44
Biochimica et Biophysica A cta, 5 8 0 ( 1 9 7 9 ) 4 4 - - 6 3 © Elsevier/North-Holland Biomedical Press
BBA 38262 PHYSICOCHEMICAL PROPERTIES OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE-CYTOCHROME P-450 REDUCTASE FROM BOVINE ADRENOCORTICAL MICROSOMES
ATSUO HIWATASHI and YOSHIYUKI
ICHIKAWA
Department of Biochemistry, Osaka University Medical School, Nakanoshima, Kita-ku, Osaka 530 (Japan) (Received December 19th, 1978)
Key words: Adrenocortical microsomes; Cytochrome P-450; NADPH-cytochrome P-450 reductase; 21-Hydroxylase; Mixed function oxidase
Summary
Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 455 nm with shoulders at 290,360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17~-hydroxyprogesterone in the presence of cytochrome P-45021 of adrenocortical microsomes. The specific activity of the 21hydroxylase of 17a-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min
Terminology: c y t o c h r o m e P-45021, c y t o c h r o m e P-450Bp A and c y t o c h r o m e P-450BZ mean t h a t the c y t o e h r o m e s P-450 have the characteristic activities of 2 1 - h y d r o x y l a t i o n of 17(~-hydroxyprogesterone, N - d e m e t h y l a t i o n of (+)-benzphetamine and h y d r o x y l a t i o n of 3,4-benzpyrene, respectively. Hepatic ferredoxin and renal ferredoxin are redox components, redoxins. The chemical, optical and i m m u n o c h e m ieal properties of these ferredoxins differ even among bovine tissues. Therefore, we na me d the nonheme iron protein from hepatic inner mitochondrial membranes, h e p a t o r e d o x i n (hepatic ferredoxin), and that from renal inner m i t o c h o n d r i a l membranes, renoredoxi n (renal ferredoxin). NADPH-ferredoxin rcductases refer to NADPH-adrenodoxin reductase, NADPH-hepatoredoxin reductase and NADPHr enored oxin reductase. Abbreviations: NADPH, a i c o t i n a m i d e adenine dinucleotide phosphate, reduced: NADH, nicotinamide dinucleotide; reduced; NADP +, nicotinamide adenine dinucleotide, oxidized; NAD +, ni c ot i na mi de adenine dinucleotide, oxidized; FAD, flavin adenine dinueleotidc; FMN, flavin m o n o n u e l e o t i d e ; SDS, sodium dodecy l sulfate; CD, circular dichroisra; 2',5'-ADP-Sepharose 4B, Sepharose 4B-bound N6-(6-amino hexyl)adenosine-2', 5'-bisphosphate.
45 per nmol of cytochrome P-4502, at 37°C. The NADPH-cytochrome P-450 reductase, like hepatic microsomal NADPH-cytochrome P-450 reductase, could directly reduce the cytochrome P-4502,. The physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated. Its molecular weight was estimated to be 80 000 + 1000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The cytochrome P-450 reductase contained 1 mol each FAD and FMN as coenzymes. Iron, manganese, molybdenum and copper were not detected. The Km values of NADPH and NADH for the NADpH-cytochrome c reductase activity and those of cytochrome c for the activity of NADPH-cytochrome P-450 reductase were determined kinetically. They were 5.3 pM for NADPH, 1.1 mM for NADH, and 9--24 pM for cytochrome c. Chemical modification of the amino acid residues showed that a histidyl and cysteinyl residue are essential for the binding site of NADPH of NADPH-cytochrome P-450 reductase.
Introduction Most steroid hormones are synthesized by cytochrome P-450-1inked mixed function oxidase systems of adrenocortical microsomes and mitochondria and these electron transport systems are dependent o n NADPH as an electron donor. The cytochrome P-450 linked mixed function oxidase system of the mitochondrial type (inner mitochondrial type) is composed of NADPH-ferredoxin reductase, ferredoxin and cytochrome P-450. The NADPH-adrenodoxin reductase (EC 1.6.7.1) and adrenodoxin were crystallized from bovine and pig adrenocortical mitochondria and their properties have been investigated in detail [1--4]. On the other hand, the cytochrome P-450-1inked mixed function oxidase system of the microsomal type of hepatocytes is composed of two components, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and cyt0chrome P-450. Systems of the microsomal type of other tissues, except the liver, are not yet known. Reconstitution of the cytochrome P-450-1inked mixed function oxidase system of bovine adrenocortical microsomes was reported by Narasimhulu [5]. However, her components were not pure enough to allow identification of the mechanism and the components of the hydroxylase system of 21hydroxylation of 17~-hydroxyprogesterone in adrenocortical microsomes. Sweat et al. [6] reported that 21-hydroxylation and ll~-hydroxylation of steroids may have a common NADPH-dependent reductase. Masters et al. [7] reported that the microsomal NADPH-cytochrome c reductase and the mitochondrial NADPH-adrenodoxin reductase are immunochemically different and independent. We attempted to resolve the ambiguity of the components of the cytochrome P-450-1inked mixed function oxidase system of adrenocortical micro° somes. Accordingly, we purified NADPH-cytochrome P-450 reductase from bovine adrenocortical microsomes. This reductase has been confirmed to be essential for the reconstitution of the cytochrome P-450-1inked mixed function
46 oxidase system (21-hydroxylase of 17a-hydroxyprogesterone) of bovine adrenocortical microsomes. In this study, the physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated and compared with those of hepatic micr0somal NADPH-cytochrome P-450 reductase. Materials and Methods Preparation o f adrenocortical microsomes Fresh bovine adrenal glands were obtained from a local slaughter house and stored whole in a dessicator at --20°C for a week, while about 400 were collected. The surrounding connective and fat tissues of the adrenal glands were removed with scissors. Next, the adrenocortices were carefully separated from the adrenal medulla and then their capsules were trimmed away with a razor. Unless otherwise indicated, all preparation procedures were carried out at below 4°C. Centrifuges with rotors and homogenizers were refrigerated at 0°C before use. The adrenocortices were homogenized with 4 vols. of ice-cold 0.25 M sucrose solution (adjusted to pH 7.4 with 1 M Tris solution) containing 0.5 mM phenylmethylsulfonyl fluoride as a protease inhibitor, in a Matsushita homogenizer, Model MX-140S, at 10 000 rev./min for 2 min. The homogenate was filtrated through a single layer of gauze to remove fat and connective tissues. The mitochondrial fraction of the adrenocortices was prepared from the post-nuclear supernatant by centrifugation at 1 0 0 0 0 rev./min (gmax = 12 000 X g) for 15 min, according to the method of Hatefi and Lester [8]. The post-mitochondrial supernatant was further centrifuged with a Hitachi 55P ultracentrifuge with a barrel rotor at 23 000 rev./min (gm~x = 55 000 X g) for 90 min. The