Purification and properties of human placental NADPH-cytochrome P-450 reductase

NORIO MUTO' AND LIAT TAN^ Department of Biochemistry, University of Sherbrooke Medical Faculty, Sherbrooke (Qut . ) , Canada J1H 5N4

Received July 19, 1985

Muto, N. &Tan, L. (1986) Purification and properties of human placental NADPH-cytochrome P-450 reductase. Biochem. Cell Biol. 64, 184-193

Human placental NADPH-cytochrome P-450 reductase (EC 1.6.2.4) was purified to electrophoretic homogeneity in two chromatographic steps with a high retention of bioactivity. After solubilization with 1% sodium cholate in a protective medium containing 20% glycerol, 10 p M 4-androstene-3,17-dione, 1 mM dithiothreitol, and 0.2 mM EDTA, a 35-60% ammonium sulfate precipitate was prepared. The crude protein mixture was then applied to a 2',5'-ADP-Sepharose 4B affinity column, followed by high-performance anion-exchange chromatography (Pharmacia Mono-Q column). Two forms of the reductase were isolated. One was eluted at higher salt concentration and had a relative mass (M,) of 79 kdaltons (kDa) as estimated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and high-performance gel permeation chromatography. A smaller size reductase with a MI of 70 kDa, eluting at lower salt concentration, was also formed by trypsinolyis of the 79-kDa reductase. It must therefore be regarded as a proteolytic artifact. The absolute spectra in the visible region of the two reductases were identical with maxima at 376 and 452 nm, typical of a flavoprotein. They also had the same specific activity of 24.7 ? 0.7 kmol/min per milligram protein towards cytochrome c. However, only the 79-kDa reductase showed aromatase-reconstitution activity. The homogeneity of these reductases was further confirmed by the appearance of a single peak when subjected to gradient, reversed-phase high-performance liquid chromatography. According to its amino acid composition, the 79-kDa reductase is a highly acidic and hydrophobic protein, composed of 695 residues.

Muto, N. &Tan, L. (1986) Purification and properties of human placental NADPH-cytochrome P-450 reductase. Biochem. Cell Biol. 64, 184-193

Deux ktapes chromatographiques nous ont permis de purifier la NADPH-cytochrome P-450 rauctase (EC 1.6.2.4) placentaire humaine jusqu'i homogknkitC tlectrophorttique avec rttention Clevte de la bioactivitk. Apr2.s solubilisation avec le cholate de sodium 1% dans un milieu protecteur contenant du glyctrol20%, du 4-androstkne-3,17-dione 10 pM, du dithiothrkitol 1 mM et de I'EDTA 0,2 mM, nous avons prCpark un prtcipitk avec le sulfate d'arnmonium 35-60%. Nous avons alors plack le mklange prottique brut dans une colome d'affinitk contenant le 2',5'-ADP-Sepharose 4B puis nous avons effectuk une chromatographie tchangeuse d'anions i haute performance (colonne Pharmacia Mono-Q). Nous avons is016 deux formes de rtductase. L'une est kluke a concentration saline klevte et nous avons CvaluC sa masse relative (MI) 1 79 kdaltons (kDa) par Clectrophor&se sur gel de polyacrylamide avec dodtcyl sulfate de sodium et chromatographie de permtation sur gel a haute performance. Une rtductase plus petite (MI de 70kDa) eluant B une concentration saline plus faible est aussi formCe par trypsinolyse de la rkductase de 79 kDa. I1 faut donc la considkrer comrne un artefact proeolytique. Dans la rkgion visible, les spectres absolus des deux rkductases sont identiques avec des maxima a 376 et 452 nm, ce qui est typique d'une flavoprotkine. Elles ont aussi la meme activitk spkcifique de 24,7 2 0,7 pmol/min par milligamme de prottine B 1'Cgard du cytochrome c. Cependant, seule la rkductase de 79 kDa montre une activite de reconstitution de la reaction d'aromatisation. L'homogtnkitk de ces rkductases est confirrnte aussi par I'apparition d'un seul pic lorsqu'elles sont soumises a la chromatographie liquide i haute performance en phase inverske avec gradient. D'aprks sa composition en acides aminks, la rtductase de 79 kDa est une protCine fortement acide et hydrophobe et elle comporte 695 rksidus.

[Traduit par la revue]

Introduction In a previous paper (I), we reported the action of the

epime& 6-hydioperoxy-4-androstene-3, 17-diones as

ABBREVIATIONS: MI, relative mass; kDa, kilodaltons; HPLC, high-performance liquid chromatography; SDS- PAGE, sodium dodecyl sulfate - polyacrylamide gel elec- trophoresis; CHAPS, 3-((3-cholamidopropy1)dimethylammo- nio)-1-propanesulfonate; D m , dithiothreitol; HPGPC, high- performance gel-permeation chromatography; HPAEC, high- performance anion-exchange chromatography; KPB, potas- sium phosphate buffer; UV, ultraviolet; TFA, trifluoroacetic acid.

