THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol. 249, No. 19, Issue of October 10, PP. 6302-6310, 1974

Printed in U.S.A.

Preparation and Properties of Partially Purified Cytochrome

P-450 and Reduced Nicotinamide Adenine Dinucleotide

Phosphate-Cytochrome P-450 Reductase

from Rabbit Liver Microsomes*

(Received for publication, January 30, 1974)

THEODORE A. VAN DER HOEVEN AND MIXOR J. Coos

From the Department of Biological Chemistry, Medical School, The University of Michigan, Ann Arbor, Michigan

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SUMMARY

The liver microsomal enzyme system which hydroxylates fatty acids, hydrocarbons, and a variety of drugs and other foreign compounds was previously solubilized and resolved into three components: cytochrome P-450, NADPH-cyto- chrome P-450 reductase, and phospholipid. The two en- zymes were partially purified from pyrophosphate-treated, cholate-solubilized liver microsomes of phenobarbital-treated rabbits by fractionation with polyethylene glycol 6000 and DEAE-cellulose column chromatography in the presence of Renex-690, a nonionic detergent. These steps were fol- lowed by treatment with Amberlite XAD-2 and calcium phos- phate gel.

Cytochrome P-450 preparations purified to a content as high as 15 nmoles per mg of protein were free of cytochrome bS and contained no significant amount of NADPH-cyto- chrome c reductase, NADH-cytochrome c reductase, non- heme iron, other metals as measured by neutron activation analysis, or labile sulfide. The molecular weight was judged to be about 280,000 by gel exclusion chroma.tography and sucrose density gradient centrifugation. Polyacryla- mide gel electrophoresis of the partially purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of two major poly- peptide bands, one with a molecular weight of 47,000 to 49,000 and the other 52,000 to 53,000. The component of lower molecular weight corresponded to the major poly- peptide induced by phenobarbital treatment, as shown by electrophoresis of the proteins in normal and induced micro- somes. The partially purified preparation accepted 2 elec- trons from dithionite per molecule of cytochrome P-450 under anaerobic conditions, indicating the presence of another elec- tron acceptor in addition to the iron atom of the hemeprotein.

The partially purified NADPH-cytochrome P-450 reductase preparations, which catalyzed the reduction of 8.6 to 10.0 pmoles of cytochrome c per min per mg of protein at 30”, were

* This research was supported by Grant GB-30419X from the National Science Foundat,ion and Grant AM-16339 from the United States Public Health Service. A preliminary report of part of this investigation has been presented (1).

free of cytochrome P-450 and contained only a trace of NADH-cytochrome c reductase. FMN and FAD were found to be present in equimolar amounts, and labile sulfide was absent.

The reconstituted enzyme system was active in the hy- droxylation of fatty acids (laurate), hydrocarbons (hexane, cyclohexane, and octane), drugs (benzphetamine, hexobarbi- tal, and ethylmorphine), and aniline. The presence of both phosphatidylcholine and deoxycholate was necessary for maximal activity.

The cytochromc I’-450.containing mixed fuuction oxidase system of liver microsomes was solubilized and resolved into three caomponents by Lu and Coon (2, 3). A heat-stable frac- tion (Factor I!), subsequently shown to cont’aiu phosphatidyl- choline as the active component (4), was fouud to be required along with fractious contairrirlg cytochrome P-450 and NAIII’H- cytoc~hrome 1’.450 reductasc for hydroxylation activity toward a variety of substrates (2, 5-8). Procedures similar to those used in the resolution and reconstitutiorl of the rabbit, liver enzyme system (2) were also applied successfully to the cyto- chrome I’-450.containing euzymr systems of Candida fropicalis

(9, 10) and rat (6, 7), mouse (ll), and human (12) liver micro- somes. The cytochromc I -448kontaiuiug enzyme system which oxidizes polycyclic aromatic carcinogens has also been resolved and reconstituted and found to require the reductase and phosphatidylcholinc for activity, as shown with 3.methyl- cholanthrene-treated rats (13) and P-naphthoflavone-treated mice of both inducible and noninducible strains (11). Several procedures have recently been rrported for the partial purifica- tion of hepatic microsomal cytochrome 1’.450 (14-18) and NADPH-cytochrome c reductase (5, 19-22) in the presence of detergents.

the present paper is concerned with the separation and partial purification of rabbit liver microsomal cytochrome l’-450 and NADPH-cytochrome I’-450 reductase using polyethylene glycol 6000 fractionation in the preseuce of cholatc and DEAE-

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cellulose column chromatography in the presence of Renex-690. The resulting cytochrome P-450 preparation is largely free of

other known electron carriers. Anaerobic titration of the

purified cytochrome r-450 by dithionite confirms the conclusion reached from earlier studies in this laboratory with somewhat

less purified preparations (23) that 2 electrons are t,aken up per

molecule of the hcrneprotein and that a previously unrecognized

electron acceptor may play a role in the function of this enzyme system.

EXPERIMEKTAL PROCELJURE

The isolation of microsomes and the enzyme fractionations were carried out at O-4”. All pH measurements were made at 23”. Protein concentrations were measured by t,he procedure of Lowry el al. (24) with bovine serum albumin as a standard. The co,,- centration of the latter was determined by the absorbance at 279 nm using an extinction coefficient of 6.67 for a lyc solution (25). When glycerol was present the solut,ions were diluted so that, this component was at a concentration of O.O5OA, or less, because it otherwise interfered with the protein determination.

Zsolaliolr of Microson~es-New Zealand male rabbit,s (2 t,o 2.5 kg in weight) were inject,ed subcutaneously wit,h 65 mg of phcno- barbit!sl per kg body weight once a day for 2 days and again, after a l-day interval, for 3 additional days. The animals were fasted on the last day. The livers were homogenized in 4 volumes of 0.1 M Tris-acet,ate buffer, pH 7.4, cont,aining 0.1 M KCI, 1.0 rnM El)TA, and 2.3 X 10M5 M butylated hydroxyt,oluene in :I Waring Blendor for two 40-s intervals, and the mixture was centrifuged at 10,000 X q for 30 min. The pellet, was homogenized in a small volume of the bufrer mixture, and the supernat ant fract,iorr ob tained upon ccnt,rifug:ttiorr was combined with the original super- natant frac,t,ion and c.errtrifuged at 105,000 X 9 for 90 min. The resulting mirrosomal pellet was suspended in a volume of 0.1 M potassium pyrophosphatc buffer, pH 7.4, cont,aining 1.0 rn~ Rl)TA and 2.3 X 1O-6 M butylated hydroxytoluene, equal I,O t,hat, of the original homogenate. The mixture was t.reat,ed for four 10-s int,ervals in a 500-wat,t MSll: ultrasonic disintcgrat,or at, full out,- put, and cnentrifuged at, 105,000 X g for 60 min. The supcrnatant, fraction was removed, and the pellet, consisting of a reddish upper layer and a reddish brown, tightly packed lower layer, was sus- pended in 0.01 M Tris-aretate buffer, pH 7.4, containing 20% glyc- erol and 0.1 mM El)TA (hereafter called Buffer A) and stored at, -20”.

