ARCHIVES

Vol.

OF BIOCHEMISTRY

185, No. 2, January

AND

BIOPHYSICS

30, pp. 362-369,

P-450 Reductase

NADPH-Cytochrome YURI

AOYAMA,

Faculty

YUZO

1978

of Yeast

YOSHIDA,’ SACHIKO KUBOTA, AND ATSUKO FURUMICHI’

ofPharmaceutical

Sciences, Received

July

Mukogawa

University,

11, 1977; revised

September

Microsomes

HIROSHI

Nishinomiya,

Hyogo

KUMAOKA,

663,

Japan

22, 1977

NADPH-cytochrome c reductase of yeast microsomes was purified to apparent homogenetty by solubilization with sodium cholate, ammonium sulfate fractionation, and chromatography with hydroxylapatite and diethylaminoethyl cellulose. The purified preparation exhibited an apparent molecular weight of 83,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The reductase contained one molecule each of flavin-adenine dinucleotide and riboflavin 5’-phosphate, though these were dissociative from the apoenzyme. The purified reductase showed a specific activity of 120 to 140 pmol/min/mg of protein for cytochrome c as the electron acceptor. The reductase could reduce yeast cytochrome P-450, though with a relatively slow rate. The reductase also reacted with rabbit liver cytochrome P-450 and supported the cytochrome P-450-dependent benzphetamine N-demethylation~ It can, therefore, be concluded that the NADPH-cytochrome c reductase is assigned for the cytochrome P450 reductase of yeast. The enzyme could also reduce the detergent-solubilized cytochrome b, of yeast. So, this reductase must contribute to the electron transfer from NADPH to cytochrome b, that observed in the yeast microsomes.

Yoshida and co-workers (l-8) have described an electron transport system containing cytochromes 6, and P-450, two flavoproteins, and a cyanide-sensitive factor in the microsomes from anaerobically grown yeast. This system seems to consist of two electron transport chains termed the “cytochrome b, pathway” and the “cytochrome P-450 pathway” (1). The former is now known to be identical with the hepatic microsomal system not only in its constitution but also in its function (6). On the other hand, the latter is less understood. The cytochrome P-450 pathway of hepatic microsomes is made up of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and cytochrome P-450 (9, 10). The reductase had previously been named NADPH-cytochrome c reductase and was known to contain one molecule each of

FAD3 and FMN as its prosthetic groups (11). In the preceding work (51, we purified an NADPH-cytochrome c reductase which contains both FAD and FMN as its prosthetic groups from the microsomes of anaerobically grown yeast. Since the cytochrome c reductase had many properties in common with its hepatic counterpart, it is highly likely that the cytochrome c reductase corresponds to the cytochrome P-450 reductase of yeast. However, the previous preparation (5) was solubilized from the microsomes by papain digestion, and we were unable to reconstitute the electron transport chain with isolated cytochrome P-450 from yeast microsomes (7). This report deals with the purification of microsomal NADPH-cytothe yeast chrome c reductase, which is able to reconstitute the cytochrome P-450 pathway.

I To whom all correspondence dressed. L Present address: Department Kansai Medical College, Moriguch, pan.

:I Abbreviations used: G-6-P, glucose &phosphate; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulfate; PCMB, paro-chloromercuribenzoic acid; FAD, flavin-adenine dinucleotide; FMN, riboflavin 5’-phosphate.

should

he

ad-

of Pharmacology, Osaka 570, Ja362

0003-9861/78/1852-0362$02.00/O Copyright All rights

0 1978 by Academic Press, of reproduction in any form

Inc. reserved.