'Present address: Faculty of Pharmaceutical Science, Josai University, Sakado, Saitama, Japan

'~u thor to whom all correspondence should be addressed.

competitive inhibitors and (or) substrates of human placental aromatase. This particulate enzyme system consists of two components: a flavoprotein and a cytochrome P-450 type hemoprotein. The aromatization reaction itself (i.e., the conversion of the a,p-con- jugated ring-A ketone of the androgens into the ring-A phenol of the estrogens) has been shown to proceed via a three-step hydroxylation sequence (2-8). To enable us to measure the accurate binding and inhibition constants of these hydroperoxysteroids and to understand the events that take place at the active site, the aromatase enzyme complex had to be purified. In turn, the progress of the placental cytochrome P-450 purification must be monitored by measuring its aromatase activity upon reconstitution with its reductase component, which thus

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MUTO AND TAN 185

must first be separated and purified. Whereas a relatively large number of papers has been published on the NADPH-cytochrome P-450 reductase from other ani- mal sources (cf. review by Masters and Okita (9)), we are aware of only one publication that has dealt specifically with the aromatase flavoprotein component from the human placenta (10). A major obstacle that had t o be overcome was the problem of how to obtain a sufficient quantity of the pure reductase which is present in the human placenta in only minute amounts, namely about 10% of those contained in rat liver. Therefore. a rapid and highly efficient purification procedure was called for, preferably making use of contemporary techniques of HPLC. These efforts and several proper- ties o f t h e cytochrome P-450 reductase that we have isolated from the human placenta are described in this paper.

Materials and methods Chemicals

Inorganic salts for buffer solutions were of reagent grade. NADP', glucose 6-phosphate, glucose-6-phosphate dehy- drogenase, EDTA, sodium cholate, equine heart cytochrome c, SDS-PAGE molecular weight protein kit, and CHAPS were all from Sigma Chemical Co. DTT was purchased from Aldrich Chemical Co. Nonlabeled 4-androstene-3,17-dione was from Steraloids, Inc., while [1~,2~-3~(~)]androstene- dione (3pecific activity, 41 Ci/mmol; 1 Ci = 37 GBq) was obtained from New England Nuclear (Dupont). Emulgen 913 was provided by Kao-Atlas Company Ltd., Tokyo. Dye reagent for protein determinations was from Bio-Rad.

Chromatographic materials 2',5'-ADP-Sepharose 4B and the Mono-Q HR 515 anion-

exchange column were from Pharmacia Fine Chemicals. The gel permeation column (TSK-3000 SW) was supplied by Varian Canada.

HPGPC chromatography The 0.75 X 30 cm column was maintained at 1O"C using an

external mantle and connected to a LDC duplex minipump, a Rheodyne injection valve, and an Altex single wavelength detector set at 280 nm. After equilibration with 0.1 M sodium sulfate- 0.02 M sodium dihydrogen phosphate buffer (pH 6.8) containing 0.5% sodium cholate, the proteins were eluted isocratically at a flow rate of 0.45 mL/min.

HPAEC A Varian model 5020 instrument was used. It was equipped

with a Valco injection valve, a Hitachi-Beckman variable wavelength detector and a Chromatronix single wavelength detector connected in tandem, a Kipp and Zonen double channel recorder, and a Pharmacia Frac- 100 fraction collector. Elution of the reductase from the Mono-Q column was carried out at room temperature.

Preparation of microsomes This was carried out at 4OC. Fresh human term-placentae

were within 30 min after birth dissected free from cords and adhering membranes. The cotyledon was severed from the chorionic plate, cut into small cubes, and rinsed several times

with 1% KC1 solution until the wash was no longer stained with blood. The cubes were kept immersed in a protective homoge- nizing buffer solution consisting of 250 mM sucrose in 50 mM Tris-HC1 (pH 7.4), 1 mM EDTA, and 20% glycerol (v/v). The washed cubes were then cut into smaller pieces, care being taken to keep them immersed at all times, prior to their homogenization in a Brinkman Polytron apparatus (10-s bursts at maximum energy for 10 times, with a 2-min cooling period between each burst). The homogenate was centrifuged at 10000 x g for 20min, the pellet was discarded, and the supernatant was pelleted at 105 000 X g for 60 min. After removing the supernatant, the microsomal pellet was rehomo- genized in 67mM potassium phosphate buffer (pH 7.4) containing 1 mM DTT and repelleted under the same condi- tions. These microsomes were suspended in preservation buffer containing 50 mM potassium phosphate (pH 7.4), 1 mM DTT, 1 mM EDTA and 20% glycerol, distributed in Eppen- dorf 1.5-mL centrifuge tubes, and stored at -80°C until use. Prepared in this manner, the microsomes retained aromatase activity after more than 9 months of storage.

Measurement of aromatase activity This assay was based on the stereospecific tritium-isotope

transfer of [1p,2P-~H]androstenedione to tritiated water ( 9 , as simplified by us (1). The aqueous samples were mixed with Aquasol in polypropylene minivials and counted in a Beckman model 8000 iquid scintillation spectrometer.