Solubilizatio?! alid I’uriJcalio,/ (1s C?/tochrome P-&&-The pyre- phosphat,e-extracted microsomal suspension (120 ml) was mixed with 380 ml of 0.1 M Tris-ac*et,at,e buffer, pH 7.4, ront,aining 0.1 M KCI, 20°j, glycerol. 1.0 mu Rl)TA, 1.0 mM dithiot,hreitol, and 2.3 X 10e6 M butylat,cd hydroxyt,olucne, and flushed wit,h nitrogen. A 10% sodium cholat,e solution was added dropwise with stirring to give a final cho1at.c to prot,ein ratio of 3.0, and t,he slightly t,ur- bid mixture was st,irred an additional 30 min. To t,his mixture, having a protein c.onc.ent,ration of G mg per ml, a SOo/, polyethylene glycol solution (w/v) was added dropwisc, and stirring was con- tinued for 10 min. The fractions prccipit,at,ing from 0 to 4(%, 4 to 6%);,, 6 t,o S7c, 8 to lo%, and 10 to 137(, polyethylene glycol were collected by centrifugation. The last three precipitates were taken up individually in Buffer A and dialyzed overnight against, 50 volumes of the same buffer mixture. The 8 to 10yc polyethylene glycol fraction usually gave a clear solution, whereas the 6 to 8% and 10 to 137? fractions were turbid. The frac.t,ion richest in cyt,ochrome P-450 (usually the 8 t,o 10% polyet,hylene glycol frac- tlon, 50 ml i.1 volume), was diluted with Buffer A, and dithiothrei- to1 and Renex-690 were added to final concentrat,ions of 1.0 m&l and l(% (v/v), respect,ively, in a tot,al volume of 150 ml. The protein concentration was then 5 to 6 mg per ml. The clear, red solution was st,irred for 30 min and then applied to a l>EAE-cellu- lose column (2.0 X 20 cm) previously equilibrated with a solut,ion of 0.01 M Tris-acetate buffer, pTI 7.4, containing 20%, glycerol, 1.0 mM EDTA, 0.1 mM dithiothreitol, and 0.5yo (v/v) Renex-690 (hereafter called Buffer B). A broad red-brown band was ob- served at the top of the column and a small red band near the middle. Buffer B (200 ml) was then passed through the column and the total effluent (340 ml) was collected.’ To decrease t,he

1 Some of the cytochrome P-450 was not eluted by this proce-

Renex concentration, 160 ml (wet volume) of Amberlite XAD-2 were added, and the mixture was stirred for 15 min, filtered through glass wool, and rinsed with small amounts of water. In order to concentrat,e the solution, which had a volume of 435 ml, 40 g of dry Bio-Gel P-60 were added, and the mixture was allowed to stand for 10 min and then filtered on a Buchner funnel with light suction. No loss of cytochrome P-450 occurred in this opera- tion. To the resulting solution (185 ml), 50 ml of calcium phos- phate gel (containing 30 mg, dry weight,, per ml) were added with stirring, and after 10 min the mixture was centrifuged. The supernatant solution, which had slightly less than one-tenth of t,he original absorbance at 416 nm, was discarded. The reddish precipitate was suspended in 120 ml of Buffer A, and after cen- trifugation the supernatant, layer was discarded. This procedure was repeated twice. The gel was then suspended in 20 ml of 0.3 M potassium phosphate buffer, pH 7.4, containing 207/, glycerol and 1.0 mM EDTA, with stirring for 10 min, and the mixture was centrifuged at 6000 X g for 10 min. This procedure was repeated twice, and the deep red supcrnat,ant, fractions were combined and dialyzed overnight, against 20 volumes of Buffer A rontnining 0.1 mM dithiot,lrreitol. The dialyzed enzyme preparat,ion was stored at, -20”.

Previously, ammonium sulfate fract,ionnt,ion was used inst,ead of column c:hromatography on I)EAE-cellulose (26, 27). The polyet,hylene glycol prec’ipit,atn was solubilized with rholat,e in t,he same manner as described for the microaomal suspension and then treat,ed wit,h ammonium sulfat,e, and t,he fract.ions preripi- tating from 37 to 42?{,, 42 to 48%) and 48 t,o 60%) sat.urat,iorr were collected, dissolved individually in 0.05 M Tris-aretat,e buffer, pH 7.4, containing 1.0 mM ISl)TA, 0.1 mM dithiothreitol, and ZOO/, glycerol, and dialyzed overnight. against 20 volumes of the same buffer mixt.ure. This st.cp generally yielded cyt,ochrome P-450 preparations containing 5.5 to 8.1 nrnolcs per mg of protein. Such preparnt,iorrs were lower in phospholipid content, than t,hose pre- pared by c*hromatography on 1 )ISAIS-c~~llulose but, contained small amounts of c.ytochromr h:, and NAI~I’II~~:ytochromc P-450 re- durtnsc.

PuriJicafio,, oJ‘~‘AI>I’Ei~c~~~io~hron~e P-450 I2edurla.se -~-The super- nat,ant solution remaining after the 10 to 13% polyethylene glycol fractionation st,ep in t,hc purification of cytochrome P-450 was found I,O contain most of the NA I )PIl-rytochrome P-450 reduc- tase. This sollltion (620 ml) was brought, to 25cj, saturation by the addition of solid ammonium sulfate, wit,11 adjustment, of the PIT t)o 7.4 as ncxcsst~ry by the :tddit,ion of small amounts of 1.0 M