microsomal pellet was homogenized with a Potter-Elvejhem homogenizer using a loosely fitting Teflon pestle revolving at 1000 rev./min in icecold 0.15 M KC1 solution containing 1 mM EDTA (pH 7.4). The solution was recentrifuged at 55 000 X gmax for 90 min. The resulting microsomes were used for purification of NADPH-cytochrome P-450 reductase as the material source. Contamination by mitochondria in the microsomal fraction was estimated from the succinate dehydrogenase activity. It was less than 4% of the mitochondrial fraction on a protein basis. The microsomal fraction was also contaminated with hemoglobin at less than 0.4 nmol/mg of protein, judging from the amount of the CO-complex form of hemoglobin. Purification o f NADPH-cytochrome P-450 reductase from adrenocortical m icroso mes (a) Step I: Solubilization o f the microsomes. The microsomal pellet was suspended at a protein concentration of 12 mg/ml in 50 mM potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and a protease inhibitor, 0.5 mM phenylmethylsulfonyl fluoride. The protease inhibitor was already used in the case of the purification of cytochromes P-450 and P-448 [9]. A 500 ml portion of this solution was mixed with 20 ml of 15% (w/v) sodium cholate and stirred gently for 30 min at 4 ° C. After incubation, the solution was centrifuged with a Hitachi 55P ultracentrifuge at 105 000 X gmax for 90 min. (b) Step H: DEAE-cellulose chromatography. A 10% Emulgen 913 solution was added dropwise to the supernatant of Step I with gentle stirring to obtain
47 a final concentration of 0.2%, and the solution was applied to a DEAE-cellulose column (5 X 35 cm) equilibrated with 0.1 M potassium phosphate buffer, pH 7.4, containing 0.2% Emulgen 913 and 0.1 mM EDTA. The column was washed with 2 1 of the equilibrating buffer, then the NADPH-cytochrome P-450 reductase was eluted with a linear gradient of KC1 ranging from 0 to 0.35 M in 1 1 of the equilibrating buffer. The enzyme fractions were pooled and fractionated between 30 and 60% saturated ammonium sulfate. The precipitate was dissolved in 10 mM potassium phosphate buffer, pH 7.4, containing 0.1% Emulgen 913 and 0.1 mM EDTA, then dialyzed overnight against 10 1 of the same buffer at 0 ° C. The dialyzed solution was confirmed to be completely free of ammonium sulfate with Nessler's reagent. (c) Step III: 2',5'-ADP-Sepharose chromatography. The dialyzed solution was applied to a 2',5'-ADP-Sepharose 4B column (1.5 X 18 cm) equilibrated with 10 mM potassium phosphate buffer, pH 7.4, containing 0.1% Emulgen 913 and 0.1 mM EDTA. The column was washed with 100 ml of 0.2 M potassium phosphate buffer, pH 7.4, containing 0.1% Emulgen 913 and 0.1 mM EDTA, then the washing buffer was changed to 100 ml of the 10 mM potassium phosphate buffer, pH 7.4, containing 0.1% Emulgen 913 and 0.1 mM EDTA. The NADPH-cytochrome P-450 reductase was eluted with 0.8 mM NADP ÷ in 60 ml of the equilibrating buffer. The main fractions of the eluted NADPH-cytochrome P-450 reductase were pooled. (d) Step IV: DEAE-Sepharose chromatography. The eluted NADPH-cytochrome P-450 reductase was applied to a DEAE-SePharose CL-6B (1.5 X 20 cm) equilibrated with 10 mM potassium phosphate buffer, pH 7.4, in order to remove NADP ÷ and Emulgen 913 in the cytochrome P-450 reductase solution. The column was washed with 800 ml of 50 mM potassium phosphate buffer, pH 7.4, then the NADPH-cytochrome P-450 reductase was eluted with 50 mM potassium phosphate buffer, pH 7.4, containing 0.30 M KC1 and 0.1% (w/v) sodium deoxycholate. The cytochrome P-450 reductase solution was dialyzed for 3 h against 3 1 of 50 mM potassium phosphate buffer, pH 7.4, at 0°C. The NADPH-cytochrome P-450 reductase solution (1.5 mg protein/ml) was stable for at least three months when stored at --20°C in the dark.
Purification of other enzymes NADPH-cytochrome P-450 reductase was purified from bovine liver microsomes by the same method used for the adrenocortical cytochrome P-450 reductase. Cytochromes P-450 of bovine adrenocortical mitochondria and microsomes and rabbit liver microsomes were purified as described in another paper. Crystalline adrenodoxin was prepared from bovine adrenocortical mitochondria [10]. Cytochrome b5 was purified from rabbit liver microsomes [11].
Optical absorption spectra Optical absorption spectra were measured with a Cary spectrophotometer, Model 219, equipped with thermostatically controlled cell holders at 25°C and a cuvette of 1 cm light path, using a neodymium glass filter as a standard of wavelength.
48
Assay of enzymatic activities Enzymatic activities were measured at 25 ° C by the following test system and methods unless otherwise stated. NADPH-cytochrome P-450 reductase activity was determined as NADPHcytochrome P-450-phenylisocyanide complex reductase activity [12]. The reductase activity was assayed by measuring the increment in absorbance at 455 nm. The reaction mixture contained 50 nM NADPH-cytochrome P-450 reductase, 1 pM cytochrome P-450, 0.1 mM phenylisocyanide and 2 pM superoxide dismutase in 0.1 M potassium phosphate buffer, pH 7.4. The reaction was started by adding 50 pM NADPH. The molar extinction coefficient of dithionite-reduced cytochrome P-450-phenylisocyanide complex minus dithionite-reduced cytochrome P-450 was estimated to be 71 • 103 M -1 • cm -1 from the absorbance between 455 and 490 nm, in 0.1 M potassium phosphate buffer, pH 7.4. 21-Hydroxylase activity of 17a-hydroxyprogesterone was assayed by the method of Porter and Silber [13]. The reaction mixture contained 2pM NADPH-cytochrome P-450 reductase, 0.01% Emulgen 913, 50 nM cytochrome P-450, 200 pg of 17a-hydroxyprogesterone, 1 mM MgC12, 0.5 unit of isocitrate dehydrogenase and 0.5 mM sodium isocitrate in 0.1 M potassium phosphate buffer, pH 7.4, in a total volume of 1 ml. The reaction was started by adding 0.5 mM NADP ÷ and performed at 37 ° C. NADPH-cytochrome c reductase activity was assayed by measuring the increment in absorbance at 550 nm of the a-band of the reduced cytochrome c. The reaction mixture contained 10 nM NADPH-cytochrome P-450 reductase and 50/aM ferricytochrome c in 0.3 M potassium phosphate buffer, pH 7.7. To initiate the reaction, 50 pM NADPH was added. When the NADPH-cytochrome c reductase activity of the microsomes was assayed, 0.5 mM KCN was added to the reaction mixture to prevent reoxidation of the ferrocytochrome c by cytochrome aa3 of contaminating mitochondria, which may have been present. The activity of NADPH-ferricyanide reductase was assayed by measuring the decrement in absorbance at 420 nm of ferricyanide. The reaction mixture contained 20 nM NADPH-cytochrome P-450 reductase and 0.6 mM ferricyanide in 0.3 M potassium phosphate buffer, pH 7.7. The reaction was started by adding 50 pM NADPH.
