyl residues of porcine cytochrome P450s~~ are also in close agreement with the numbers of these amino acids found in the amino acid sequence deduced from a cDNA sequence of bovine cytochrome P-450s~~. We determined the amino acid sequence in the amino terminal region of the porcine cytochrome P-450s~~. The sequence of the cytochrome P-450s~~ was compared with those of the bovine and human

CO-reduced minus reduced state

CO-reduced state f (mM_’ cm-‘)

131.8 19.6

1mu (“m)

f (mM-’ cm-‘)

450490

98.2

Table 4. CornposItIons of amino acid residues of cytochromes P-450s~ from various mammalian adrenocortical mitochondria Numbers of amino acid residues determined by various methods Amino acid analysis Amino acid residuesa As” > Asp Asx Ala Arg Ile Gly g;; > Glx CYS Ser TY~ Trp Thr Val His Phe Pro Met LYS LLX

Total numbers of amino acid residues

Porcine

cDNA

Bovineb

BovineC

47 28 29 24 34 52

42 22 28 28 24 50

20 24 22 29 32 23 19

Hum& 15 26 29 31 27 24 22

(2) 23 17 8 21 32 12 27 20 11 30 56

(2) 20 19 7 22 28 14 27 26 9 31 49

2 22 20 9 23 29 15 30 30 14 33 51

1 23 18 11 25 33 15 29 26 15 24 52

413

448

481

474

‘Values are amino acid residues per mole of cytochrome P-450s~~. bFrom M. Tsubaki er al., unpublished results. ‘From Ref. Morohashi er al., 1984. dFrom Ref. Chung er al., 1986. ‘Optical measurement.

enzymes deduced from cDNA up to the 25th amino acid residue. The results are shown in Fig. 5. In spite of species differences, the sequences of these three cytochromes P-450s~~ were highly homologous and showed a broad consensus sequence. The amino acid residue at the carboxyl terminal of the porcine cytochrome P-450s~ was alanine, like that of bovine cytochrome P-450s~~. The amino acid residue at the carboxyl terminal of the human cytochrome P-450s~~ has been found to be substituted to glutamine at position 521. Bovine serum albumin was used to construct a standard protein concentration curve. The content of

Table 3. Enzymatic actwtles of the porcrne and bovme cytochrome P-450swreconstltuted (v/v) Tween 20

monooxygenase systems I” the presence of 0.1%

Substrates Species

Cholesterol

Porcme Bovme

7.8 5 1.9 7.7 * 0 5

20(R),22(R)-dlhydroxycholesterol 165.3 + 18.4 172.1 f 28.0

22(R)-hydroxycholesterol 29.0 f 3.8 24.3 f 1.7

20(S)-hydroxycholesterol 19.7 f 3.4 14.7 * 0.5

The activities are expressed as “mole of pregnenolone formed/mi” pe.r nmol of cytochrome P-450s~~. The values are the averages of five esttmations f SD.

907

Porcine cytochrome P-450s~: hj,no-terminal

sequences

of amino acid residues of cytochromes P-4SOscc from

adrenocorticalmitochondria

Carboxyl terminal amino acid residues References

Mammals

This work

Porcine

A

Bovine

A

Morohashibtei..1984

Q

Chung ctnl ,1986

Human

Fig. 5. Amino-terminal amino acid sequences and the carboxyl terminal amino residue of cytochromes P-4SOscc from adrenal ~tochond~a of various mammal. A single letter code is used for amino acid residues. Data on the sequences of the bovine and human cytochromes P-450s~~ were taken from Refs respectively.

bovine serum albumin was measured using the published molar coefficient of 6.68 x 103M-l cm-’ at 280nm (King and Spencer, 1970). The molecular weight of porcine cytochrome P-45Oscc was calculated on a protoheme basis from each molar coefficient value for bovine serum albumin and the amino acid analysis. The results are summarized in Table 5. The table indicated that there is one protoheme per SO-55 kDa molecular mass. Differences in the amino acid compositions of porcine, bovine and human cytochromes P-45Oscc were observed with respect to hydrophobic amino acid residues such as Ala, Ile and Pro, and other amino acid residues, such as Glu, Cys and Lys. The compositions for other neutral and hydrop~lic amino acid residues were apparently similar among these species. Figure 6 shows the immunopr~pitin lines of porcine, ovine and bovine cytochromes P-450s~ against antibodies to porcine cytochrome P-450s~~ in an Ouchterlony double-diffusion plate. The precipitin lines of porcine and bovine cytochromes P-45Oscc showed a spur formation, but not between porcine and ovine cytochromes P-4SOscc. The results indicate that the porcine cytochrome P-450s~~ was immunochemically similar, but not identical with bovine and ovine cytochromes P45Oscc.