NADPH-CYTOCHROME MATERIALS

AND

P-450

METHODS

Materials. Purified preparations of cytochrome P-450 from phenobarbital-treated rabbit liver microsomes and NADPH-cytochrome P-450 reductase from phenobarbital-treated rat liver microsomes were kindly supplied by Dr. Y. Imai of Osaka University. Detergent-solubilized cytochromes P450 and 6, of yeast microsomes were purified according to the methods of Yoshida et al. (7) and Tamura et al. (81, respectively. A purified preparation of yeast cytochrome c was supplied by Sankyo Co., Tokyo. NADP, NADH, NADPH, G-6-P, and G-6-P dehydrogenase (EC 1.1.1.49) from yeast were obtained from Oriental Yeast Co., Tokyo. FAD and FMN were purchased from Nakarai Chemicals Co., Kyoto. Crystalline catalase (EC 1.11.1.6) from beef liver and glucose oxidase (EC 1.1.3.4) of fungal origin, grade III, were the products of BoehringerManheim GmbH. Standard proteins for molecular weight determination were the products of Schwarz/ Mann, Orangeburg, New York. Cholic acid (Nissui Seiyaku Co., Tokyo) was purified by decolorization with active charcoal followed by recrystallization from 50% aqueous ethanol. Benzphetamine was the gift of Dr. Y. Imai of Osaka University. Hydroxylapatite (Hypatite C) was the product of Clarkson Chemical Co., Williamsport, Pennsylvania. Other chemicals were commercial products of reagent grade. Cultivation of yeast and preparation of microsomes. The wild-type bakers’ yeast, Saccharomyces cereuisiae, was cultivated anaerobically as described previously (1). Microsomes were prepared by the large-scale method of the preceding paper (1) and were stored at -20°C under nitrogen until use. Purification of NADPH-cytochrome c reductase. All manipulations were carried out at 2 to 4°C. Microsomes (2 to 3 g of protein) were suspended in 0.1 M potassium phosphate buffer, pH 7.2, containing 1 mM EDTA, 1 PM FAD, and 1 PM FMN to give a protein concentration of 10 mg/ml. A 10% solution of sodium cholate, pH 7.2, was added to the mixture with gentle stirring to give a final concentration of 0.5% (w/v). After 15 min of stirring, solid ammonium sulfate was added (22.8 g/l00 ml of the suspension), and the mixture was stirred for an additional 30 min. The suspension was then centrifuged. Ammonium sulfate was further added to the supernatant (15.2 g/100 ml) and left to stand for 30 min. The reductase-containing precipitate thus formed was collected by centrifugation at 10,OOOg for 20 min. The reductase was extracted from the precipitate with 40 ml of 10 mM buffer’ containing ’ Since potassium phosphate buffers, pH 7.0, containing 1 PM FAD, 1 ELM FMN, and 1 rnM EDTA were used after this step, these were expressed simply as 10 rnM buffer, etc.

REDUCTASE

OF

YEAST

363

1.0% sodium cholate and was dialyzed overnight against 10 mM buffer containing 0.5% sodium cholate. The dialyzed solution was usually turbid, and the insoluble materials were removed by centrifugation at 65,000g for 30 min. The clear supernatant containing the reductase thus obtained was applied to a column (1.6 x 15 cm) of hydroxylapatite equilibrated with the dialyzing buffer. The column was washed with the equilibrating buffer and eluted with a linear concentration gradient of potassium phosphate made of 80 ml of 10 and 180 mM buffers both containing 0.5% sodium cholate. The reductase-containing eluate from the hydroxylapatite column was diluted twofold with a 1.0% solution of sodium cholate containing 1 mM EDTA, 1 PM FAD, and 1 PM FMN, pH 7.0. The diluted solution was then applied to a DE-52 column (1.2 x 10 cm) equilbrated with 10 mM buffer containing 0.75% sodium cholate. The reductase was adsorbed at the top of the column forming a yellow band. The column was washed extensively with the equilibrating buffer and eluted with a linear concentration gradient of KC1 of from 0 to 0.35 M in the equilibrating buffer. The reductase was eluted from the column at about 0.2 M KC1 forming a single peak. The fractions around the peak were collected and are referred to as the purified preparation. Determination of enzyme activities. The activity of the NADPH-cytochrome c reductase was determined according to a previous method (5). The amount of the reductase was expressed by using “unit.” One unit was defined as the amount of the reductase reducing 1.0 pmol of cytochrome c per minute. The reduction of cytochrome P-450 was measured by following the increment of the CO complex of the reduced cytochrome P-450. The reaction medium contained 25 mM potassium phosphate buffer, pH 7.2, 7.5 mM glucose, 2.0 units of glucose oxidase, 2600 units of catalase, 0.15 mM NADPH, and suitable amounts of yeast cytochrome P-450 and the reductase preparation in a total volume of 2.0 ml. The mixture without NADPH was saturated with CO and incubated at 30°C for 2 min to achieve anaerobiosis. The reaction was then initiated with NADPH and the reduction of the cytochrome P-450 was followed at 30°C in a Hitachi 156 dual-wavelength spectrophotometer at 450 and 490 nm. The reduction of cytochrome b, was measured in a reaction medium consisting of 50 mM potassium phosphate buffer, pH 7.2, 0.15 mM NADPH, and suitable amounts of the detergent-solubilized cytochrome bj of yeast and the reductase preparation. The reaction was started by the addition of NADPH and the reduction of the cytochrome b, was followed at 30°C in a Hitachi 156 spectrophotometer at 423 and 409 nm. Benzphetamine V-demethylase activity was esti-