Electrophoresis Thin-layer SDS-polyacrylamide gel electrophoresis was performed by the method of Weber and Osborn [14], using 7.5% acrylamide. The molecular weight of NADPH-cytochrome P-450 reductase was estimated by SDS-polyacrylamide gel electrophoresis, using molecular weight markers of proteins such as RNA polymerase a-subunit (Mr -- 39 000), catalase (Mr = 60 000), bovine serum albumin (Mr -- 67 000), phosphorylase a of rabbit muscle (Mr = 95 000) and RNA polymerase/3-subunit (Mr = 155 000).
Analytical ultracentrifugation The molecular weight of NADPH-cytochrome P-450 reductase was also estimated by sedimentation equilibrium using interference optics, according to Yphantis' method [15]. A 0.5 ml sample of cytochrome P-450 reductase
49 (2.5 mg/ml in 0.1 M potassium phosphate buffer, pH 7.4) was incubated in 6 M guanidine HC1 and 0.1 M sodium iodoacetate at 80°C for 10 min. The sample was dialyzed against 100 ml o f 6 M guanidine HC1 for 30 h with three changes o f th e dialysis buffer. A 20 h period was allowed for equilibrium t o be reached at 20°C in a Hitachi analytical ultracentrifuge, Model 282, at a speed o f 10 000 rev./min. A blank pattern for t he cell was obtained using water for correction o f optical irregularities. Measurements o f fringe elevation and radial distance were d o ne with a Nikon com pa r at or .
Fluorescence measurement Fluorescence measurements were made in a Hitachi fluorescence spectrop h o t o m e t e r , Model MPF-2A, equipped with a thermostatically controlled cell holder using a Coolnics Model CTR-120 and circular Model CTE-120 K o mats u - Yamato Co. with slits o f 1.3 m m .
Circular dichroism spectra Circular dichroism (CD) spectra were measured with a Union Giken, Dichrograph Mark III-J, calibrated with D-camphor-10-sulfonic acid. T he measurements were made in fused silica cells: a 1.0 cm light path cell was used in the visible range and a 0.2 cm light path cell in the ultraviolet range. T he temperature o f th e solution was kept at 25°C.
Analytical procedures The molar ex t i nct i on coefficients o f NADPH and NADH were 6.30 • 103 M -1 • cm -1 at 340 nm (pH 7.6 and 25°C) [16] and those o f NADP ÷ and NAD ÷ were 1 8 . 0 . 1 0 3 M -1. cm -1 at 260 nm (pH 7.6 and 25°C) [17]. T he molar e x t i n ctio n coefficients o f c y t o c h r o m e c in the reduced minus oxidized forms and o f ferricyanide were 21 • 103 M -1. cm -1 at 550 nm [18] and 1 . 0 2 . 1 0 3 M -1 • cm -~ at 420 nm [ 19] , respectively. The c o n t e n t o f a d r e n o d o x i n was estim a t ed f r o m the molar ext i nct i on coefficient o f 9.8 • 103 M -~ • cm -t at 414 nm [10]. Th e c o n t e n t o f c y t o c h r o m e P-450 was det erm i ned from the CO difference spectrum o f t he dithionite-reduced f o rm , by assuming a value o f 91.0 • 103 M -1 • cm -1 as the molar e xt i nc t i on i nc rem ent o f the absorbance between 450 and 490 nm [20]. C y t o c h r o m e bs was det erm i ned, taking a value o f 163 • 103 M -~ • cm-' as the molar extinction coefficient between 424 and 409 nm in th e difference spectrum between t h e dithionite-reduced and t h e oxidized forms [21]. To calculate the r e duc t i on rates o f acceptors by NADPH-cytoc h r o m e P-450 reductase, the follo~ving e xt i n ct i on coefficients were used: 2,6d i c h l o r o p h e n o l i n d o p h e n o l , 21 • 103 M -~ • cm -1 at 600 nm [22] ; nitrobluetetrazolium, 28.6 • 103 M -1 • cm -1 at 550 nm [ 2 3 ] ; and neot et razol i um , 15.5 • 103 M -1 • cm -1 at 505 nm. The c o n t e n t o f hemoglobin was estimated from a CO difference spectrum using the molar e x t i n c t i o n coefficient o f 191 • 103 M -t • cm -1 at 419 nm [24]. Protein c o n t e n t was det erm i ned by the m e t h o d o f L o w r y et al. [25] and by the biuret reaction [26] using bovine serum albumin as a standard. Th e c o n c e n t r a t i o n o f t he albumin was determined using the e x t i n c t i o n coefficient, Elcm 1 at 280 nm, o f 6.6 [27].
Analyses o f compositions o f amino acids, sugars and metals Amino acid residues were analyzed by t he m e t h o d o f Moore and Stein [28]
50 in the absence and presence of 2% thioglycolic acid [29], using a Hitachi amino acid analyzer. Model KLA-3B. The contents of serinyl and threonyl residues were estimated exactly by the decomposition at various hydrolysis times for 24, 48 and 72 h. The number of sulfhydryl groups was determined by Ellman's m e t h o d [30] in the absence and presence of 8 M guanidine HC1. The amount of tryptophanyl residue present was also determined fluorometrically by the Pajot's method [31]. Neutral and amino sugars were separated b y the method of Haberman et al. [32] then measured b y the Sinohara's m e t h o d [33]. The sialic acid content was determined b y the Aminoff's m e t h o d [34]. The contents of iron, copper, manganese and m o l y b d e n u m were determined with a Hitachi atomic absorption spectrophotometer, Model 208. To determine and detect the metals in NADPH-cytochrome P-450 reductase, a 0.5 pM sample was used for atomic absorption spectrophotometry, which was sufficient to allow detection of even one atom of m o l y b d e n u m / m o l of the cytochrome P-450 reductase. Molybdenum was the most difficult metal to detect by the atomic absorption spectrophotometer.
Analyses of flavins FAD and FMN were obtained from Nakarai Chemical Co. FAD and FMN were purified by DEAE-cellulose chromatography [35]. Both were confirmed to produce a single spot on a paper chromatography with three different solvent systems (n-butanol/acetic acid/H20 (4 : 1 : 5), pyridine/H20 (2 : 1) and 5% Na2HPO4) and R F values o f the flavins in the various solvents [36]. The concentration of flavin solutions were determined spectrophotometrically using the extinction coefficients of 11.3 • 103 M -1 • cm -1 at 450 nm (pH 7.0) for FAD and 12.5 • 103 M -1 • cm -1 at 450 nm (pH 7.0) for FMN [37]. To release the flavin, NADPH-cytochrome P-450 reductase was digested with 0.2% (w/v) protease for 30 min for 37°C, then the solution was heated for 10 min at 80°C, rapidly cooled to 0°C with ice, and centrifuged at 9000 × g for 20 min to remove the heat~lenatured proteins [38]. The fluorescence of the supernatant was determined at 25°C and at both pH 7.7 and 2.6 as described b y Faeder and Siegel [39].