Assay for NADPH-cytochrome P 4 5 0 reductase Enzyme activity was determined using cytochrome c as

substrate (11) by a slightly modified procedure. Briefly, the assay mixture consisted of 0.1 M potassium phosphate buffer (pH 7 3 , 1 mM KCN, 40 p M equine heart cytochrome c, and 0.2 mM NADPH, freshly generated. The reaction was carried out at 22OC and monitored at 550 nm using a Beckman model 24 spectrophotometer. Reductase activities were calculated from the initial velocity rates and expressed as the amount of enzyme that catalyses the reduction of 1 pmol/min of cyto- chrome c. The protein content of these fractions was deter- mined with the Bio-Rad dye-binding assay (12).

SDS-PAGE Electrophoresis was carried out with a Gelman apparatus

and 0.5 x 7.5 cm glass tubes (13). Each sample was maintained for 5 min at 100°C in 10 mM sodium phosphate buffer containing 25% glycerol, 1% SDS, and 5% 2-mercap- toethanol. To each tube was then added 5 pL of 0.05% bromphenol blue. The gel concentration was 7.5%. Samples (50 pL) were layered onto the gel and electrophoresed for 3 h at a constant current of 7mAltube. Protein bands were visualized by staining for 2 h with a 0.025% solution of Coomassie brilliant blue R 250 in 50% methanol - 10% acetic acid, followed by destaining overnight in an aqueous solution of 5% methanol - 7.5% acetic acid.

Purification of human plancental NADPH-cytochrome P-450 reductase

All manipulations, with the exception of HPAEC at the final stage of purification, were conducted in the cold room.

Solubilization and ammonium sulfate fractionation Frozen microsomes (about 1 g of protein) were suspended in

50 mM KPB (pH 7.5) containing 20% (v/v) glycerol, 1 mM

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186 BIOCHEM. CELL BIOL. VOL. 64, 1986

DTT, 0.2 mM EDTA, and 10 p M androstenedione (solubil- ization buffer) to give a concentration of 10-15 mg pro- tein/mL. A 10% (w/v) solution of sodium cholate in the same buffer was added to give a final concentration of 1 % detergent. After 30 min of stirring, the solubilized mixture was centri- fuged at 105 000 x g for 60 min. To the supernatant was added ammonium sulfate to obtain 35% saturation and the solution was stirred for 30min. The precipitate was removed by centrifugation at 12 000 X g for 15 min. It was devoid of reductase and aromatase activity and was therefore discarded. The supernatant was brought to 60% saturation by further addition of ammonium sulfate and stirred for another 20 min. After centrifugation at 12 000 X g for 15 min, this 35-60% ammonium sulfate fraction was dissolved in the solubilization buffer containing 0.5% sodium cholate. It was dialyzed overnight against the same buffer.

AfJinity chromatography The dialyzed solution was applied to a 2',5'-ADP-Sepharose

4B (1.5 x 10 cm) column which was previously equilibrated with 20mM KPB (pH 7.5) containing 20% glycerol, 1 mM DTT, 0.2 mM EDTA, and 0.5% sodium cholate. The column was then thoroughly washed with 100 mM KPB containing the same ingredients as above. Elution was started by pumping through the column 20 rnM KPB (pH 7.5) containing 20% glycerol, 0.1 mM D'IT, 0.2 mM EDTA, 0.1% Emulgen 913, and 2 mM 2'-AMP. The reductase-active fractions (40 mL) were pooled and dialyzed against 20 mM Tris-acetate buffer (pH 7.5) containing 20% glycerol, 0.1 mM DTT, 0.2 mM EDTA, and 0.05% Emulgen 9 13. The dialyzed solution was then concentrated using Amicon Centricon-30 membrane tubes by centrifugation for 20 min at 4000 X g, yielding about 4 mL of concentrate.

HPAEC HPAEC was carried out at room temperature. The reduc-

tase-active concentrate (3.6 mL) was then loaded on a Phar- macia Mono-Q anion-exchange column which was previously equilibrated with 20 mM Tris-acetate buffer (pH 7.5) contain- ing 20% glycerol, 0.2 mM EDTA, and 0.5% CHAPS. The proteins were eluted by applying a salt gradient (see Figure legends). The reductase-active fractions were collected, con- centrated, and subjected to a second HPAEC using the same conditions as above.

Amino acid analysis The purified reductases were dissolved in 0.2 mL of 6 M,

constant boiling hydrochloric acid containing 2% thioglycolic acid (14). The hydrolysis tube was then evacuated, sealed, and heated at 1lO"C for 24 h. Analyses were performed using a Beckman model 119CL instrument.