364

AOYAMA

mated by either NADPH oxidation or HCHO formation. The reaction mixture for determining the NADPH oxidation consisted of 0.1 M potassium phosphate buffer, pH 7.2, 0.15 mM NADPH, 1.25 mM benzphetamine, 0.16 PM cytochrome P-450 from phenobarbital-induced rabbit liver, and the reductase preparation. The total volume of the reaction mixture was made up to 2.0 ml. The reaction was carried out at 30°C under aerobic conditions. The rate of oxidation of NADPH was followed by a Hitachi 156 spectrophotometer at 340 and 374 nm. HCHO formation was measured in 2.0 ml of reaction medium consisting of 0.1 M potassium phosphate buffer, pH 7.2, 1.25 mM benzphetamine, 1.5 mM NADPH, 10 mM G-6-P, 10 units of G-6-P dehydrogenase, 0.16 PM rabbit liver cytochrome P-450, and the reduetase preparation. The reaction was carried out at 30°C with constant shaking for 20 min. The reaction was terminated by 5% trichloroacetic acid, and HCHO was determined by the method of Nash (12). Other analytical methods. Protein was determined by the method of Lowry et al. (13) using bovine serum albumin as the standard. Flavins were determined fluorometrically according to the method of Bassay et al. (14). The molecular weight of the reductase preparation was estimated by SDSpolyacrylamide gel electrophoresis according to the method of Neville (15). RESULT’s

NADPH-cytochrome c reductase could be extracted from yeast microsomes with 0.5% of sodium cholate and was purified in the presence of the detergent. A typical result of the purification is summarized in Table I. The specific activity of the purified reductase was usually in the range of 120 to 140 units/mg of protein and the overall yield in the range of 30 to 50%. Note that the buffers used in the purification conTABLE PURIFICATION

OF NADPH-CYTOCHROME

ET

AL.

tained 1 FM FAD and 1 PM FMN. The addition of flavins was indispensable to yield the preparation having high specific activity with good recovery. When FAD and FMN were omitted from the buffers, the specific activity of the preparation was not more than 60 unitslmg of protein, and the overall yield was less than 20%. When the purified preparation was analyzed by electrophoresis with a polyacrylamide gel column in the presence of 0.1% SDS, a single protein band was observed, as shown in Fig. 1, indicating high purity of the preparation. The mobility of the reductase in the polyacrylamide gel column gave an apparent molecular weight of 83,000. The gel filtration of the same reductase preparation through a column of Sephadex G-150 in the presence of 0.75% cholate gave a similar molecular weight of 82,000. However, the reductase preparation showed a higher molecular weight of about 160,000 when cholate was omitted from the Sephadex G-150 column. As already reported, the molecular weight of the yeast microsomal NADPH-cytochrome c reductase solubilized by papain digestion was determined to be 70,000 by both SDS-polyacrylamide gel electrophoresis and gel filtration with a detergentfree Sephadex G-150 column (51, and the value was markedly lower than that of the present preparation. These facts seem to suggest that the NADPH-cytochrome c reductase preparation obtained here contained a hydrophobic tail and tended to associate in an aqueous solution in the absence of a detergent. I c REDUCTASE

FROM YEAST

NADPH-cytochrome Step

Protein

Microsomes Ammonium sulfate precipitate Eluate from hydroxylapatite column Eluate from DE-52 column * The

reductase

was activated

(mg)

1820 177 16.0 1.66 20 to 30% over

Units

438 4470

Yield

(%)

Units per milligram of protein

100 102”

336

76.8

219

50.1

the microsomes

MICRWOMES c reductase

upon

0.24 2.53 21.0 132 solubilization.