Tris base. The mixture was stirred for 10 min and centrifuged at) 12,ooO X g for 10 niirr to give a rolorlcss upper phase containing polyethylene gly~ol and a slightly yellowish to reddish lower phase c*ont.aining the rcductasc. The upper layer was removed by aspiration, an d solid ammonium sulfate was added to the lower phase (400 ml) t.o 60(x, saturation. The result,ing mixture was stirred for 10 rnirl and centrifugrd at 20,000 X g for 1.5 min. The slightly reddish floating pellet, was dissolved in 80 ml of Buffer A and dialyzed overnight, against 2 lit,crs of the same solution. A lO(%, (v/v) Rcncx solution was added to the dialyzed preparat,ion t,o bring the final concentrat,ion to 0.4”/;, and the solution was applied to a l)EAE-cellulose caolumn (4.2 X 9 cm) previously equilibrat,ed with 0.05 M Tris-chloride buffer, pH 7.4, rontaining 10yo glycerol, 0.1 mM El>TA, 0.1 mM dithiothreitol, and 0.4% Renex (hereafter ralled Buffer C). A reddish fraction containing no redurtase was eluted when 350 ml of Buffer C containing 0.1 M KC1 were passed through the c*olumn, and t,he reduct,ase was elutcd as a thin yellow band with 200 ml of Buffer C containing 0.3 M KCl. Fractions containing reductasc activity greater t,han 5.0 AA,,, per min per ml were pooled to give 114 ml of solution, t,o which a suspension of calcium phosphate (30 mg, dry weight,, per ml) was added dropwise in the amount of 1.0 mg per 10.0 AA550 per min. The mixture was stirred for 15 min and rentri- fuged at 6,000 X g for 10 min. The supernatant fraction was dis- carded, and the yellowish brown precipitate was suspended in 150 ml of Buffer C without Renex. The precipitate recovered by centrifugation was suspended in 140 ml of 0.1 M potassium phos-

dure. When the column was then treated with Buffer B contain- ing 0.25 M KCI, cptorhrome P-450 was eluted along with cyto- chrome P-420 and cytochrome hb, and when the KC1 concent,ra- tion was increased to 0.3 M, some NADPH-cytochrome c reductase was eluted.

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phate buffer, pH 7.7, containing 20y0 glycerol, 0.1 mM EDTA, raphy-The molecular weight of cytochrome P-450 was estimated and 0.1 mM dithiothreitol, with stirring for 15 min. The mixture by gel permeation chromatography at 4’ on a Bio-Gel A-1.5m was centrifuged, and the yellowish supernatant fraction was con- column (2.8 X 52.2 cm) according to Andrews (40). The column centrated in an Amicon ultrafiltration cell with a PM-30 filter was calibrated with standard proteins in 0.1 M Tris-acetate buffer, and stored at -20”. DH 7.4. containing 0.1 M KC1 and 1 rnM EDTA. Fractions of 2.2

Assau of Fattu Acid. Alkane. and Drua Hudroxvlation-Sub- ” ”

st,rate hydroxylation was determined at 30” Ln a l-ml reaction mixture containing, unless stated otherwise, cytochrome P-450 (0.1 nmole), NADPH-cytochrome P-450 reductase (at a saturating level), dilauroyl-GPC? (30 rg), 0.05 M Hepes buffer, pH 7.4, 0.015 M MgCls, sodium deoxycholate (100 pg),, and 0.10 mM NADPH. The two enzyme fractions and the phospholipid were mixed, the remaining components were then added in the order given, and the mixture was incubated for 3 min at 30” before the reaction was initiated with NADPH. Unless stated otherwise, cytochrome P-450 was the rate-limiting component. The substrate-dependent disappearance of NAI)PH was determined at 340 nm as described previously (6, 7) in a Gilford spectrophotometer equipped with a multiple sample absorbance recorder. In some instances, form- aldehyde formation from ethylmorphine was measured by the method of Nash (28) as modified by Co&in and Axelrod (29). The hydroxylation of radioactive laurate was det,ermined accord- ing to Kusunose et al. (30) and that of radioactive octane accord- ing to Gholson et al. (31), except that the alumina used for chro- matographic separation of octanol from octane was activated by heating for 24 hours at 130”.

Assay of Cytochrome P-450-A sample of cytocbrome P-450 in a buffered solution containing 207, glycerol was bubbled with CO for about 20 s and a few grains of dithionite were then added to the cuvette as well as to a cuvette containing cytochrome P-450 not exposed to CO. The difference spectrum was recorded in a Cary model 14 recording spectrophotometer, and the cytochrome P-450 concentration was calculated with the use of an extinction coefficient of 91 cm-’ mM-1 for Adsa minus A,,, (32). Turbid mix- tures were clarified by the addition of Renex-690 to a concentration of 1%. Under these conditions, no conversion to cytochrome P- 420 could be detected, nor did the detergent otherwise interfere with the assay procedure. Cytochrome P-420 was also determined by the CO difference spectrum (32).

Assay of Reductase-~-The NAl)PH-cytochrome P-450 reductase was assayed by its activity toward cyt,ochrome 2 as determined spectrophotometrically at, 550 nm. The procedure, based on that of Masters et al. (33), was the same as described previously (5) except that 0.3 M potassium phosphate buffer, pH 7.7, was used and the rat,e was determined at 30”. The specific activity of the reductase is defined as the number of enzyme units (micromoles of cvtochrome c reduced per min) per mg of protein. NADH- cytochrome c reductase activity was measured in a similar manner, and NADPH-ferricvanide reductase and NAl)H-ferricyanide re- ductase activities ,ere determined at 30’ as described-elsewhere (34).

Disc Gel Electrophoresis -Polyacrylamide gel electrophoresis was performed in t,he presence of sodium dodecyl sulfate with a discontinuous buffer system according t,o the procedure described by Ornstein (35) and Davis (36) as modified by Laemmli (37) and with the lower buffer at 20”. Protein fractions were first treated for 2 min at 100” in 0.06 M Tris-chloride buffer, p1-I 6.8, containing 2y0 sodium dodecyl sulfate and 57; mercaptoethanol. The sep- arating gel (10.5 X 0.6 cm, or 7.5 X 0.6 cm in the molecular weight determinations) contained 7.5yL acrylamide. Proteins were de- tected by st,aining with Coomassie blue R250 (38), followed by diffusion destaining of the gel in 207, methanol-7% acetic arid, and mobilities were measured relative to the tracking dye band marked by a stainless steel wire.

&‘ucro.sa Densitu Gradient CentrifuantiotL-The molecular weight of cytochrome P-450 was determine2 by sucrose density gradient centrifugation as described by Martin and Ames (39). The sample (1.5 ml) was layered onto 38 ml of a 5 to 15c/, linear gradient, of sucrose in 0.05 M Tris buffer, pH 7.4, containing 0.1 mM EDTA. After centrifugation at 150,000 X 9 for 18 hours at, 5” in a Spinco SW 27 rotor, the tubes were punctured and 1.0.ml fractions were collected for assay.