Results Solubilization

The concentration of NADPH-cytochrome P-450 re- ductase that is present in the human placenta is very low. We found a reductase specific activity of 0.0117 2 0.0015 pmol/min per milligram protein ( n = 6) for preparations that had been stored at -80°C for more than 6 months. Fresh preparations had a specific activity of 0.031. This is still much lower than the values

TABLE 1. Comparison of the efficiency of sodium cholate and CHAPS as solubilizers of human placental NADPH-cyto-

chrome P-450 reductasea -

Specific activityb Preparation (kmol/min per milligram)

Nonsolubilized microsomes 0.01 1720.0015 1 % CHAPS supernatant 0.040020.0108 1 % Sodium cholate supernatant 0 .0349~0.0120

"Microsomal preparations stored for more than 6 months at -80°C were used for these experiments.

bValues are the means ? SE of three determinations.

reported h r n other microsoma1 sources such as normal and phenobarbital-treated rat liver that have reductase specific activities of 0.1 18 and 0.46 prnol/min per milligram protein, respectively (15, 16). Our objective was to develop a rapid purification procedure, yet entailing a minimal loss of activity. To achieve this goal, we had to make sure that the very first step of the purification procedure, i.e., the solubilization of the membrane-bound enzyme, be carried out under optimal conditions. Reasoning that binding of the natural sub- strate to the aromatase enzyme system should help in stabilizing the native conformation of the enzyme, we added androstenedione during the solubilization pro- cess. To select the most suitable detergent, we have compared the relative solubilizing capacity of two chemically related surfactants, i.e., CHAPS (17) and cholic acid (see Table 1). At the same concentration of 1%, CHAPS enhanced the specific activity of the crude reductase 3.4 1 -fold and cholate enhanced it 2.98-fold. Although CHAPS is clearly the more efficient solubiliz- ing agent, because of its high price, we nevertheless chose sodium cholate to carry out this step of the purification procedure.

Ammonium sulfate fictionation The highest activity of the reductase was found

between 50 and 60% saturation. However, the 35-50% precipitate also showed some reductase activity, al- though the major protein component of this fraction consisted of cytochrome P-450. In order not to lose any reductase, we decided to pool these two fractions. In this way over 70% of the reductase and mare than 50% of cytmhrorne P-450 were recovered at this carly stage of the purification procedure. To prevent loss of enzyme activity, the pH of the buffer medium had to be carefulIy maintained between 7.0 and 7.5 during the fraaionation by addition of dilute ammonia.

Afinity chromatography Rather than starting the actual purification procedure

from a solution of solubilized microsomes in which the reductase concentration is very low (lo), we have

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MUTO AND TAN 187

TUBE NUMBER

FIG. 1. Affinity chromatography of the 35-60% ammo- nium sulfate precipitate. In the cold room, the crude reductase- active fraction obtained by ammonium sulfate fractionation was applied to a 1.5 X 10 cm, 2',5'-ADP-Sepharose 4B column, equilibrated with 20 mM KPB (pH 7.5) containing 20% glycerol, 1 mM DTT, 0.2 mM EDTA, and 0.5% sodium cholate. After sampling, the column was eluted successively with the following: (a) 100 mM KPB containing the same components, (b) as in a but with 20 mM KPB and addition of 2 mM 2'-AMP, (c) as in b but sodium cholate substituted by 0.1 % Emulgen 913. The flow rate was 15 rnL/h.

applied the concentrated, 35-60% ammonium sulfate dialyzate on a short 2'3'-ADP-Sepharose column. When we tried to displace the bound reductase from the column with 2 m M 2'-AMP in the presence of up to 0.5% sodium cholate or CHAPS, only a small amount of reductase activity was found in the fractions collected. It should be noted that higher concentrations of detergent could not be used as this was found to be detrimental for preserving enzyme activity. However, elution of the reductase proceeded smoothly with over 90% recovery when 0.1 % Emulgen 9 13 was used as the detergent. As shown in Fig. 1, we obtained two reductase-active peaks. A minor peak was collected between fractions 35 and 42, while the major peak eluted between fractions 44 and 64. The minor peak was obtained in insufficient quantities to warrant subsequent purification and was not further examined. At this purification stage, the major peak had a specific activity of 7.14 p,mol/min per milligram protein (determined at 22°C in 0.1 M potas- sium phosphate buffer, pH 7.5).

TIME (min)

FIG. 2. HPAEC of NADPH-cytochrome P-450 reductase. The major peak of Fig. 1 was, after concentration and dialysis, directly injected into a Pharmacia Mono-Q column equili- brated with 20mM Tris buffer (pH 7.5), containing 20% glycerol, 0.2mM EDTA, and 0.6% CHAPS. Elution was started at 22OC by application of a linear sodium chloride gradient in the same buffer, as indicated. The flow rate was 0.3 ml/min and 1.5-min fractions were collected. Fractions 1 and 2 corresponding respectively to reductases I and I1 were pooled and concentrated, and each reductase was then rechromato- graphed under the same conditions.