Kinetic analysis of NADPH-cytochrome P-450 reductase The NADPH-cytochrome P-450 reductase catalyzes the reaction according to the following equations, described with respect to the hepatic NADPH-cytochrome c reductase [40]. NAD(P)H + NADPH-cytochrome P-450 reductase (E) ~ EH2 + NAD(P) ÷
(1)
EH2 + 2 Ferricytochrome c (cyt. c 3÷) ~ E + 2 Ferrocytochrome c (cyt. c 2÷) + 2n÷
(2)
These reactions can be studied kinetically by measuring the increase in absorbance at 550 n m of the a-band of ferrocytochrome c. In the kinetic investigation of sequential binary reactions [41], the K m values of the NADPH-cytochrome P-450 reductase for N A D P H , N A D H and cytochrome e can be obtained Line-
51 weaver-Burk plots: 1Iv = 1 I V . [1 + KNAD(P)H/NAD(P)H + Kcyt.c3+/cyt. c 3÷]
(3)
where v is the initial velocity, V is the maximal velocity in the forward direction and KNAD(P)Hand Kcyt.c 3÷ are the Km values of the NADPH-cytochrome P-450 reductase for NADPH or NADH and ferricytochrome c, respectively. Chemicals NADPH, NADH and their oxidized forms were kind gifts from Oriental Yeast Co. Cytochrome c (type III, horse heart), 5,5'-dithiobis(2-nitrobenzoate), diethyl pyrocarbonate, phenylhydrazine hydrochloride and protease (type XIII, Bacillus subtilis) were purchased from Sigma Chemical Co. Sepharose 4B and DEAE-Sepharose CL-6B were purchased from Pharmacia Fine Chemicals. 2',5'-ADP-Sepharose 4B was synthesized by the method of Brodelius et al. [42]. Standard proteins for the estimation of molecular weights were purchased from Boehringer Mannheim GmbH. Emulgen 913 was a kind gift from Kao Atlas Co. Superoxide dismutase was purified from bovine erythrocytes by the method of McCord and Fridovich [43] and catalase was crystallized from bovine livers by Sirakawa's method [44]. Crystalline phenylisocyanide was synthesized by the method of Prager et al. [45]. All other reagents were of the highest grade available commercially. Results
General properties NADPH-cytochrome P-450 reductase was purified from bovine adrenocortical microsomes. A typical purification scheme is summarized in Table I. As shown in Table I, the ratio of activities toward cytochrome c and cytochrome P-450 does not vary significantly throughout the purification procedure. The yield of the NADPH-cytochrome P-450 reductase was 28%. It was confirmed to be a single protein band on SDS-polyacrylamide gel electrophoresis, thus being homogeneous and pure. Fig. 1 shows the electrophoretograms of NADPH-cytochrome P-450 reductases of the bovine adrenocortical and liver microsomes from SDS-polyacrylamide gel electrophoresis. The NADPH-cytochrome P-450 reductase, like liver NADPH-cytochrome P-450 reductase, could dirgctly reduce cytochrome P-450 and cytochrome c. These reductions were not due to superoxide or hydrogen peroxide, because superoxide dismutase and catalase did not influence the reducing rate of cytochrome P-450 by NADPH-cytochrome P-450 reductase. The specific activities of NADPH-cytochrome P-450 reductase and NADPH-cytochrome c reductase of our purified reductase preparation were 2.28 and 29.5 tlmol/min per mg protein at 25°C, respectively. Fig. 2 shows the optical absorption spectra of NADPH-cytochrome P-450 reductase of bovine adrenocortical microsomes in the oxidized and reduced forms. The absorption peaks of the cytochrome P-450 reductase were at 274, 380 and 455 nm with shoulders at 290, 360 and 480 nm in the oxidized form at 25°C. The absorption spectrum is that of a typical flavoprotein. The ratio of the intensity of the peak at 274 nm to that at 455 nm was 8.3, and that at
52 TABLE I P U R I F I C A T I O N OF N A D P H - C Y T O C H R O M E P-450 R E D U C T A S E FROM BOVINE A D R E N O C O R T I C AL MICROSOMES P r o t e i n c o n c e n t r a t i o n w a s d e t e r m i n e d b y the b i u r e t r e a c t i o n a n d t h e m e t h o d of L o w r y et al. [ 2 5 ] , a f t e r r e m o v a l o f t h e i n f l u e n c e o f nucleic acids. Specific a c t i v i t y o f c y t . c is t h e N A D P H - c y t o c h r o m e c r e d u c tase a c t i v i t y . This a c t i v i t y was a s s a y e d in t h e p r e s e n c e o f 0.5 m M KCN a n d 0.3 M p o t a s s i u m p h o s p h a t e b u f f e r , p H 7.7 ( 2 5 ° C ) . Specific a c t i v i t y of cyt. P - 4 5 0 is t h e N A D P H - c y t o c h r o m e P - 4 5 0 r e d u c t a s e a c t i v i t y . This a c t i v i t y was d e t e r m i n e d b y m e a s u r i n g t h e N A D P H - c y t o c h r o m e P - 4 5 0 - p h e n y l i s o c y a n i d e c o m p l e x r e d u c t a s e a c t i v i t y . T h e r a t i o of activities is e x p r e s s e d as N A D P H - c y t o c h r o m e c r e d u c t a s e / N A D P H - c y t o c h r o m e P - 4 5 0 r e d u c t a s e . P u r i f i c a t i o n a n d t o t a l a c t i v i t y are b a s e d o n t h e specific a c t i v i t y of N A D P H - c y t o c h r o m e c r e d u c t a s e . Yield is b a s e d o n t h e t o t a l a c t i v i t y of N A D P H - c y t o c h r o m e c r e d u c t a s e . Purification step
Microsomes Solubilized microsomes DEAE-cellulose c o l u m n eluate 2'5'-ADP Sepharose column eluate DEAE-Sepharose c o l u m n eluate
Volume (ml)
Protein concn, (mg/ml)
Total protein (mg)
500 495
12.1 7.6
6050 3762
38
5.4
205
Specific a c t i v i t y cyt. c
Ratio
cyt. P-450
0.12 0.19
---
2.6
Total activity (~tmol/ min)
Purification (-fold)
1 1,6
Yield (%)
---
726 715
100 98
0.24
10.8
533
22
73
4.6
3.3
15.2
26.8
2.10
12.8
407
223
56
4,5
1.5
6.8
29.5
2,28
12.9
201
246
28
Fig. 1. E l e c t r o p h o r e t o g r a m o f N A D P H - c y t o c h r o m e P - 4 5 0 r e d u c t a s e f r o m b o v i n e a d r e n o c o r t i c a l a n d h e p a t i c m i c r o s o m e s o n S D S - p o l y a c r y l a r n i d e gel. Samples of 1 0 ~g p r o t e i n o f t h e c y t o c h r o m e P - 4 5 0 r e d u c t a s e s w e r e u s e d o n t h e gel. A, A d r e n o c o r t i c a l N A D P H - c y t o c h r o m e P - 4 5 0 r e d u c t a s e ; H , h e p a t i c NADPH-cytochrome P-450 reductase.