Molecular Weight Estimation by Gel Permeation Chromatog-

2 The abbreviations used are: dilauroyl-GPC, dilauroylglyceryl- 3-phosphorylcholine; Hepes, A’-2.hydroxyet,hylpiperazine-A”-2-

E$ect of Pyrophosphafe Treatment on &\iicrosomal Componenfs-

Hepatic microsomes isolated as the 105,000 x g pellet are usually

contaminated by hemoglobin unless the organ is perfused before homogenization. It was found that pyrophosphate treatment

not only increased the content of some of the electron carriers in

the microsomal fraction, as stated by Welton and Aust (48), but also removed almost all of the contaminating hemoglobin. In a typical experiment hemoglobin was reduced from 0.75 to

0.01 nmole per mg of protein. This procedure resulted in the purification of cytochrome I’-450 from 2.7 to 3.4 nmoles per mg

of protein with 85 to 90% recovery, as well as of cytochrome be from 1.2 to 1.4 nmoles per mg of protein with about 80y0 rccov- cry. The NADPH-cytochrome c reductase specific activity was increased from 0.07 to 0.13. The amount of reductase recovered was found to be about 20% greater than present in the untreated microsomes. The cause for this apparent ill- crease, which has been observed consistently, is not known.

ethanesulfonate. Cyfochrome P-450 Purification-The results of a representative

ml each were collected and assayed for the various enzyme activ- ities and for protein by the absorbance at 230 nm. Partially purified cytochrome P-450 (112 nmoles, 10.5 nmoles per mg of protein) was applied to the column and the fractions were assayed for absorbance at 416 nm.

Other Procedures-Total heme was determined by the pyridine hemochrome method (32), which gave the expected values with hemoglobin and myoglobin. Total iron was estimated by atomic absorption spect,rophotometry and FMN and FAD by the proce- dure of King (41). Cytochrome b, was determined by enzymatic reduction with NADH as the electron donor (32) in the presence of Triton X-lOO-solubilized NADH-cytochrome bg reductase (42) and deoxyrholate (100 pg per ml, final concentration). Hemo- globin was est,imated as the CO complex using an extinction co- efficient of 154 rnM-l cm-l at 418 nm. Phospholipids were ex- tracted by the method of Bligh and Dyer (43) and estimated by determination of the phosphorus content according to King (44).

Materials-d-Benzphetamine was generously provided by Dr. J. W. Hinman of the Upjohn Company, xanthine oxidase by 1)r. V. Massev. of this department. Amberlite XAD-2 bv the Rohm and Haas &mpany, and Renex-690, a nonionic detergent, by the Atlas Chemical Company. Polyethylene glycol, obtained from Baker, was purified to remove impurities absorbing in the ultraviolet re- gion by treatment with organic solvents (45). A 5070 (w/v) solu- tion of the puritied polyethylene glycol was prepared by gentle heating and allowed to cool and then filtered through a Buchner funnel prior to use; a 170 solution exhibited no significant absorb- ance at 288 nm. NADPH, NADH, Tris, horse heart cytochrome c (type III), tetrapotassium pyrophosphate, sodium cholate, and DEAE-cellulose were obtained from Sigma. The pyrophosphate salt was neutralized with HCl. The sodium cholate was converted to the free acid and recrystallized from 95y0 ethanol. The DEAE- cellulose (medium mesh; capacity 0.85 meq per g) was thoroughly washed before use with the following solutions in the order given: 0.5 N NaOH, 0.1 N HCl, 0.5 N NaOH, and water. Aldolase and ovalbumin were obtained from Pharmacin, glutamate dehydro- genase from Nutrit,ional Biochemicals, bovine serum albumin (Fraction V) from Pentex, sodium deoxycholate, human r-globu- lin, and myoglobin from Schwarz-Mann,.Hepes buffer and crystal- line bovine liver catalase from Calbiochem. Bio-Gel P-60 (100 to 200 mesh) and Rio-Gel A-1.5m from Bio-Rid, 11.14C]laurate and [1-‘%]octane from New England Nuclear, 6.galactosidase from Boehringer, and butylat,ed hydroxytoluene from Pfhalta and Bauer. Calcium phosphate gel was prepared according to Keilin and Hartree (46); dilauroyl-GPC, was synthesized by a published procedure (47) by Dr. H. JS. Radtke, of this department, and an aqueous suspension of the compound was sonicated before use.

RESULTS

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TABLE I TABLE II

Partial purification of liver microsomal cytochrome P-450 Analysis of partially purified cytochrome P-450

A cytochrome P-450 preparation containing 13.0 nmoles per mg of protein was analyzed for the various components. Preparation Volume

ml

Pyrophosphate-extracted, sonicated microsomes 120

Polyethylene glycol pre- cipitate (8 to 10%). 50

l>EAIS-cellulose column eluate (wit,h 0.5% Renex) ; calcium phos- phate gel eluate. 55

Protein

-

_-

Cytochrome P-450 contentQ

nmnles/mg prolein

3,000 3.0 (2.6-3.6)

820 6.3 (5.4-7.0)

155b 12.9 (9.0-15.0)h

-

7

-

Geld

YO

100

57

22

n The vsll~es in parentheses indicate the range of cytochrome P-450 concent rat ions found in a series of such experiments.

* The method used routinely for protein determination (24) gave erroneously high values wit,h the most purified enzyme preparations. The protein content was dotermincd by amino acid analysis, which gave a value 70°jo as great,.

experiment on the purification of rytochromc 1’.450 from pyro- phosphatt:~cstrac,t(,(l liver rnicrosomcs are shown in Table I. The range of specific activitic,s obtained at cacl1 step in a series of such cxpcrimcrits is also givtll; as indicated, ryto&romc~ I’-450 c,oil~c,lrtratiolIs as high as 15.0 Iunolcs per mg of protcGt1 11nvc bc,cn obtainc~tl by this proccdurr. I’olyrthylrrlc~ &~l, which has burl usrtl prGously for tl1c purification of several other protc% (49), provrtl to bc useful in the sqaration of cytorhromct I’-450 from thr rcductasc and in tlir purification of both of thcsc, protc%s, 111 thr cxpcrimcnt shower tl1c\ protein fractioli prclcil)itatirig bc>twcrn 8 and lo%, ~~olyc~tllyl~~~lc~ glycY11 was usrtl for furthor frac6onatiori of c*ytoc~hronic~ I’-450. 111 sorm illstariccs, hovirvcyr, tht fractions prc~cipitatirig from (i to 8% arid 10 t,o 139, l~olyrthylcnc~ glycol contained apprc&bl~~ amounts of t,his c~ytochrornc~ (4.0 to 5.8 nmolcs prr rrrg of protcGn). Thr total rrcovcry of c~ytoclironrc~ I’-450 in t,hr threr frac:t,iolls rangrd from 80 to OO(j$, a11d little or IIO cytochrome I’-420 mns detected. FVllc~rl thcl proc(‘durc was carried out at higher tcm- pcraturrs (26) (e.g. 200), the 8 to 10% and 10 to 13% polyrt~hyl- cm glycol I)rcGpitatcls rontainrtl cytochromr I’-450 at a slightl) higher c,o1lcrr1tratiorl, but the tot,al rccovcry was only 30 to 450/,. The rholate to protrin ratio of 3 was chosen since it gave the best rrsults iii the polyet,liylcnc glycol frartioiiation; at lower ratios the cytoc~hrome I’-450 prccipitatrd at, a lower polycttiylene glycol concentratiol1 with less purification, and at hi&r ratios t,he cytochromc I’-450 \vas part,ly converted to cytochromc 1’.420.