Purification (n-fold) 1.0 10.5 87.5 551

NADPH-CYTOCHROME

P-450

REDUCTASE

OF

365

YEAST

K, of the FMN was assumed to be 0.2 PM from Fig. 2. The inactivation was not observed when the reductase was diluted in the presence of NADPH. This fact seems to suggest that the affinity of FMN for the apoenzyme was increased by the interaction of the enzyme with NADPH, though the exact reason for this is not known. It was reported in the preceding paper (5) that the FAD of papain-solubilized NADPH-cytochrome c. reductase binds tightly to the apoprotein. However, when the flavins of the present reductase preparation were determined after the removal of excess flavins by Sephadex G-50 gel filtration, both FAD and FMN contents were found in the range of 5 to 7 nmol/mg of protein. These values were

FIG. 1. SDS-polyacrylamide gel electrophoresis of the purified NADPH-cytochrome c reductase. Twenty-six micrograms of protein was loaded on a polyacrylamide gel column prepared according to the method of Nevill (15). Protein was stained with Coomassie blue. The migration of protein was from top to bottom.

The reductase preparation was very unstable in a highly diluted solution; when 10 ~1 of the reductase solution containing 0.03 unit of the reductase, 1 PM FAD, and 1 PM FMN was diluted with 3 ml of 50 mM potassium phosphate buffer, pH 7.5, 70 to 80% of the activity was lost within 4 min of incubation at 25°C. This inactivation was prevented by the enrichment of the dilution buffer with FMN, as shown in Fig. 2. As shown in the legend to Fig. 2, the cytochrome c reductase was diluted with buffers containing 1 FM FAD in this experiment. The FAD was, however, practically ineffective. Furthermore, the inactivated reductase could be reactivated by the addition of 1 pM FMN alone to the diluted enzyme solution. These observations indicate that the inactivation should be due to the reversible dissociation of the FMN prosthetic group, and the apparent

oLfl

-i

-i log

[FMN]

-i CM)

2. Protection of the NADPH-cytochrome c reductase by FMN from the dilution-induced inactivation. Ten microliters of the reductase solution (9.42 units/ml) containing 1 PM FAD and 1 PLM FMN was diluted with 3.0 ml of the reaction medium (containing 150 pmol of potassium phosphate buffer, pH 7.5, 0.2 pmol of ferricytochrome c, 3.0 pmol of FAD, and various amounts of FMN) and incubated at 25°C for 5 min. When the reaction medium did not contain FMN, more than 70% of the reductase activity was lost during this incubation period. The reaction was then started by the addition of 0.3 pmol of NADPH. The FMN concentration on the abscissa includes the FMN due to the enzyme solution. FIG.

366

AOYAMA

considerably lower than those expected from the apparent molecular weight of the reductase, and more than 60% of the reductase activity was lost by gel filtration. These facts seem to suggest that the FAD as well as FMN of the cholate-solubilized preparation was partially removed by the gel filtration. Unfortunately, the decreased reductase activity of the gel-filtrated preparation could not be restored by the addition of FAD and FMN. As described above, the FMN prosthetic group of the reductase dissociates from the apoenzyme reversibly and promptly. It can, therefore, be considered that the irreversible inactivation of the reductase caused by the gel filtration may be due to the irreversible and time-dependent dissociation of the FAD prosthetic group from the apoenzyme. The NADPH-cytochrome c reductase could catalyze the 2-methyl-1,4-naphthoquinone (vitamin K&mediated NADPH oxidation. This activity is known as one of the characteristics of the NADPH-cytochrome c (P-450) reductase of hepatic microsomes (161, and the papain-solubilized preparation of the NADPH-cytochrome c reductase of yeast also showed this activity (5). The vitamin K,-dependent NADPH oxidase activity of the present preparation, 39 nmol of NADPH oxidized/min/unit of the cytochrome c reductase, was comparable to that of the papain-solubilized preparation (5). The NADPH-cytochrome c reductase could reduce 2,6-dichloroindophenol and ferricyanide, though the activities were about 60% lower than the cytochrome c reducing activity. The Michaelis constant of the NADPHcytochrome c reductase for NADPH was determined to be 17.2 pM, and this value was slightly lower than that reported for the papain-solubilized preparation of the cytochrome c reductase, 32.4 PM (5). The optimum pH and ionic strength of the medium for the cytochrome c reductase activity were determined to be 7.5 and 0.2, respectively, and the values were comparable to those reported for the previous preparation of the cytochrome c reductase (5). PCMB and HgCl, inhibited the NADPH-

ET

AL.