HPAEC We have selected the Pharmacia Mono-Q anion-

exchange column not only for its high degree of monodispersity and loading capacity, but also because the all-glass column enabled us to follow visually the elution of any colored material. Moreover, with our viscous solvent system that must contain 20% glycerol at all times to protect the enzyme, the short 5-cm-long Mono-Q column generates at a flow rate of 0.5 mL/min a back pressure of only 34-38 atm (1 atm = 101.325 kPa), thus greatly diminishing any possible change of protein conformation due to mechanical compression. The major peak collected earlier by affinity chromatog- raphy was resolved into two reductase-active peaks by this anion-exchange column. The first peak (reductase I) eluted at a 0.53 M NaCl concentration (89% solvent B), while the second peak (reductase 11) eluted at 0.57 M NaCl(95% solvent B), as shown in Fig. 2. Reductases I and I1 showed practically the same specific activity towards cytochrome c (i.e., 24.4 and 23.9 p,mol/min

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188 BIOCHEM. CELL BIOL. VOL. 64, 1986

TABLE 2. Scheme of NADPH-cytochrome P-450 reductase purification from human placental microsomes

Protein Activitya Specific activity Recovery Purification Step (mg) (@mol/min) (kmol/rnin per milligram) (%) (n-fold)

Crude rnicrosomes 884 27.4 0.031 100 1 Cholate supernatant 437 25.8 0.059 94 1.9 35-60% Ammonium sulfate precipitate 233 19.4 0.083 7 1 2.7 Affinity chromatography 2.69 19.2 7.14 70 230 Concentration, dialysis 2.47 14.0 5.67 51 183 First HPAEC reductase I 0.163 3.98 24.4 14.5 787 First HPAEC reductase I1 0.294 7.02 23.9 25.6 77 1 Second HPAEC reductase I 0.139 3.50 25.2 12.8 813 Second HPAEC reductase I1 0.201 5.13 25.5b 18.7 823 -- - - -

"Reductase activity was assayed at 22°C in 0.1 M potassium phosphate buffer (pH 7.5). b31.8 pmol/min milligram protein when assayed in 0.3 M KPB at 22°C.

per milligram protein, respectively). After a second chromatography on the Mono-Q column carried out under indentical conditions, reductases I and I1 were recovered retaining, respectively, 88 and 73% of en- zyme activity (cf. Table 2). The reproducibility of these high-performance chromatographic purifications was excellent. Reductases I and I1 were again eluted at the same NaCl concentrations of 0.53 and 0.57 M, re- spectively.

Properties of human placental reductases I and 11 The molecular mass of the two reductases as esti-

mated by the method of Weber and Osborn gave a value of 70 kDa for reductase I and 79 kDa for reductase 11, as shown in Fig. 3. The molecular mass of reductase II was also estimated by HPGPC on a TSK-3000SW column in the presence of myoglobin, ovalbumin, y-globulin, and thyroglobulin as protein standards. When no detergent was present, reductase activity was only found in the void volume, meaning that under these conditions aggregation had occurred. Aggregation can be pre- vented to some extent by adding 0.5% sodium cholate in the eluent. In this case, a minor and major reductase- active fraction of molecular masses in the order of 165 (dimer) and 76kDa (monomer), respectively, were obtained. Whereas reductases I and I1 showed no difference in their action towards cytochrome c , only reductase I1 had a high capacity to restore full aromatase activity upon recombination with a partially purified, aromatase-active, and cytochrome P-450 containing fraction from human placental rnicrosomes. Reductase I showed merely a trace of aromatase-reconstitution activity, probably owing to slight contamination with reductase I1 (see Table 3). The homogeneous state of the two reductases, after being subjected twice to purifica- tion by the Mono-Q anion-exchange column, was verified by SDS-PAGE. Both reductases I and I1 gave a single band (Fig. 4). As shown in Fig. 5, the absolute absorption spectra of the two reductases differ only in

lo' I I I 0.2 0.4 0.6 Rf

R f

FIG. 3. Molecular mass estimation by SDS-PAGE of reductases I and 11, purified twice by HPAEC. (a) P-Galac- tosidase; (b) phosphorylase b; ( c ) reductase 11; (d) reductase I; ( e ) bovine serum albumin; (f) ovalbumin; (g) carbonic anhydrase.

their extinction coefficients. In the visible region, both reductases have characteristic broad band maximums at 452 (with a shoulder at 476 nm) and 378 nm, typical of flavoproteins, while there is a high-intensity band at 270 nm in the UV region of the spectrum.

Membrane-bound proteins are notoriously very diffi- cult to obtain in their nonaggregated state by means of high-resolution liquid chromatographic techniques that rely on hydrophobic interactions. This problem is even more compounded when dealing with enzymes that must retain their native conformation. In this respect, the behaviour of the two purified placental reductases that we have subjected to reversed-phase HPLC, in the presence of 0.1% TFA and a 2: 1 mixture of acetonitrile and 2-propanol as organic modifying agents, is reveal- ing. By applying a linear gradient at an increase of 1 % solvent B/rnin and a flow rate of l.OmL/min, both reductases I and I1 were eluted as single peaks at 30.6

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MUTO AND TAN 189

TABLE 3. Arornatase reconstitution activity of homogeneous reductases I and Il

Aromatase activity

pmol/min per 0.5 mL nmol/min Assay components reaction mixture per nanomole P-450