53
0.8
0.8
0.4
0.6
0.2 v ¢ 0.4 o w
0.0
350
Biochimica et Biophysica A cta, 5 8 0 ( 1 9 7 9 ) 4 4 - - 6 3 © Elsevier/North-Holland Biomedical Press
BBA 38262 PHYSICOCHEMICAL PROPERTIES OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE-CYTOCHROME P-450 REDUCTASE FROM BOVINE ADRENOCORTICAL MICROSOMES
ATSUO HIWATASHI and YOSHIYUKI
ICHIKAWA
Department of Biochemistry, Osaka University Medical School, Nakanoshima, Kita-ku, Osaka 530 (Japan) (Received December 19th, 1978)
Key words: Adrenocortical microsomes; Cytochrome P-450; NADPH-cytochrome P-450 reductase; 21-Hydroxylase; Mixed function oxidase
Summary
Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 455 nm with shoulders at 290,360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17~-hydroxyprogesterone in the presence of cytochrome P-45021 of adrenocortical microsomes. The specific activity of the 21hydroxylase of 17a-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min
Terminology: c y t o c h r o m e P-45021, c y t o c h r o m e P-450Bp A and c y t o c h r o m e P-450BZ mean t h a t the c y t o e h r o m e s P-450 have the characteristic activities of 2 1 - h y d r o x y l a t i o n of 17(~-hydroxyprogesterone, N - d e m e t h y l a t i o n of (+)-benzphetamine and h y d r o x y l a t i o n of 3,4-benzpyrene, respectively. Hepatic ferredoxin and renal ferredoxin are redox components, redoxins. The chemical, optical and i m m u n o c h e m ieal properties of these ferredoxins differ even among bovine tissues. Therefore, we na me d the nonheme iron protein from hepatic inner mitochondrial membranes, h e p a t o r e d o x i n (hepatic ferredoxin), and that from renal inner m i t o c h o n d r i a l membranes, renoredoxi n (renal ferredoxin). NADPH-ferredoxin rcductases refer to NADPH-adrenodoxin reductase, NADPH-hepatoredoxin reductase and NADPHr enored oxin reductase. Abbreviations: NADPH, a i c o t i n a m i d e adenine dinucleotide phosphate, reduced: NADH, nicotinamide dinucleotide; reduced; NADP +, nicotinamide adenine dinucleotide, oxidized; NAD +, ni c ot i na mi de adenine dinucleotide, oxidized; FAD, flavin adenine dinueleotidc; FMN, flavin m o n o n u e l e o t i d e ; SDS, sodium dodecy l sulfate; CD, circular dichroisra; 2',5'-ADP-Sepharose 4B, Sepharose 4B-bound N6-(6-amino hexyl)adenosine-2', 5'-bisphosphate.
45 per nmol of cytochrome P-4502, at 37°C. The NADPH-cytochrome P-450 reductase, like hepatic microsomal NADPH-cytochrome P-450 reductase, could directly reduce the cytochrome P-4502,. The physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated. Its molecular weight was estimated to be 80 000 + 1000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The cytochrome P-450 reductase contained 1 mol each FAD and FMN as coenzymes. Iron, manganese, molybdenum and copper were not detected. The Km values of NADPH and NADH for the NADpH-cytochrome c reductase activity and those of cytochrome c for the activity of NADPH-cytochrome P-450 reductase were determined kinetically. They were 5.3 pM for NADPH, 1.1 mM for NADH, and 9--24 pM for cytochrome c. Chemical modification of the amino acid residues showed that a histidyl and cysteinyl residue are essential for the binding site of NADPH of NADPH-cytochrome P-450 reductase.
Introduction Most steroid hormones are synthesized by cytochrome P-450-1inked mixed function oxidase systems of adrenocortical microsomes and mitochondria and these electron transport systems are dependent o n NADPH as an electron donor. The cytochrome P-450 linked mixed function oxidase system of the mitochondrial type (inner mitochondrial type) is composed of NADPH-ferredoxin reductase, ferredoxin and cytochrome P-450. The NADPH-adrenodoxin reductase (EC 1.6.7.1) and adrenodoxin were crystallized from bovine and pig adrenocortical mitochondria and their properties have been investigated in detail [1--4]. On the other hand, the cytochrome P-450-1inked mixed function oxidase system of the microsomal type of hepatocytes is composed of two components, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and cyt0chrome P-450. Systems of the microsomal type of other tissues, except the liver, are not yet known. Reconstitution of the cytochrome P-450-1inked mixed function oxidase system of bovine adrenocortical microsomes was reported by Narasimhulu [5]. However, her components were not pure enough to allow identification of the mechanism and the components of the hydroxylase system of 21hydroxylation of 17~-hydroxyprogesterone in adrenocortical microsomes. Sweat et al. [6] reported that 21-hydroxylation and ll~-hydroxylation of steroids may have a common NADPH-dependent reductase. Masters et al. [7] reported that the microsomal NADPH-cytochrome c reductase and the mitochondrial NADPH-adrenodoxin reductase are immunochemically different and independent. We attempted to resolve the ambiguity of the components of the cytochrome P-450-1inked mixed function oxidase system of adrenocortical micro° somes. Accordingly, we purified NADPH-cytochrome P-450 reductase from bovine adrenocortical microsomes. This reductase has been confirmed to be essential for the reconstitution of the cytochrome P-450-1inked mixed function
46 oxidase system (21-hydroxylase of 17a-hydroxyprogesterone) of bovine adrenocortical microsomes. In this study, the physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated and compared with those of hepatic micr0somal NADPH-cytochrome P-450 reductase. Materials and Methods Preparation o f adrenocortical microsomes Fresh bovine adrenal glands were obtained from a local slaughter house and stored whole in a dessicator at --20°C for a week, while about 400 were collected. The surrounding connective and fat tissues of the adrenal glands were removed with scissors. Next, the adrenocortices were carefully separated from the adrenal medulla and then their capsules were trimmed away with a razor. Unless otherwise indicated, all preparation procedures were carried out at below 4°C. Centrifuges with rotors and homogenizers were refrigerated at 0°C before use. The adrenocortices were homogenized with 4 vols. of ice-cold 0.25 M sucrose solution (adjusted to pH 7.4 with 1 M Tris solution) containing 0.5 mM phenylmethylsulfonyl fluoride as a protease inhibitor, in a Matsushita homogenizer, Model MX-140S, at 10 000 rev./min for 2 min. The homogenate was filtrated through a single layer of gauze to remove fat and connective tissues. The mitochondrial fraction of the adrenocortices was prepared from the post-nuclear supernatant by centrifugation at 1 0 0 0 0 rev./min (gmax = 12 000 X g) for 15 min, according to the method of Hatefi and Lester [8]. The post-mitochondrial supernatant was further centrifuged with a Hitachi 55P ultracentrifuge with a barrel rotor at 23 000 rev./min (gm~x = 55 000 