DISCLJSSION

Porcine cytochrome P-450s~~ was purified from adrenal mitochondria. The properties of the cytochrome P45Oscc were compared with those of bovine and human cytochromes P-450s~~. The minimum molecular weight and optical absorption spectrum of the porcine cytochrome P-450s~~ were somewhat different from those of bovine and human cytochromes P-450s~ (Tsubaki et al., 1986). The molar extinction coefficients of the cytochrome P-450s~~ were clearly different from those of the microsomal cytochrome P-450 (Omura and Sato, 1964; Tsubaki and Ichikawa, 1985). Although the extinction coefficient value for the CO-difference spectrum of the microsomal cytochrome P-450 has been generally accepted to be 91 mM-’ cm-’ for the optical intensity difference between 450 and 490 nm, we found that the corresponding value for the porcine cytochrome P450s~~ was 97.8 mM-‘cm-’ (pH 7.4 and ZYC), which agreed closely with that of the bovine cytochrome P-45Oscc from adrenocortical mitochondria (Tsubaki et al., 1986). The enzymatic activity of the r~onstituted porcine cytochrome P-450scc-linked monooxygenase system with cholesterol (and hydroxycholesterol) as a substrate was also compared with that of the bovine system (Stein, 1968; Larroque et al., 1981; Hume et al., 1984). Previously, several groups determined Table 5. Heme content of minimum molecular weight of porcine cvtochrome P-45oscC the enzymatic activity, for the reconstituted cytochrome P-450~ system, using nonionic detergent Heme content Minims molecuiar weight (mnol/mg protein) Method (such as Tween 20) or artificial phospholipid vesicles. Takigawa et al. (1978) reported that the reconstituted Lowry’s method, with bovine serum albumin bovine cytochrome P-450s~~ enzyme system showed concentration for a maximum activity in the presence of 0.3% Tween standard curves determined 20 with cholesterol as a substrate. The Y,, of by: conversion of cholesterol to pregnenolone under A,, = 6.68’ 20.6 52,632 Am-?&l? 19.0 57,766 these conditions was reported to be 30 ~ol/min per S~~~iyac~i~ide nmol P-450s~ (37°C). Kido et al. (1979) also regel electrophoresis 53,000 ported that Tween 20 is capable of triggering the 41,954 Amino acid seauence cholesterol side-chain cleavage activity toward ‘The optical absorption values for bovine serum albumin are for 1% cholesterol as a substrate, but the optimal concensolutions at 28Onm and are from Ryan and Chopra, 19X-tration of Tween 20 was, however, around 0.21 mM A, = 6.68; King and Spencer, 1970-A,, = 6.17.

908

KAzumKo IW.4BASBr et al.

(0.023%) and V,,,, under these conditions was 28 nmol/min per nmol P-450s~~ (Kido et al., 1979). Later, Hanukoglu et al. (1981) reported that at 0.3% Tween 20 the reconstituted enzyme system showed V,,, of between 20 and 30 nmol/min per nmol P4.505~~ with cholesterol as a substrate. They also found that V,,,,, was considerably dependent on the ionic strength of the buffer (Hanukoglu et al., 1981). On the other hand, Lambeth et al. (1982) showed that for artificial phospholipid vesicle-reconstituted cytochrome P-450s~~ (37°C) each of the three hydroxylations required for the cholesterol side-chain cleavage reaction occurs at approximately the same turnover rate if the number of hydroxylations required for pregnenolone formation from each substrate is taken into account [i.e. 9.5 min-’ for cholesterol, 15 min-’ for 22(R)-hydroxycholesterol and 31 mitt-’ for 20(R), 22(R)-dihydroxycholesterol (Lambeth et al., 1982)]. Tuckey and Stevenson (1984) also showed that at 37°C the relative turnover rates for 20(R), 22(R)dihydroxycholesterol (217 min-‘), 22(R)-hydroxycholesterol (102 mitt-‘) and cholesterol (55 min-I) were almost 1: 0.5 : 0.33 for bovine luteal cytochrome P-450s~~ in phospholipid vesicles, indicating that each of the three hydroxylations occurs at approximately the same rates. However, the turnover rate

values were 6-7-fold higher than those reported by Lambeth et al. (1982). In the present study, we measured the turnover rates for various hydroxycholesterols in the presence of 0. I % (v/v) Tween 20 for the first time. Our present data established that, first, the turnover rate for 20(R), 22(R)-dihydroxycholesterol is far higher than those for cholesterol, 22(R)-hydroxycholesterol and 20(S)-hydroxycholesterol, and, second, a plausible intermediate in the side-chain cleavage reaction, 22(R)-hydroxycholesterol, shows a higher turnover rate than 20(S)-hydroxycholesterol. Although the turnover rate for cholesterol in our study was considerably lower than those previously reported [except for that by Lambeth et al. (1982), it was not due to the partial denaturation of the reconstituted system since the same system also showed a very high turnover rate for 20(R), 22(R)-dihydroxycholesterol, close to the value reported by Tuckey and Stevenson (1984). In conclusion, we did not obtain any evidence to indicate that the three hydroxylations occur at approximately the same rates, as suggested by Lambeth et al. (1982) and Tuckey and Stevenson (1984). The amino acid compositions of residues of these cytochromes P-450~ were highly homologous to each other but not identical, as shown in Table 4. The primary structure of the porcine cytochrome P-450s~~ in the amino-terminal region was compared with those of other mammalian cytochromes P450s~~ (Morohashi et al., 1984). A broad consensus sequence was found among these cytochromes P450s~~ (Morohashi et al., 1984). The comparison of the primary structures of the several mammalian cytochromes P-450 is very important for an understanding of the individual functional domains of the structure of cytochromes P-450s~~. Bovine and ovine cytochromes P-450s~~ showed an immunochemical precipitin reaction against antibodies to the purified porcine cytochrome P-45Oscc, and a spur formation was observed between porcine

and bovine cytochromes P-45Oscc, but not between porcine and ovine cytochromes P-450s~~. This demonstrated that these cytochromes P-450s~~ are immun~he~~lly similar to each other but not identical. Acknowledgements-We are grateful to Miss Chiharu Shimizu, Research Center of Medical Science, for measuring the amino acid sequences. We are also grateful to Dr Orino, from the Orino Hospital REFE~NCES

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