Reductase I + P-450 + NADPH 0.281 0.012 Reductase II + P-450 + NADPH 36.2 1.554 Reductase I1 + NADPH NDa - P-450 + NADPH ND - Reductase I1 + P-450 ND - NOTE: The complete reconstitution system for measuring aromatase activity consisted of 0.023 nmol of

partially purified, human placental cytochrome P-450 (specific content, 1.02nmol/mg protein), 0.05U of purified reductase I or 11, 5 ~ m o l of glucose 6-phosphate. 2 U of glucose-6-phosphate dehydrogenase, 0.5 wmol of NADP', and about 40000cpm of [lp,2P-3H]androstenedione in a total volume of 0.5 mL of 50mM potassium phosphate buffer (pH 7.5). After lOmin of incubation at 37°C and removal of excess substrate by active charcoal, the radioactivity present as tritiated water was counted.

WD, not detectable.

In addition, a spectroscopic scan of the reductase I1 fraction taken after undergoing reversed-phase HPLC indicated a bathochromic shift of 8-14nm of its

I original, 270-nm high-intensity absorption peak (figure 7 1 not shown). This spectral shift could be due to protein

unfolding caused by the denaturing effect of the organic - modifier, exposing the active site to the acid medium, - ' and entailing the loss of one or both of the prosthetic

--w groups (i.e., FMN and FAD) from the reductase I1 molecule. These coenzymes appear not to be too tightly

* 9 bound, as under much less drastic conditions (simply by dilution with buffer at pH 7.5) a similar dissociation of the prosthetic group has been observed with yeast NADPH-cytochrome P-450 reductase (1 8).

I Y Although reductase I1 is chemically stable at pH 2.2, B b c d we conclude that it cannot be isolated in its active

conformation by reversed-phase HPLC. We can also

FIG. 4 . SDS-PAGE of purified reductases from human placental microsomes. The proteins were treated with 1% SDS and 5% 2-mercaptaethanol at 100°C for 5 mio and subjected to electrophoresis on a 7.5% polyacrylmide gel. GeIs were stained with Coomassie blue R 250. (a) 2.5 pg ductase I: ( h ) 2.5 pg reductare II; ( c ) a mixture of 2.5 pg each of reductase 1 and 11; (4 a mixture of P-galactosidase, phosphorylase b, albumin, ovalbumin, and carbonic anhydrase with Ma of 1 16, 97.4,66, 45. and 29, kDa, respectively.

(63% solvent B) and 36.1 min (68% solvent B), respec- tively. Upon injection of a mixture of both proteins, two peaks with identical retention times corresponding to those of reductases I and II were obtained (Fig. 6). At first glance, it seemed as if the two reductases could be purified by reversed-phase HPLC. However, when the collected fractions were assayed, neither reductase turned out to possess any significant activity towards cytochrome c.

state that reductases I and i1 are hydrophobic proteins since, already with the diphenylsilyl column of low hydrophobicity, a high concentration of the polar mixture of organic modifier was necessary for their elution. In fact, both reductases I and I1 could not be stripped off a C18 protein column (Vydac 21 8TP), even at very high concentrations of acetonitrile.

The results of these HPLC analyses clearly indicate that reductase I1 does not undergo acid hydrolysis at low pH to yield reductase I. On the other hand, our findings (a) that reductase I is already present at an early stage of the purification process, namely after affinity chroma- tography and perhaps even earlier during the solubiliza- tion step, and (b ) that it is devoid of any significant aromatase reconstitution activity support the hypothesis that reductase I is a product of enzymic proteolysis. We believe, therefore, that reductase I1 is the real comple- ment of the microsomal cytochrome P-450, forming with it the complete aromatase enzyme complex of the

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BIOCHEM. CELL BIOL. VOL. 64. 1986

WAVELENGTH ( n m )

FIG. 5. Absolute spectra of reductases I and II. After the second, final purification on the Mono-Q column, fractions corresponding to reductases I (173 pg/mL) and I1 (70 pg/rnL) were scanned at 22OC using a Cary 210 spectrophotometer, operated in the automatic base-line correction mode. The sample and reference cuvettes contained the same mobile phase. The following maxima in nanometres and extinction coefficients, in parentheses, expressed as per millimolar concentration per centimetre were obtained. Reductase I: 270 (186.2), 378 (14.3), and 452 (15.1). Reductase 11: 270 (302.6), 378 (13.5), and 452 (13.2).

human placenta. The presence in the liver of phenobar- bital-treated rats and likewise in rabbit liver of two forms of NADPH-cytochrome P-450 reductase, of which the lower molecular weight form is viewed as a proteolysis product of the higher molecular weight enzyme, has been discussed by other workers also (15, 19-21). However, we would like to point out that, apart from Bellino's paper (lo), there has been no other report in the literature on the presence in human placental microsomes of two distinct forms of NADPH-cyto- chrome P-450 reductase. Experimental evidence sup-