X g) for 90 min. The microsomal pellet was homogenized with a Potter-Elvejhem homogenizer using a loosely fitting Teflon pestle revolving at 1000 rev./min in icecold 0.15 M KC1 solution containing 1 mM EDTA (pH 7.4). The solution was recentrifuged at 55 000 X gmax for 90 min. The resulting microsomes were used for purification of NADPH-cytochrome P-450 reductase as the material source. Contamination by mitochondria in the microsomal fraction was estimated from the succinate dehydrogenase activity. It was less than 4% of the mitochondrial fraction on a protein basis. The microsomal fraction was also contaminated with hemoglobin at less than 0.4 nmol/mg of protein, judging from the amount of the CO-complex form of hemoglobin. Purification o f NADPH-cytochrome P-450 reductase from adrenocortical m icroso mes (a) Step I: Solubilization o f the microsomes. The microsomal pellet was suspended at a protein concentration of 12 mg/ml in 50 mM potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and a protease inhibitor, 0.5 mM phenylmethylsulfonyl fluoride. The protease inhibitor was already used in the case of the purification of cytochromes P-450 and P-448 [9]. A 500 ml portion of this solution was mixed with 20 ml of 15% (w/v) sodium cholate and stirred gently for 30 min at 4 ° C. After incubation, the solution was centrifuged with a Hitachi 55P ultracentrifuge at 105 000 X gmax for 90 min. (b) Step H: DEAE-cellulose chromatography. A 10% Emulgen 913 solution was added dropwise to the supernatant of Step I with gentle stirring to obtain
47 a final concentration of 0.2%, and the solution was applied to a DEAE-cellulose column (5 X 35 cm) equilibrated with 0.1 M potassium phosphate buffer, pH 7.4, containing 0.2% Emulgen 913 and 0.1 mM EDTA. The column was washed with 2 1 of the equilibrating buffer, then the NADPH-cytochrome P-450 reductase was eluted with a linear gradient of KC1 ranging from 0 to 0.35 M in 1 1 of the equilibrating buffer. The enzyme fractions were pooled and fractionated between 30 and 60% saturated ammonium sulfate. The precipitate was dissolved in 10 mM potassium phosphate buffer, pH 7.4, containing 0.1% Emulgen 913 and 0.1 mM EDTA, then dialyzed overnight against 10 1 of the same buffer at 0 ° C. The dialyzed solution was confirmed to be completely free of ammonium sulfate with Nessler's reagent. (c) Step III: 2',5'-ADP-Sepharose chromatography. The dialyzed solution was applied to a 2',5'-ADP-Sepharose 4B column (1.5 X 18 cm) equilibrated with 10 mM potassium phosphate buffer, pH 7.4, containing 0.1% Emulgen 913 and 0.1 mM EDTA. The column was washed with 100 ml of 0.2 M potassium phosphate buffer, pH 7.4, containing 0.1% Emulgen 913 and 0.1 mM EDTA, then the washing buffer was changed to 100 ml of the 10 mM potassium phosphate buffer, pH 7.4, containing 0.1% Emulgen 913 and 0.1 mM EDTA. The NADPH-cytochrome P-450 reductase was eluted with 0.8 mM NADP ÷ in 60 ml of the equilibrating buffer. The main fractions of the eluted NADPH-cytochrome P-450 reductase were pooled. (d) Step IV: DEAE-Sepharose chromatography. The eluted NADPH-cytochrome P-450 reductase was applied to a DEAE-SePharose CL-6B (1.5 X 20 cm) equilibrated with 10 mM potassium phosphate buffer, pH 7.4, in order to remove NADP ÷ and Emulgen 913 in the cytochrome P-450 reductase solution. The column was washed with 800 ml of 50 mM potassium phosphate buffer, pH 7.4, then the NADPH-cytochrome P-450 reductase was eluted with 50 mM potassium phosphate buffer, pH 7.4, containing 0.30 M KC1 and 0.1% (w/v) sodium deoxycholate. The cytochrome P-450 reductase solution was dialyzed for 3 h against 3 1 of 50 mM potassium phosphate buffer, pH 7.4, at 0°C. The NADPH-cytochrome P-450 reductase solution (1.5 mg protein/ml) was stable for at least three months when stored at --20°C in the dark.
Purification of other enzymes NADPH-cytochrome P-450 reductase was purified from bovine liver microsomes by the same method used for the adrenocortical cytochrome P-450 reductase. Cytochromes P-450 of bovine adrenocortical mitochondria and microsomes and rabbit liver microsomes were purified as described in another paper. Crystalline adrenodoxin was prepared from bovine adrenocortical mitochondria [10]. Cytochrome b5 was purified from rabbit liver microsomes [11].
Optical absorption spectra Optical absorption spectra were measured with a Cary spectrophotometer, Model 219, equipped with thermostatically controlled cell holders at 25°C and a cuvette of 1 cm light path, using a neodymium glass filter as a standard of wavelength.
48
Assay of enzymatic activities Enzymatic activities were measured at 25 ° C by the following test system and methods unless otherwise stated. NADPH-cytochrome P-450 reductase activity was determined as NADPHcytochrome P-450-phenylisocyanide complex reductase activity [12]. The reductase activity was assayed by measuring the increment in absorbance at 455 nm. The reaction mixture contained 50 nM NADPH-cytochrome P-450 reductase, 1 pM cytochrome P-450, 0.1 mM phenylisocyanide and 2 pM superoxide dismutase in 0.1 M potassium phosphate buffer, pH 7.4. The reaction was started by adding 50 pM NADPH. The molar extinction coefficient of dithionite-reduced cytochrome P-450-phenylisocyanide complex minus dithionite-reduced cytochrome P-450 was estimated to be 71 • 103 M -1 • cm -1 from the absorbance between 455 and 490 nm, in 0.1 M potassium phosphate buffer, pH 7.4. 21-Hydroxylase activity of 17a-hydroxyprogesterone was assayed by the method of Porter and Silber [13]. The reaction mixture contained 2pM NADPH-cytochrome P-450 reductase, 0.01% Emulgen 913, 50 nM cytochrome P-450, 200 pg of 17a-hydroxyprogesterone, 1 mM MgC12, 0.5 unit of isocitrate dehydrogenase and 0.5 mM sodium isocitrate in 0.1 M potassium phosphate buffer, pH 7.4, in a total volume of 1 ml. The reaction was started by adding 0.5 mM NADP ÷ and performed at 37 ° C. NADPH-cytochrome c reductase activity was assayed by measuring the increment in absorbance at 550 nm of the a-band of the reduced cytochrome c. The reaction mixture contained 10 nM NADPH-cytochrome P-450 reductase and 50/aM ferricytochrome c in 0.3 M potassium phosphate buffer, pH 7.7. To initiate the reaction, 50 pM NADPH was added. When the NADPH-cytochrome c reductase activity of the microsomes was assayed, 0.5 mM KCN was added to the reaction mixture to prevent reoxidation of the ferrocytochrome c by cytochrome aa3 of contaminating mitochondria, which may have been present. The activity of NADPH-ferricyanide reductase was assayed by measuring the decrement in absorbance at 420 nm of ferricyanide. The reaction mixture contained 20 nM NADPH-cytochrome P-450 reductase and 0.6 mM ferricyanide in 0.3 M potassium phosphate buffer, pH 7.7. The reaction was started by adding 50 pM NADPH.