cytochrome c reductase and the concentrations giving half-maximal inhibition were 3.0 and 0.2 PM, respectively. NADPH could prevent the enzyme from inhibition by the mercurials when added prior to the addition of the inhibitors. NADP inhibited the reductase competitively with a K, of 16.8 FM. These effects of the inhibitors were comparable to those reported for the previous cytochrome c reductase preparation (5). As shown in Fig. 3, the NADPH-cytochrome c reductase purified by the present method could reduce cytochrome P-450 isolated from the yeast microsomes (7), and the reduction depended exclusively on the reductase added (Fig. 3, inset). However, the rate of reduction was very slow. Phospholip was not added to the reaction system of the experiment shown in Fig. 3, but the addition of micelles of the phospholipids extracted from the yeast microsomes did not enhance the reduction rate. The cytochrome c reductase could also react with the cytochrome P-450 from phenobarbital-treated rabbit liver microsomes and supported the cytochrome P-450-dependent oxidative demethylation of benzphetamine, as shown in Table II. The data indicate that the apparent activity of the yeast reductase to support the demethylation was about one-third of that of the rat liver cytochrome P-450 reductase. However, the specific activity of the yeast cytochrome c reductase (120 to 140 unitslmg of protein) was almost three times as high as that of liver cytochrome P-450 reductase [less than 50 units/mg of protein as the NADPH-cytochrome c reductase (17) I, and the apparent molecular weights of these reductases were nearly the same. So, it can be said that the reactivity of the yeast reductase with the rabbit liver cytochrome P-450 was comparable to that of the rat liver enzyme when compared on a molecular basis. It is pointed out in Table II that the NADPH consumption and the HCHO production in the demethylation by these reconstituted systems were not stoichiometric. Since the values of NADPH oxidation shown in Table II were corrected by the endogenous NADPH oxidations, the discrepancy indicates the uncoupling of

NADPH-CYTOCHROME

P-450

REDUCTASE

OF

367

YEAST

NADPH

NQS%O~

FIG. 3. Reduction of yeast cytochrome P-450 with NADPH-cytochrome c reductase. An aliquot (0.6 ml) of the reductase solution containing 5.77 units of the enzyme, 1 PM FAD, and 1 FM FMN was placed in a spectrophotometer cuvette and mixed with 0.1 ml of the yeast cytochrome P-450 solution containing 0.62 nmol of the cytochrome. The mixture was diluted with 1.2 ml of the reaction medium containing 50 pmol of potassium phosphate buffer, pH 7.2, 15 pmol of glucose, 2 units of glucose oxidase, and 2600 units of catalase. The cuvette was then bubbled with CO and incubated at 30°C for 2 min to achieve anaerobiosis. The reaction was then started by the addition of 0.3 Fmol of NADPH as indicated, and the increasing absorbance difference between 450 and 490 nm was recorded at 30°C. Inset: Experiments of the type described above were performed with the indicated amounts of reductase, and the observed first-order rate constants of the cytochrome P-450 reduction were plotted against the enzyme amount. TABLE RECONSTITUTION

OF BENZPHETAMINE THE

Cytochrome

Rabbit Rabbit

liver liver

Rabbit Rabbit

liver liver

CYTOCHROME

N-DEMETHYLASE

P-450

FROM

II WITH

THE NADPH-CYTOCHROME

PHENOBARBITAL-TREATED

P-450

Reductase

(0.32 nmol) (0.32 nmol)

Yeast -

(0.32 nmol) (0.32 nmol)

Yeast (1.70 units) Rat liver (0.43 unit) Yeast, papain solubilized

-

c REDUCTASE

RABBIT

NADPH oxidation (nmol/min)

(1.70 units)

’ Cytochrome P-450-dependent N-demethylation of benzphetamine Materials and Methods using the indicated reductase preparations,

the demethylation with the electrons transferred to cytochrome P-450. The uncoupling rates were nearly the same regardless of the origin of the reductase. The NADPH-cytochrome c reductase solubilized by papain digestion (5) could not support the demethylation (Table II), The NADPH-cytochrome c reductase also catalyzed the reduction of cytochrome 6, purified from yeast microsomes using Triton X-100 solubilization (81, as shown in Fig. 4. This activity was never observed when either the reductase cr the cyto-

AND

LIVER”

(1.60 units) was determined

HCHO production (nmol/min)