Column chromatography of the enxymc solution on I)I2UG

ccllulosc ill the prcsrnce of 0.5% Kenex further purifitd the cytocllrome I’-450 and removrd all but traces of the rcmainirrg cytochrome bs arid NADI’H-cytochromc c reductase. The preparation was usually not assayed at this stage but irlstead promptly treated \vitl1 Amberlite XAI)-2 in a batch procedure or passrd through an Ambcrlite column in tar&m with the l)EAE-cellulose column, thereby lowering the Iienex conce~ tration. This procedure was necessary since cytochromc I’-450 is otherwise not adsorbed or1 calcium phosphate grl. Adsorption

onto and elution from calcium phosphate gel yielded an erlzyrne preparation containing less Kenex and havir1g 12.9 nmoles of cytochrome K450 per mg of protein in the experiment shown and as high as 15.0 in other experiments. Unless the detergent concentration is lowered by these latter steps, the hydrosylation activity of the cytochrome I’-450 is inhibited.

Component

Cyt,ochrome P-450. _. Cytochrorml P-420.. Cytochrome bs Heme. FAD.. FMN... Phospholipid.. NAl>PB-cytochrome c reductnse, NAL)H-cytochrome c reductase.. ltenex. Labile sulfide.

Concentration 01 activity (per mg of protein)

13 .O nmoles 0.3 nmole 0

14.4 nmoles 0.07 nmole 0.27 nmole

50.0 nmoles 2.5 X 1OP Imoles per min 4.3 X lo-’ gnoles l,er min

0.43 mg 0

Anal@ of Purified Cytochrome P-450 Jar Various Compo-

nents-Sonic aiialytiral data on the final c~ytorhromc 1’.450 prrparat,ion arc’ given in Table 1 I. Since low levels of ryto- chrornr l’-420 arr diflicult to &xtrrrninc in the prcscnc*c of qto- cliromr I’-450, tlit> latter was totally converted to c*gtochromc I’-420 by illcubatiorr for 4 min in 0.0 M urra at room tempera- turr, and by diffcrc~iirc thr original lcvrl was rstimatcd to be 0.2 Iimolc pc’r mg of protciii. No cgtochromc bs roultl be dc- tc,ctc:d using KAI)II RS the cxlrrtron donor undrr appropriate coiitlitions in tl1cj pros(‘nr(~ of d(~tc~rgrllt-solul)ilizrtl NASH- rytochrornc~ bs rrtluctasc: alId d~~oxgc~l1olatc~. The hcmc content was slightly highc,r thaii cqlc~rtc~tl from tlic c~ytoc~hrornrs prcsrnt, possibly hausc~ of thus I)rcxsrnc*cl of hcrnc: dissociated from thcsc protc+is, but in othcxr preparations no clstra hemtx was present (23). NAl~II-~~toc~l~roi~1~~ c rc&ctasr ai~tl NAl)l’II-cytoc:hromc c rcldurtascl wcro prc,scxllt mly at, vctry low lrvcls. 111 ot lier cytoc~liromc~ I’-450 Iq)aratioiis purific~d iii this mann(‘r and tlialyzc~tl (~stc~tisivolj il@illSt glass~tlistill~~tl wat(xr, flo c’oppc’r, rnangairc~sc, mol~l~tlc~ium, s~antliurn, or srlellium coultl b(l drtcc~ted by ncut,ron artivatioil analysis in the ~lichigau Ford nuclear rractor, :LJI~ 01lly traces of cobalt ard chromium w(lrc found. As dcscribcd clscwhc~rc (23), the prcsencr of noll-hcmc iron can be rulctl out bocausc the iron content, detcrr11iuetl b> atomic absorption, is equal t,o tlir heme content. No labilr sulfide v1 as clrtrc~trtl in the cytochromcx I’-450 preparation when it was analgzcd by a slight modification of published methods (50, 51).

Absorption Spectra of PuriJied Cyiochrome P-450-The absolutr spectra of various forms of the partially purified cytochrornc 1’.450 are shown in Fig. I, ln the osidized spectrum, maxima were seen at 568, 534, arid 417 run. up011 reduction of the protein by dithionitr the Soret band shifted to lower wave- lcngtli (414 nm) with a decrease in absorbance, and a single band at 542 ilm replaced the (Y and /3 bands seen in the osidized spectrum. The shift of the Sorct band toward the blue arid the absence of a tlistirlct peak or shoulder at 558 r1m up011 reduc- tion llrovided confirmatory cviderice for the absence of cyto-

chrome bg. The spect,rum of the CO c,omplex of the reduced cytochrome showed the typical peak at 450 nm. The slight shoulder at about 424 nm in the absolute spectrum may bc attributed to a low level of cytochromc I’-420 not detected in the CO difference sllectrurn of the reduced hemeprotcin. The extinction coefficients, determined from the absolute spectra in Fig. 1, are given in Table III.

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0’ I I 1 / I

390 420 450 480 510 540 570

WAVELENGTH (ml

FIG. 1. Spectra of partially purified cytochrome P-450. A preparation of cytochrome P-450 containing 9.0 nmoles per mg of protein was diluted to a concentration of 5.4 nmoles per ml in 0.05 M Tris-acetate buffer, pH 7.4, containing 200j0 glycerol and 1.0 mM EDTA, and the spectra were recorded at 20”. Curve 1, oxidized form; Curve 2, dithionite-reduced form; Curve 5, dithio- nite-reduced form after exposure to carbon monoxide. Inset, spectrum at higher wavelengths with a lo-fold expanded absorb- ance scale.

TABLE III Spectral characteristics of liver microsomal cytochrome P-450

Form of cytochrome P-450 Amax I c

Oxidized

Reduced

Reduced CO complex

nm

568 534 417 542 414 552 450

?nN-1 cm-’

14.2 13.6

124 17.4 94.2 15.2

117

Estimation of Slolecular Weight of Cytochrome P-&O--The apparent molecular weight of cytochrome F-450 was determined by gel permeation chromatography with glutamate dehydrogen- ase (52), xanthine oxidase, human y-globulin, and aldolase as standards to be 280,000, and sucrose density gradient centrifu- gation yielded an SZO,~ value of 12.2 corresponding to a molecular weight of 270,000. During gel chromatography a small amount of the cytochrome p-450 was converted to cytochrome p-420, whereas no such denaturation was observed during the centrifu- gation experiment. These values must be considered provi- sional since the cytochrome P-450 preparations contain more than one polypeptide component, as described below.