Electrophoresis Thin-layer SDS-polyacrylamide gel electrophoresis was performed by the method of Weber and Osborn [14], using 7.5% acrylamide. The molecular weight of NADPH-cytochrome P-450 reductase was estimated by SDS-polyacrylamide gel electrophoresis, using molecular weight markers of proteins such as RNA polymerase a-subunit (Mr -- 39 000), catalase (Mr = 60 000), bovine serum albumin (Mr -- 67 000), phosphorylase a of rabbit muscle (Mr = 95 000) and RNA polymerase/3-subunit (Mr = 155 000).
Analytical ultracentrifugation The molecular weight of NADPH-cytochrome P-450 reductase was also estimated by sedimentation equilibrium using interference optics, according to Yphantis' method [15]. A 0.5 ml sample of cytochrome P-450 reductase
49 (2.5 mg/ml in 0.1 M potassium phosphate buffer, pH 7.4) was incubated in 6 M guanidine HC1 and 0.1 M sodium iodoacetate at 80°C for 10 min. The sample was dialyzed against 100 ml o f 6 M guanidine HC1 for 30 h with three changes o f th e dialysis buffer. A 20 h period was allowed for equilibrium t o be reached at 20°C in a Hitachi analytical ultracentrifuge, Model 282, at a speed o f 10 000 rev./min. A blank pattern for t he cell was obtained using water for correction o f optical irregularities. Measurements o f fringe elevation and radial distance were d o ne with a Nikon com pa r at or .
Fluorescence measurement Fluorescence measurements were made in a Hitachi fluorescence spectrop h o t o m e t e r , Model MPF-2A, equipped with a thermostatically controlled cell holder using a Coolnics Model CTR-120 and circular Model CTE-120 K o mats u - Yamato Co. with slits o f 1.3 m m .
Circular dichroism spectra Circular dichroism (CD) spectra were measured with a Union Giken, Dichrograph Mark III-J, calibrated with D-camphor-10-sulfonic acid. T he measurements were made in fused silica cells: a 1.0 cm light path cell was used in the visible range and a 0.2 cm light path cell in the ultraviolet range. T he temperature o f th e solution was kept at 25°C.
Analytical procedures The molar ex t i nct i on coefficients o f NADPH and NADH were 6.30 • 103 M -1 • cm -1 at 340 nm (pH 7.6 and 25°C) [16] and those o f NADP ÷ and NAD ÷ were 1 8 . 0 . 1 0 3 M -1. cm -1 at 260 nm (pH 7.6 and 25°C) [17]. T he molar e x t i n ctio n coefficients o f c y t o c h r o m e c in the reduced minus oxidized forms and o f ferricyanide were 21 • 103 M -1. cm -1 at 550 nm [18] and 1 . 0 2 . 1 0 3 M -1 • cm -~ at 420 nm [ 19] , respectively. The c o n t e n t o f a d r e n o d o x i n was estim a t ed f r o m the molar ext i nct i on coefficient o f 9.8 • 103 M -~ • cm -t at 414 nm [10]. Th e c o n t e n t o f c y t o c h r o m e P-450 was det erm i ned from the CO difference spectrum o f t he dithionite-reduced f o rm , by assuming a value o f 91.0 • 103 M -1 • cm -1 as the molar e xt i nc t i on i nc rem ent o f the absorbance between 450 and 490 nm [20]. C y t o c h r o m e bs was det erm i ned, taking a value o f 163 • 103 M -~ • cm-' as the molar extinction coefficient between 424 and 409 nm in th e difference spectrum between t h e dithionite-reduced and t h e oxidized forms [21]. To calculate the r e duc t i on rates o f acceptors by NADPH-cytoc h r o m e P-450 reductase, the follo~ving e xt i n ct i on coefficients were used: 2,6d i c h l o r o p h e n o l i n d o p h e n o l , 21 • 103 M -~ • cm -1 at 600 nm [22] ; nitrobluetetrazolium, 28.6 • 103 M -1 • cm -1 at 550 nm [ 2 3 ] ; and neot et razol i um , 15.5 • 103 M -1 • cm -1 at 505 nm. The c o n t e n t o f hemoglobin was estimated from a CO difference spectrum using the molar e x t i n c t i o n coefficient o f 191 • 103 M -t • cm -1 at 419 nm [24]. Protein c o n t e n t was det erm i ned by the m e t h o d o f L o w r y et al. [25] and by the biuret reaction [26] using bovine serum albumin as a standard. Th e c o n c e n t r a t i o n o f t he albumin was determined using the e x t i n c t i o n coefficient, Elcm 1 at 280 nm, o f 6.6 [27].
Analyses o f compositions o f amino acids, sugars and metals Amino acid residues were analyzed by t he m e t h o d o f Moore and Stein [28]
50 in the absence and presence of 2% thioglycolic acid [29], using a Hitachi amino acid analyzer. Model KLA-3B. The contents of serinyl and threonyl residues were estimated exactly by the decomposition at various hydrolysis times for 24, 48 and 72 h. The number of sulfhydryl groups was determined by Ellman's m e t h o d [30] in the absence and presence of 8 M guanidine HC1. The amount of tryptophanyl residue present was also determined fluorometrically by the Pajot's method [31]. Neutral and amino sugars were separated b y the method of Haberman et al. [32] then measured b y the Sinohara's m e t h o d [33]. The sialic acid content was determined b y the Aminoff's m e t h o d [34]. The contents of iron, copper, manganese and m o l y b d e n u m were determined with a Hitachi atomic absorption spectrophotometer, Model 208. To determine and detect the metals in NADPH-cytochrome P-450 reductase, a 0.5 pM sample was used for atomic absorption spectrophotometry, which was sufficient to allow detection of even one atom of m o l y b d e n u m / m o l of the cytochrome P-450 reductase. Molybdenum was the most difficult metal to detect by the atomic absorption spectrophotometer.
Analyses of flavins FAD and FMN were obtained from Nakarai Chemical Co. FAD and FMN were purified by DEAE-cellulose chromatography [35]. Both were confirmed to produce a single spot on a paper chromatography with three different solvent systems (n-butanol/acetic acid/H20 (4 : 1 : 5), pyridine/H20 (2 : 1) and 5% Na2HPO4) and R F values o f the flavins in the various solvents [36]. The concentration of flavin solutions were determined spectrophotometrically using the extinction coefficients of 11.3 • 103 M -1 • cm -1 at 450 nm (pH 7.0) for FAD and 12.5 • 103 M -1 • cm -1 at 450 nm (pH 7.0) for FMN [37]. To release the flavin, NADPH-cytochrome P-450 reductase was digested with 0.2% (w/v) protease for 30 min for 37°C, then the solution was heated for 10 min at 80°C, rapidly cooled to 0°C with ice, and centrifuged at 9000 × g for 20 min to remove the heat~lenatured proteins [38]. The fluorescence of the supernatant was determined at 25°C and at both pH 7.7 and 2.6 as described b y Faeder and Siegel [39].