10.3 9.3 Trace as described

4.10 0.34 0.27 3.19 under

chrome was prepared using solubilization by proteinase digestion. Therefore, it is considered that the reduction of cytochrome bj with the cytochrome c reductase depends on the formation of a sort of complex micelles or aggregates between the reductase and the cytochrome by hydrophobic interactions. DISCUSSION

NADPH-cytochrome c reductase of yeast microsomes had already been purified and characterized (5). The prepara-

368

AOYAMA

ET AL

tase showed the vitamin K,-dependent NADPH oxidase activity. From the evidence described above, it is concluded that the present NADPH-cytochrome c reductase preparation was the same enzyme as the previous NADPH-cytochrome c reductase (51, but retained a hydrophobic tail, as described in detail by Spatz and Strittmatter (18, 19) on cytochrome b, and NADH-cytochrome b5 reductase of hepatic microsomes. tlmn i It is now known that the NADPH-cytoI * chrome c reductase which acts as the cytonAq*3m40q= 331 chrome P-450 reductase in hepatic microsomes contains one molecule each of FAD -1 and FMN as its prosthetic groups (11). FIG. 4. Reduction of detergent-solubilized yeast The previous preparation of the yeast micytochrome b, with NADPH-cytochrome c reduccrosomal NADPH-cytochrome c reductase tase. An aliquot (0.2 ml) of the reductase solution containing 1.92 units of the enzyme, 1 PM FAD, also contained one molecule each of FAD and 1 PM FMN was placed in a spectrophotometer and FMN, but the FMN was dissociable cuvette and mixed with 0.2 ml of the solution of from the apoenzyme (5). The NADPH-cydetergent-solubilized cytochrome b, of yeast contochrome c reductase obtained here had taining 0.43 nmol of the cytochrome. The mixture both FAD and FMN as its prosthetic was diluted with 1.6 ml of 62.5 mM potassium groups, and the FMN was dissociable, as phosphate buffer, pH 7.2. The reaction was then shown in Fig. 2. Regarding the present started by the addition of 0.3 Fmol of NADPH as cholate-solubilized preparation, it seems indicated, and the increasing absorbance difference likely that the FAD was also dissociable, between 424 and 409 nm was recorded at 30°C. and good results for purification were obtion, however, seemed to lose the hydrotained by the addition of FAD, together phobic tail, because papain digestion of with FMN, to the buffers used in the the microsomes was used to solubilize the purification. Dissociation of the FAD prosreductase. In the present study, we have thetic group from the apoenzyme was not established a procedure obtaining the observed in the papain-solubilized prepaNADPH-cytochrome c reductase prepararation (5). So, it can be said that the tion without proteinase treatment. The dissociation of the FAD prosthetic group molecular weight of the NADPH-cytofrom the apoenzyme is a unique character chrome c reductase preparation obtained of the cholate-solubilized preparation. In by the present method, 83,000, was markaddition, it seems likely that the dissociaedly higher than that of the papain-solution of FAD occurred in a manner different bilized preparation, 70,000 (5). The present from that of the dissociation of FMN, as reductase preparation tended to associate pointed out under Results. Although the in an aqueous solution in the absence of a reason for the above is not yet known, it is detergent, but such a tendency had not of interest that the addition of FAD and been observed with the papain-solubilized FMN to the buffers was also effective for reductase preparation (5). Despite these the purification of the detergent-solubidiscrepancies, most of the catalytic proplized hepatic NADPH-cytochrome c reducerties of the present cytochrome c reduc- tase (Dr. H. Satake, personal communicatase closely resembled those of the pre- tion). vious preparation (5). These include speciThe present preparation of NADPH-cyficity for electron donors and acceptors, tochrome c reductase from yeast microMichaelis constant for NADPH, competisomes could reconstitute benzphetamine N-demethylase activity when combined tive inhibition by NADP, and protection by NADPH from mercurials inhibition. with rabbit liver cytochrome P-450 (Table II), and the ability of the yeast reductase Moreover, the present cytochrome c reduc-