Disc Gel Electrophoresis of Cytochrome P-450 Preparation- Sodium dodecyl sulfate polyacrylamide electrophoresis was used to examine the pattern of polypeptides in the various cytochrome P-450 preparations, as shown in Fig. 2. The various enzyme preparations were treated with mercaptoethanol and sodium dodecyl sulfate to yield the individual polypeptides prior to electrophoresis. In Experiment 1, the microsomes from pheno- barbital-induced rabbits Kere found to contain a group of major polypeptides with mobilities of 0.5 to 0.7 along with a number of other less prominent bands. Upon polyethylene glycol fractionation several of the peptides were removed or decreased in amount, and in the most purified preparation only two major

FIG. 2. Polyacrylamide gel electrophoresis of cytochrome P-450-containing enzyme preparat,ions in the presence of dodecyl sulfate. Electrophoresis was carried out as described in the text, with migration from top to bottom. In Experiment 1, the follow- ing samples were used: A, pyrophosphate-extracted microsomes from phenobarbital-induced animals (34 pg of protein); B, 8 to 10% polyethylene glycol precipitate (16 pg of protein); C and D, purified preparation containing 9.0 nmoles of cytochrome P-450 per mg of protein (5 and 20 pg of protein, respectively) ; in Experi- ment 2: h’, pyrophosphate-extracted microsomes from normal animals (20 fig of protein); P, pyrophosphat,e-extracted micro- somes from phenobarbital-induced animals (20 pg of protein); G and H, purified preparation containing 10.2 nmoles of cyto- chrome P-450 per mg of protein (7 and 14 rg of protein, respec- tively) .

bands were seen. In Experiment 2, a comparison was made of liver microsomes from normal and induced animals, and it is apparent that treatment with phenobarbital greatly increased the concentration of the polypeptide band indicated by the arrow.

This band is presumably due to cytochrome P-450, which is seen as the major component in the purified preparation of cytochrome I’-450 (tubes G and H). These experiments indicate that the best preparations are not yet homogeneous, but the purity may be greater than 70%.

Other investigators have examined the polypeptides in normal and phenobarbital-induced rat liver microsomes by this tech- nique. They have concluded that either one (16, 53) or two peptides (54) in the 50,000 molecular weight range are increased in amount by the administration of this drug.

Molecular Weight Estimated by Calibrated Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis-The molecular weight of the polypeptide chain of the partially purified cyto- chrome was estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis experiments in which standard proteins were included. The cytochrome P-450 preparation used contained a major band and two relatively minor bands, as already shown in Fig. 2 (tubes G and H). The results, presented in Fig. 3, indicate that the major polypeptide in the cytochrome P-450 preparation had a molecular weight of about 47,000 and two other minor components had molecular weights of about 52,000

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and 56,000. From this and other experiments it appears that of the two major polypeptide bands seen upon gel electrophoresis of the purified cytochrome P-450, one has a molecular weight of about 47,000 to 49,000 and the other 52,000 to 53,000.

Anaerobic Titration of Cylochrome P-450 Preparation by Dithionite-As reported recently (23), the titration of a cyto-

I I I I I 0 0.2 0.4 0.6 0.8 1.0 1.2

Relative Mobility

FIG. 3. Estimation of molecular weight of polypeptide chains in purified cytochrome P-450 from electrophoretic mobility in polyacrylamide gel in the presence of sodium dodecyl sulfate.

Dithlomte Added (nmoles)

FIG. 4. Cytochrome P-450 reduction as a function of dithionite added. The titration with dithionite was carried out in the ab- sence of oxygen under conditions described previously (23). A solution of cytochrome P-450 (21.2 nmoles; 1.60 mg of protein) was dialyzed overnight against 100 volumes of 0.013 M Tris buffer, pH 7.4, containing 1.0 X 10Y4 M EDTA and 10yO glycerol, with a change of the buffer solution at the end of about 6 hours. The dithionite was standardized against a solution of lumiflavin-3- acetate.

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chrome P-450 preparation containing 6.0 nmoles per mg of protein by dithionite in an oxygen-free atmosphere of carbon monoxide revealed the presence of an electron acceptor distinct from the iron atom of the protein. Following the reduction of small amounts of other electron acceptors during a lag phase, the reduction of cytochrome P-450 proceeded in a linear phase characterized by the consumption of approximately 1 mole of dithionite per mole of cytochrome P-450. Since dithionite is a 2.electron donor, it was evident that 2 electrons were taken up for each molecule of cytochrome P-450 reduced. In other experiments carried out under similar conditions, cytochrome c and cytochrome P-450cam (kindly provided by Dr. I. C. Gunsalus, University of Illinois) exhibited the expected uptake of only 1 electron per molecule (23). Peterson (55) has reached the same conclusion for the bacterial protein using dithionite, and Gun- salus et al. (56) have reported that only 1 electron is donated to the bacterial protein under anaerobic conditions by NADH.

Cytochrome P-450 purified to a concentration of 13.2 nmoles per mg of protein by the procedures described in the present paper was titrated with dithionite under anaerobic conditions with CO present, and the formation of the reduced CO complex was followed by the appearance of the characteristic peak at 450 nm. The results are presented in Fig. 4. After a brief lag phase, cytochrome P-450 reduction was found to be propor- tional to the amount of dithionite added. A total of 22.1 nmoles of dithionite were required for the reduction of 20.6 nmoles of cytochrome P-450 in the linear phase, corresponding to 2.1 electrons per molecule. Clearly, therefore, the more purified preparations of cytochrome P-450 still contain an electron ac- ceptor in addition to the iron atom of the heme. The results suggest that this additional acceptor has an oxidation-reduction potential similar to that of cytochrome P-450.

Purification of NADPH-Cytochrome P-450 Reductase-Table IV summarizes the results of a typical experiment in which the reductase was purified about 25.fold as measured by cytochrome c reduction. The polyethylene glycol fractionation step led to a 3-fold purification wit,h about 50% recovery of the reductase. The resulting preparation contained cytochrome P-450 and cytochrome P-420, which were removed by the DEAE-cellulose column chromatography step in the presence of Renex because they were eluted before the reductase. The presence of glycerol was important since in its absence a large loss of enzymatic activity occurred. After elution of the reductase from the ion exchange column, the enzyme was adsorbed onto calcium phos- phate, and the gel was then washed uith buffer to remove most of t,he detergent. The reductase was readily bound to calcium phosphate, whereas cytochrome P-450, as already described, was not bound unless treatment with Amberlite XAD-2 was carried out to further lower the Renex concentration. The calcium phosphate strep effect,ively decreased the concentration of the nonionic detergent in the reductase preparation.