Kinetic analysis of NADPH-cytochrome P-450 reductase The NADPH-cytochrome P-450 reductase catalyzes the reaction according to the following equations, described with respect to the hepatic NADPH-cytochrome c reductase [40]. NAD(P)H + NADPH-cytochrome P-450 reductase (E) ~ EH2 + NAD(P) ÷
(1)
EH2 + 2 Ferricytochrome c (cyt. c 3÷) ~ E + 2 Ferrocytochrome c (cyt. c 2÷) + 2n÷
(2)
These reactions can be studied kinetically by measuring the increase in absorbance at 550 n m of the a-band of ferrocytochrome c. In the kinetic investigation of sequential binary reactions [41], the K m values of the NADPH-cytochrome P-450 reductase for N A D P H , N A D H and cytochrome e can be obtained Line-
51 weaver-Burk plots: 1Iv = 1 I V . [1 + KNAD(P)H/NAD(P)H + Kcyt.c3+/cyt. c 3÷]
(3)
where v is the initial velocity, V is the maximal velocity in the forward direction and KNAD(P)Hand Kcyt.c 3÷ are the Km values of the NADPH-cytochrome P-450 reductase for NADPH or NADH and ferricytochrome c, respectively. Chemicals NADPH, NADH and their oxidized forms were kind gifts from Oriental Yeast Co. Cytochrome c (type III, horse heart), 5,5'-dithiobis(2-nitrobenzoate), diethyl pyrocarbonate, phenylhydrazine hydrochloride and protease (type XIII, Bacillus subtilis) were purchased from Sigma Chemical Co. Sepharose 4B and DEAE-Sepharose CL-6B were purchased from Pharmacia Fine Chemicals. 2',5'-ADP-Sepharose 4B was synthesized by the method of Brodelius et al. [42]. Standard proteins for the estimation of molecular weights were purchased from Boehringer Mannheim GmbH. Emulgen 913 was a kind gift from Kao Atlas Co. Superoxide dismutase was purified from bovine erythrocytes by the method of McCord and Fridovich [43] and catalase was crystallized from bovine livers by Sirakawa's method [44]. Crystalline phenylisocyanide was synthesized by the method of Prager et al. [45]. All other reagents were of the highest grade available commercially. Results
General properties NADPH-cytochrome P-450 reductase was purified from bovine adrenocortical microsomes. A typical purification scheme is summarized in Table I. As shown in Table I, the ratio of activities toward cytochrome c and cytochrome P-450 does not vary significantly throughout the purification procedure. The yield of the NADPH-cytochrome P-450 reductase was 28%. It was confirmed to be a single protein band on SDS-polyacrylamide gel electrophoresis, thus being homogeneous and pure. Fig. 1 shows the electrophoretograms of NADPH-cytochrome P-450 reductases of the bovine adrenocortical and liver microsomes from SDS-polyacrylamide gel electrophoresis. The NADPH-cytochrome P-450 reductase, like liver NADPH-cytochrome P-450 reductase, could dirgctly reduce cytochrome P-450 and cytochrome c. These reductions were not due to superoxide or hydrogen peroxide, because superoxide dismutase and catalase did not influence the reducing rate of cytochrome P-450 by NADPH-cytochrome P-450 reductase. The specific activities of NADPH-cytochrome P-450 reductase and NADPH-cytochrome c reductase of our purified reductase preparation were 2.28 and 29.5 tlmol/min per mg protein at 25°C, respectively. Fig. 2 shows the optical absorption spectra of NADPH-cytochrome P-450 reductase of bovine adrenocortical microsomes in the oxidized and reduced forms. The absorption peaks of the cytochrome P-450 reductase were at 274, 380 and 455 nm with shoulders at 290, 360 and 480 nm in the oxidized form at 25°C. The absorption spectrum is that of a typical flavoprotein. The ratio of the intensity of the peak at 274 nm to that at 455 nm was 8.3, and that at
52 TABLE I P U R I F I C A T I O N OF N A D P H - C Y T O C H R O M E P-450 R E D U C T A S E FROM BOVINE A D R E N O C O R T I C AL MICROSOMES P r o t e i n c o n c e n t r a t i o n w a s d e t e r m i n e d b y the b i u r e t r e a c t i o n a n d t h e m e t h o d of L o w r y et al. [ 2 5 ] , a f t e r r e m o v a l o f t h e i n f l u e n c e o f nucleic acids. Specific a c t i v i t y o f c y t . c is t h e N A D P H - c y t o c h r o m e c r e d u c tase a c t i v i t y . This a c t i v i t y was a s s a y e d in t h e p r e s e n c e o f 0.5 m M KCN a n d 0.3 M p o t a s s i u m p h o s p h a t e b u f f e r , p H 7.7 ( 2 5 ° C ) . Specific a c t i v i t y of cyt. P - 4 5 0 is t h e N A D P H - c y t o c h r o m e P - 4 5 0 r e d u c t a s e a c t i v i t y . This a c t i v i t y was d e t e r m i n e d b y m e a s u r i n g t h e N A D P H - c y t o c h r o m e P - 4 5 0 - p h e n y l i s o c y a n i d e c o m p l e x r e d u c t a s e a c t i v i t y . T h e r a t i o of activities is e x p r e s s e d as N A D P H - c y t o c h r o m e c r e d u c t a s e / N A D P H - c y t o c h r o m e P - 4 5 0 r e d u c t a s e . P u r i f i c a t i o n a n d t o t a l a c t i v i t y are b a s e d o n t h e specific a c t i v i t y of N A D P H - c y t o c h r o m e c r e d u c t a s e . Yield is b a s e d o n t h e t o t a l a c t i v i t y of N A D P H - c y t o c h r o m e c r e d u c t a s e . Purification step
Microsomes Solubilized microsomes DEAE-cellulose c o l u m n eluate 2'5'-ADP Sepharose column eluate DEAE-Sepharose c o l u m n eluate
Volume (ml)
Protein concn, (mg/ml)
Total protein (mg)
500 495
12.1 7.6
6050 3762
38
5.4
205
Specific a c t i v i t y cyt. c
Ratio
cyt. P-450
0.12 0.19
---
2.6
Total activity (~tmol/ min)
Purification (-fold)
1 1,6
Yield (%)
---
726 715
100 98
0.24
10.8
533
22
73
4.6
3.3
15.2
26.8
2.10
12.8
407
223
56
4,5
1.5
6.8
29.5
2,28
12.9
201
246
28
Fig. 1. E l e c t r o p h o r e t o g r a m o f N A D P H - c y t o c h r o m e P - 4 5 0 r e d u c t a s e f r o m b o v i n e a d r e n o c o r t i c a l a n d h e p a t i c m i c r o s o m e s o n S D S - p o l y a c r y l a r n i d e gel. Samples of 1 0 ~g p r o t e i n o f t h e c y t o c h r o m e P - 4 5 0 r e d u c t a s e s w e r e u s e d o n t h e gel. A, A d r e n o c o r t i c a l N A D P H - c y t o c h r o m e P - 4 5 0 r e d u c t a s e ; H , h e p a t i c NADPH-cytochrome P-450 reductase.
53
0.8
0.8
0.4
0.6
0.2 v ¢ 0.4 o w
0.0
350