TABLE 4. Comparison of the amino acid com- positions of purified human placental reductase I1 and the higher molecular weight reductase

from rat liver

Rat Amino acid Reductase I1 liver reductasea

Asx 72 71 Thr 37 38 Ser 50 43 Glx 88 87 Pro 25 32 G ~ Y 53 44 Ala 53 50 Val 42 45 Met 17 19 Ile 23 24 Leu 63 58 T Y ~ 30 33 Phe 28 26 His 2 1 19 L Y ~ 39 33 '4% 38 37 TT 9 6 CYS 7 6 Total 695 67 1

"Data taken from Ref. 21.

porting the suggestion that the lower molecular weight reductase I is a proteolysis-derived product from reduc- tase I1 is shown in Fig. 7B. Treatment of reductase I1 with trypsin gave within 9 min rise to a 70-kDa protein band on SDS-PAGE. After 120min, its intensity equalled that of the reductase I1 band. Concurrently, the original reductase 11, while retaining full reductase activity, lost aromatase-reconstitution activity during the course of this proteolytic treatment with trypsin (Fig. 7A).

Discussion The work presented in this paper differs in several

important aspects from Bellino's, First, our claim of the homogeneity of the two reductases that we have isolated is based not jusr upon SDS-PAGE analysis, but also upon the elution of a single peak for each reductase by reversed-phase HPLC. The latter criterion is more stringent because of the high degree of resolution that is inherent with this chromatographic technique. In fact, the Whatman Protesil-diphenyl column used had an efficiency of 14400 theoretical plates. Second, we found an appreciable difference in molecular mass of about 6kDa for the aromatase-active form of the reductase, namely 73 (Bellino's) versus 79 kDa (ours). This may be due to the fact that we have used fast HPAEC as the final purification step rather than the

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RETENTION TIME [min)

FIG. 6. Analysis by reversed-phase HPLC. Human placental reductases I and I1 were, after purification to homogeneity by HPAEC, injected directly into a Whatman-Protesil300 - diphenylsilylsilica (0.4 X 25 cm) column. To prevent aggregation, no guard column was installed. Solvent reservoir A consisted of 15% acetonitrile and 0.1% TFA; reservoir B was a 2: 1 mixture of 95% acetonitrile - 2-propanol and 0.1% TFA. Three minutes after the injection, a linear gradient from 0 to 40% solvent B for 5 min was started, followed by an increase of 1% solvent B/min for the next 35 min. The flow rate was 1.0 mL/min and the column temperature was 24°C. Retention times and percent solvent B (in parentheses) are indicated.

slower size-exclusion chromatography. It has enabled us to obtain the pure reductase within 40 min after injection of the sample by effectively minimizing any possible conformational change and proteolytic frag- mentation. This is reflected in the high recovery of reductase activity. Finally, we have achieved in three steps over a 800-fold purification of reductase I1 with an overall yield of between 18.7 and 25.6% (see Table 2).

According to its amino acid composition (Table 4), reductase I1 is a highly acidic and hydrophobic protein. In fact, 23% of the enzyme consists of Asx and Glx. Excluding Glu, the total amount of hydrophobic resi-

dues (Pro, Ala, Met, Ile, Leu, Phe, and Trp) accounts for 31.7% of the entire molecule. If Glu is included in the calculation, its hydrophobic content rises to 44%. This high degree of hydrophobicity explains why reductase I1 was irreversibly retained by the Vydac-C18 column in the presence of TFA as a counter anion: in es- sence, under reversed-phase HPLC conditions whereby the protein is fully protonated and hydrophobic interac- tions with the support are maximized.

Human placental reductase I1 appears to be slightly larger than its detergent-solubilized counterpart from rat liver (21), which has 671 residues and a M, of 76509

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BIOCHEM. CELL BIOL. VOL. 64. 1986

lncubat,ion(min) M r

REDUCTASE Stds ' 0 8 30 60 120' Stds ( kDa)

Incubation (h) FIG. 7. Conversion of human placental reductase I1 to reductase I by enzymic proteolysis. Reductase II (specific activity,

12.8 pmol/min per milligram; 123.6 pg/mL) was dialyzed against 10 rnM KPB (pH 7.5) containing 10% glycerol and 0.1 mM EDTA. To the dialyzate was added trypsin (bovine pancreas, Boehringer Mannheim) at a substrate:enzyme ratio of 30:l. The digest was performed at 4OC. At the indicated time intervals, aliquots were withdrawn and transferred to Eppendorf tubes containing a solution of trypsin inhibitor (0.2 mg/mL). (A) For measurement of reductase activity, 10 pL of the treated reaction medium was used, while for the aromatase assay 50 pL was mixed with 0.023 nmol placental cytochrome P-450 (specific content, 1.02nrnol/mg). (B) Results of SDS-PAGE analysis. At times 0, 8, 30, 60,'and 120min, 2.5-pL samples were withdrawn and applied to a 1.5-rnrn-thick, 7.5% acrylamide slab gel. A mixture of the same protein standards (Stds) used in Figs. 3 and 4 was applied in the extreme left and right lanes. Bands were stained with Coomassie blue R 250.