TABLE IV Partial puri$calion of NADPH-eytochrome c reductase

Preparation

Pyrophosphate-extracted microsomes Polyethylene glycol (13%) supernatant fraction; am-

monium sulfate precipitate (607, saturation). DEAE-cellulose column eluate (with 0.4oj, Renex); cal-

cium phosphate gel eluate ---

VOIU~~ Protein

ml mg

120 3000

80 480

40 32

Units Specific activity

1050 0.35 (0.13-0.35)

557 1.16 (1.06-1.66)

275 8.6 (5.1-10.0)

Yield

%

100

52

26

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TABLE V Analysis of partially purified reductase

Component

NADPH-cytochrome c reductase.. NADPH-ferricyanide reductase.. NADH-cytochrome c reductase.. NADH-ferricyanide reductase. FAD.... FMN... Cytochrome P-450’. 1. Cytochrome P-420.. . . . Cytochrome ba. Heme... Labile sulfide. Phospholipid. Detergent (Renex)

-

_-

-

Concentration or activity (per mg of protein)

8.6 pmoles per min 11.2 rmoles per min 0.027 hmole per min 0.12 rmole per min 2.5 nmoles 2.8 nmoles 0 2.3 nmoles 0.6 nmole 3.5 nmoles 0

83.8 nmoles 1.15 mg

TABLE VI

Requirements for benzphetamine hydroxylation The complete reaction mixture included a cytochrome P-450

preparation containing 9.0 nmoles per mg of protein (0.10 nmole), reductase having a specific activity of 7.0 (0.09 mg of protein), and the other usual components. The values given have all been corrected for the rate when benephetamine was omitted (0.006 per min), which represents the NADPH oxidase activity of the enzyme system in the absence of substrate. ----.-. -

Conditions I

Rate

Complete. . No cytochrome P-450. No reductase. No dilauroyl-GPC .‘. . : No deoxycholate. No dilauroyl-GPC and no deoxycholate

-AAtro/min

0.024 0.002 0 0.005 0.012 0.004

Properties of Partially PuriJied NADPH-cytochrome P-450 Reductase-The results of the analysis of a typical NADFH- cytochrome P-450 reductase preparation, made as described above, are presented in Table V. The preparation was free of cytochrome P-450 but still contained cytochromes p-420 and bs. Only negligible amounts of NADH-ferricyanide reductase ac- tivity and NADH-cytochrome c reductase activity remained. In other experiments it was shown that such preparations slowly reduced added cytochrome 65 in the presence of NADI H and even more slowly in the presence of NADH. The reductase preparation contained a total of 5.3 nmoles of flavin per mg of protein, with FAD and FMN contributing about equally; Iyanagi and Mason (21) have previously reported the presence of both flavin nucleotides in purified reductase preparations active toward cytochrome c. The phospholipid content of the purified reductase was approximately %a of that present in microsomes, and no labile sulfide was detectable in the prepa- ration Sodium dodecyl sulfate polyacrylamide gel electro- phoresis of such a purified preparation revealed a major poly- peptide band having a molecular weight of about 80,000 and several bands of greater and lesser mobility.

Substrate Hydroxylation in Partially Purijied, Reconstituted Enzyme System-The effect of omitting individual components from the reaction mixture is shown in Table VI. The hydroxyl- ation of benephetamine, measured by the spectrophotometric

Cytochrome P450 Added (nmole) Reductose Fraction Added (mg Proten)

FIG. 5. Benzphetamine hydroxylation as a function of cyto- chrome P-450 or NADPH-cytochrome P-450 reductase concentra- tion. In Experiment A the reductase preparation (specific activ- ity, 8.6) was present in the amount of 0.016 mg of protein and the cytochrome P-450 was varied, and in Experiment B the cyto- chrome P-450 preparation (10.5 nmoles per mg of protein) was present in the amount of 0.10 nmole and the reductase was varied.

assay, did not occur at a significant rate when either the cyto- chrome l’-450 or reductase was omitted. We have recently found that under some circumstances detergents partially replace the phospholipid requirement for substrate hydroxylation. In many purified enzyme preparations, dilauroyl-GPC and deoxy- cholate are both required for maximal activity. In contrast, in the less purified preparations used earlier, which contained deoxycholate as the only added detergent, an almost complete dependence upon added phospholipid for the hydroxylation of various substrates was observed (7).

In Fig. 5, benzphetamine hydroxylat’ion (measured by the substrate-dependent disappearance of NADPH) is shown as a function of the concentration of cytochrome Y-450 or the reduc- tase. At lower levels of the enzyme component being varied, a linear relationship was seen, and it is apparent that within this region each of the enzymes may be assayed by its effect in sup- porting substrate hydroxylation. For maximal activity the enzyme components must apparently be present in a particular ratio; evidence has been presented earlier that a dissociable complex may be formed from these two enzymes and the phos- pholipid in the reconstituted system but that membrane-like structures are not formed (57).

The turnover numbers for various substrates at 30” are pre- sented in Table VII. The partially purified cytochrome P-450 retains the ability to hydroxylate drugs, of which d-benzpheta- mine is the most active, as well as alkanes and fatty acids. For comparison, the activities of several of the substrates in intact microsomes are also given. The data indicate that with all substrates except laurate the reconstituted system was more active than the intact microsomal system, as judged by turnover numbers related to cytochrome P-450. It should be noted that in the reconstituted enzyme system the experimental conditions may be readily adjusted so that cytochrome P-450 is the rate- limiting component, whereas this is not the case with microsomal suspensions. Nonetheless, the data presented indicate a simi- larity in substrate specificity in the microsomal and purified preparations. Under the conditions employed, including optimal dilauroyl-GPC and detergent concentrations, a turnover number of over 40 was determined for benzphetamine. Even higher

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TABLE VII

Substrate hydroxylation in rabbit liver enzyme system

The usual assay conditions were used with cytochrome P-450 as the rate-limiting component. Lauratc and octane hydroxyla- tion were determined by the radioactive assay, aniline hydroxyl- ation by a published method (58, 59), and hydroxylation of the other substrates by NADPH disappearance. A similar value was obtained for ethylmorphine when formaldehyde formation was determined.