NADPH-CYTOCHROME

P-450

to support the demethylation was almost comparable to that of the rat liver cytochrome P-450 reductase, as pointed out under Results. In this reconstituted system, only about 40% of the electrons from NADPH were coupled with the demethylation, and a similar coupling rate was also observed in the system using rat liver cytochrome P-450 reductase, as shown in Table II. It was suggested by Imai and Sato (20) that the coupling of the cytochrome P-450-linked monooxygenation with the electrons from NADPH was affected by the efficiency of the “second-electron” transfer to the ferricytochrome P450-oxygen-substrate ternary complex. It can, therefore, be suggested that the yeast NADPH-cytochrome c reductase was as effective as the rat liver cytochrome P-450 reductase in transferring the “second electron” to the ternary complex. Taken together, the NADPH-cytochrome c reductase of yeast microsomes can be identified as NADPH-cytochrome P-450 reductase. However, the rate of reduction of yeast cytochrome P-450 with yeast reductase was very slow, as shown in Fig. 3. Three possibilities may be considered to account for the reason that yeast cytochrome P-450 reductase ill-reacted with its intrinsic associate in the reconstituted system. First, the preparation of yeast cytochrome P-450 was somewhat damaged, though the spectral characterisitcs were not altered. Second, the reconstitution conditions were not refined. Finally, the reduction rate of yeast cytochrome P-450 was slow unless a suitable substrate bound to the cytochrome. These possibilities are now being examined in our laboratory. It is known that the cytochrome b, in yeast microsomes is readily reduced by NADPH (11, and NADPH can act as the electron donor for the cytochrome bjlinked oxidative desaturation of palmitoylCoA (6). Since the NADPH-cytochrome c (P-450) reductase could reduce detergentsolubilized cytochrome bg, as shown in Fig. 4, the reductase should also act as

REDUCTASE

OF

369

YEAST

the NADPH-cytochrome yeast microsomes.

b, reductase

in

ACKNOWLEDGMENTS We wish to thank Dr. Y. Imai of the Institute for Protein Research, Osaka University, for gifts of purified preparations of rabbit liver cytochrome P450 and rat liver NADPH-cytochrome P-450 reductase and of benzphetamine. REFERENCES 1. YOSHIDA, Y., KUMAOKA, J. Biochem. (Tokyo) 2.

3. 4. 5.

6.

7.

8. 9. 10. 11. 12. 13.

14. 15. 16. 17.

18. 19. 20.

H., ANDSATO, R. (1974) 75, 1201-1210. YOSHIDA, Y., KUMAOKA, H., AND SATO, R. (1974) J. Biochem. (Tokyo) 75, 1211-1219. YOSHIDA, Y., AND KUMAOKA, H. (1975) J. Biothem. (Tokyo) 78, 785-794. KUBOTA, S., YOSHIDA, Y., AND KUMAOKA, H. (1977) J. Biochem. (Tokyo) 81, 187-195. KUBOTA, S., YOSHIDA, Y., KUMAOKA, H., AND FURUMICHI, A. (1977). J. Biochem. (Tokyo) 81, 197-205. TAMURA, Y., YOSHIDA, Y., SATO, R., AND KuMAOKA, H. (1976) Arch. Biochem. Biophys. 175, 284-294. YOSHIDA, Y., AOYAMA, Y., KUMAOKA, H., AND KUBOTA, S. (1977) Biochem. Biophys. Res. Common. 78, 1005-1010. TAMURA, Y., YOSHIDA, Y., AND KUMAOKA, H. (1976) Seikaguku (Tokyo) 48, 512. Lu, A. Y. H., AND LEVIN, W. (1974) Biochem. Biophys. Acta 344, 205-240. IMAI, Y. (1976) J. Biochem. (Tokyo) 80, 267-276. IYANAGI, T., AND MASON, H. S. (1973) Biochemistry 12, 2297-2308. NASH, T. (1953) Biochem. J. 55, 416-421. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, F. J. (1951) J. Biol. Chem. 193, 265-275. BASSAY, 0. A., LOWRY, 0. H., AND LOVE, R. H. (1949) J. Biol. Chem. 180, 755-769. NEVILLE, D. M., JR. (1971) J. Biol. Chem. 346, 6328-6334. NISHIBAYASHI-YAMASHITA, H., AND SATO, R. (1970) J. B&hem. (Tokyo) 67, 199-210. VERMILLION, J. L., AND CONN, M. J. (1974) Biochem. Biophys. Res. Commun. 60, 13151322. SPATZ, L., AND STRITTMATTER, P. (1971) Proc. Nat. Acad. Sci. USA 68, 1042-1046. SPATZ, L., AND STRITTMATTER, P. (1973) J. Biol. Chem. 248, 793-799. IMAI, Y., AND SATO, R. (1977)Biochem. Biophys. Res. Commun. 75, 420-426.