Da. We have obtained a total of 695 residues, account- ing for a M, of 78 500 Da. Also, the amino acid composition of human placental and rat liver reductase show significant differences in their contents of Ser, Pro, Gly, Leu, and Lys. To our knowledge, the amino acid composition of neither the aromatase-active nor the inactive form of human placental NADPH-cyto- chrome P-450 reductase has been reported before in the literature. As demonstrated in this work, only the higher molecular weight reductase 11 exhibits aromatase-recon- stitution activity. The 70-kDa reductase I is probably a proteolytic artifact with no apparent biological function, although it is active towards cytochrome c.

The purification of membrane-bound proteins by high-performance ion-exchange chromctography using supports with wide pores of 300 A (1 A = 0.1 nm) or larger has found more and more adherents in recent years (22-28). As shown in this work, fast HPAEC with a column of only 5 cm in length can indeed be applied with success for the efficient purification of a membrane-

bound hydrophobic, and acidic enzyme with high recovery of enzyme activity. Reversed-phase HPLC, on the other hand, while inactivating the enzyme, can and should be used as an additional criterion of verifying its chemical purity.

Acknowledgements We thank the Medical Research Council of Canada

for financial support and Josai University for awarding a leave of absence to N.M.

1. Tan, L., Hrycay, E. G. & Matsumoto, K. (1983) J. Steroid Biochem. 19, 1329-1338

2. Ryan, K. J. (1959) J. Biol. Chem. 234,268-272 3. Engel, L. L. (1973) in Handbook of Physiology (Greep,

R. O . , ed.), sect. 7, pp. 467-483, The American Physiological Society, Bethesda, MD

4. Braselton, W. E., Jr., Engel, L. L. & Om, J. C. (1974) Eur. J. Biochem. 48, 35-43

5. Thompson, E. A., Jr. & Siiteri, P. K. (1974) J. Biol. Chem. 249,5364-5372

Bio

chem

. Cel

l Bio

l. D

ownl

oade

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ww

.nrc

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s.co

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20/1

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MUTO AND TAN 193

6. Kelley, W. G., Judd, D. & Stolee, A. (1977) Biochemis- try 16, 140-145

7. Fishman, J. & Goto, J. (1981) J. Biol. Chem. 256, 4466-447 1

8. Akhtar, M., Calder, M. R., Corina, D. L. &Wright, J. N. (1981) J. Chem. Soc. Chem. Commun., 129-130

9. Masters, B. S. S. & Okita, R. T. (1982) in Hepatic Cytochrome P-450 Monoonygenase System (Schenkman, J . B. & Kupfer, D., eds.), pp. 343-360, Pergamon Press, Oxford

10. Bellino, F. L. (1982) J. Steroid Biochem. 17, 261-270 11. Yoshinaga, T., Sassa, S. & Kappas, A. (1982) J. Biol.

Chem. 257, 7778-7785 12. Bradford, M. (1976) Anal. Biochem. 72, 248-254 13. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244,

4406-4412 14. Matsubara, H. & Sasaki, R. (1969) Biochem. Biophys.

Res. Commun. 35, 175-181 15. Yasukochi, Y. &Masters, B. S. S. (1976) J . Biol. Chem.

251,5337-5344 16. Vermilion, J. L. & Coon, M. J. (1978) J. Biol. Chem.

253, 2694-2704 17. Hjelmeland, L. M. (1980) Proc. Natl. Acad. Sci. U.S.A.

77,6368-6370

18. Aoyama, Y., Yoshida, Y., Kubota, S., Kumaoka, H. & Furumichi, A. (1978) Arch. Biochem. Biophys. 185, 362-369

19. Iyanagi, T., Anan, F. K., Imai, Y. & Mason, H. S. (1978) Biochemistry 17,2224-2230

20. Black, S. D., French, J. S., Williams, C. H., Jr. &Coon, M. J. (1979) Biochem. Biophys. Res. Commun. 91, 1528-1535

21. Gum, J. R. & Strobel, H. W. (1979) J. Biol. Chem. 254, 4177-4185

22. Kotake, A. & Funae, Y. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6473-6475

23. Bansal, S. K., Love, J. & Guaoo, H. L. (1983) Biochem. Biophys. Res. Commun. 117, 268-274

24. Tarr, G. E. & Crabb, J. W. (1983) Anal. Biochem. 131, 99- 107

25. Flashner, M., Ramsden, H. & Crane, L. J. (1983) Anal. Biochem . 135, 340-344

26. Gupta, S., Pfannkoch, E. & Regnier, F. E. (1983) Anal. Biochem. 128, 196-201

27. Gooding, K. M. & Schmuck, M. N. (1984) J . Chroma- togr. 296, 321-328

28. Lundahl, P., Greijer, E., Lindblom, H. & Fagerstam, L. G. (1984) J. Chromatogr. 297, 129-137

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