Substrate

Henzphetamine. Cyclohexane.. Hexane Octane. Hexobarbital. I<:t hylmorphinc. Laurate.. Aniline

Apparent turnover number

MicrosomeP Purified system

nnoles/nmole P-450/?&

9.4 (14.9)

1.7 (2.4) 1.6 0.3 (0.3)

41.8 40.3 35.7 14.5 7.8 3.1 0.9 0.5

a The values in pnrcnthrscs indicntc the activities measured when the rnicrosomal suspension was supplement.ed with t hr liver supcrnatant, fraction (0.6 mg of protein) prepared by ex- traction of an acetone powder according to Nelson el al. (GO).

turnovcsr numhcrs have \~fsfw dcterminc~tl in othrr cxpc~rirnc~nts (cf. Fig. 5R). m’hc+hc~r thfl highrr turnover numbers see~l with

the lmrificd ~IIZ~YYW ~(1 duct to the removal of an inhibitory

substnncc or duo to the q)urifi&ion of an accessory factor is unccrtaill.

The values given in parclnthrscs in Table VI 1 for th(l micro- somes wf’rf’ obtained ill the prrsrllcr of a supcrnatant fraction which, nfarordirlg to r\‘c~lson et al. (60), contains a stimulating

factor. ‘I’hc> h)drosylation activity t,owartl bcrlzphctarninc> and cthylmorphinc was rnhanccd in the proscncc of t,his fraction, but no f~tmrigr was fhsfwfd irl miiliric liyflrosylatiori. IIowvever,

cont,rary to the, obsrrvation of Gandolfi ard Van 1 )yke (cl), no

stimulation of bonzphrtaminr hydroxglation was obscrvcd upon the addition of the supernatant fraction to the rcconstitutrd cnzymr system.

R11cw the liver microsomal ellzyrnc system was first solubilizcd and resolved by column (ShromatoRraphy OII 1 IEAE-c~~llulosc, two fractions, designated A and I%, were found to be necessary for hytlrosglation activity (2). Fraction A contained both cgtochrornc II-450 and NXDYH-cytochrome c reductase, which were partially separated because the latter was eluted from the column at higher ionic strength, and Fraction 1% contained a heat-stable componrnt which was called Factor 13 and subse- quently identified as phosphatidylcholine (4). The present paper describes the separation of the two enzymes by the use of polyethylene &co1 and procedures for the partial purification of each enzyme to a point where it is completely free of the other. The data givrn in the present paper show that cytochromc 1’.450 free of cytochrome bs and containing YIO appreciable amount of other known cltctron acceptors such as noll-heme iron, other metals, or Ilavms accepts 2 electrons from dithionite. This finding, which confirms results reported elsewhere on somewhat less purified preparations of cytochrome I’-450 (23), clearly indicates the presence of an electron acceptor distinct from the

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iron atom of the hemeprotein. The unidentified acceptor, which will be referred to as Factor C, appears to have an oxida- tion-reduction potential similar to that of cytochrome l>-450. Whether t’his component is closely associated with or separate from the heme of the cytochrome P-450 molecule, or even in a separate polypeptide chain, and whet,her it plays a unique role in the hydroxylation reaction remain to be established.

The solubilized cytochrome r-450 from liver microsomes exhibits substrate difference spectra and electron paramagnetic resonance spectra characteristic of the membrane-bound form, as rcportrd rarlier (3, 5). The extinction coefficients of the oxi- dized, reduced, and CO-reduced spectra of the partially purified protein, as shown in the present paper, are highly similar to those reported for this cytochrome isolated from Pseudomonas putida by Lipscomb and Gunsalus (62). Additional experiments not included in the present paper indicate that the purified liver microsomal rytochromc I’-450 gives characteristic type I and type I I diffcrrncc spectra with benzphetamine and aniline, respectively.

As reported elsewhere, the drtergrnt-solubilized NADPEI- cytochromc c rrductase is capablr of reducing cytorhromc 1’.450 (4) and of functioning in substrate hydroxylation (2). The purified, rcronstitutrd rnzymc system described in the prrsent paper retains the ability to hydroxylatc a variety of type I substratc,s, such as fatty acids, alkanes, and drugs, as well as a typcx I I substrate such as aniline, but a conclusion OII the cause of this rrmarkably broad specificity cannot bc drawn at this timr. It appears that neither the reductasc nor the phospho- lipid cnonfcrs substrate specificity on the system, particularly since the rat liver reductasc fraction and the synthetically pre- pared dilauroyl~G1’C function (Brctivrly with various cyto- rhromc I’-450 prrparations having different substrate spccifici- tics. Thus, cytochrornc P-450 from rat (7), rabbit (6), mouse (II), and human liver (12) and from a yeast (9, lo), as well as the c~arcillogc,tl-illtluciblc cytochromr l-‘-448 from mouse (11) and rat liver (13)) havr all been couplctl cffcctivcly with the rat liver microsomal rcductasc alltl with rat liver microsomal phospho- lipids or synthetically prcparcd dilauroyl-GI’C. It remains to b(l cstablishrd, however, wh(thrr multiple forms of cytochromc I’-450 occur in liver microsomrs. lsozymcs differing only slightly in their amino acid sequence mi&t not have been scpa- rated by t,he fractionation procrdures or clectrophoretic methods used in the present studies.

Acknou;ledgnLents-Wr wish to cxprcss our appreciation to Dr. I)avid A. ilaugen for carrying out the polyacrylamide clectro- phorctic experiments and t,o Dr. Frederick 1’. Guengerirh and Dr. David 1’. I<allou for the anaerobic titration of the purified cyto- chrome 1’.450. We also wish to acknowledgr the skillful assis- tance of Joanne K. Hciderna, Barbara M. Michnirwicz, and Sylvia 1%. Dahl.

Note Added in Proof-Liver microsomal cytochrome r-450 has been further purified recently by hydroxylapatitc column chro- matography to a concentration of 17.5 nmoles per mg of protein. Such preparations are apparently homogeneous as judged by polyacrylamide gel elect,rophoresis in the presence of sodium dodecyl sulfate. 1 he results obtained confirm the identification of the major electrophoretic band in partially purified prepara- tions (see Fig. 2) as the polypeptide chain of cytochrome 1’.450.

A paper from another laboratory describing the purification of rat liver microsomal cytochrome I’-450 to a concentration of 9 to 11 nmoles per mg of protein has recently appeared (63).

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Theodore A. van der Hoeven and Minor J. CoonRabbit Liver Microsomes

Nicotinamide Adenine Dinucleotide Phosphate-Cytochrome P-450 Reductase from Preparation and Properties of Partially Purified Cytochrome P-450 and Reduced

1974, 249:6302-6310.J. Biol. Chem. 

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