The Journal of Biochemistry, Vol. 67, No.

2, 1970

A New Method for Simultaneous Purification

of Cytochrome b5 and NADPH-cytochrome c
Reductase from Rat Liver Microsomes

By Tse NEO OMURA and SACHIKO TAKESUE

(Front Institute for Protein Research, Osaka University, Osaka)

(Received for publication, July 14, 1969)

1. A new procedure for the purification of cytochrome bs and NADPH-cytochrome c

reductase from rat liver microsomes is described. This procedure is a preparative
application of a small-scale procedure reported earlier, and is suitable for purifying
both enzymes simultaneously starting from several hundred grams or more of rat liver.
The method is applicable to liver microsomes of other animals.
2. The recovery of cytochrome b3 and NADPH-cytochrome c reductase from micro
somes was 35-45%, and the homogeneity of purified preparations was confirmed by
sedimentation analysis and disc electrophoresis. The high purity of the preparations
obtained by the previous small-scale procedure was also confirmed.
3. Purified NADPH-cytochrome c reductase was reducible by either NADPH or
NADII, and had a very weak NADH-linked reductase activity.

Cytochrome b5 was first isolated from rab

MATERIALS AND METHODS
bit liver microsomes by STRITTMATTERand
VELICK (1). The cytochrome has been puri Rat Liver-Adult male rats of the Sprague-Dawley

fied from bovine, swine, or rabbit liver micro strain were generously supplied by Takeda Phar

maceutical Co., Osaka. The animals were killed by

somes by several workers later (2-6). Liver
decapitation, and the livers were quickly excised and
NADPH-cytochrome c reductase (7) is also a
rinsed with ice-cold 0.15M KCI. The livers were
microsomal enzyme, and was first purified
stored frozen at -15--20•Ž until use.
from calf liver microsomes by WILLIAMSand Assay of NADPH-cytochrome c Reductase-NADPH-
KAMIN (8) followed by PHILLIPS and LANGDON eytochrome c reductase activity of microsomes and

(9 ), NISHIBAYASHIet al. (10), and HERNANDEZ purified enzyme preparations was assayed by measuring
et al. (11). the rate of reduction of yeast cytochrome c by NADPH.

This paper presents a new procedure by The reaction mixture contained 2•~10-5M yeast cyto

which both cytochrome b5 and NADPH-cyto chrome c, l•~l0-4M NADPH, and enzyme in 2 ml

chrome c reductase can be easily purified from of 0.1 M potassium phosphate buffer (pH 7.5). The

reaction was started by the addition of NADPH, and

rat liver. This method is a preparative ap
the increment of optical density at 550 mo was meas
plication of a small-scale procedure developed ured at 25°C by a Cary model 14 recording spectro
previously in order to study the turnover of
photometer. When the reductase activity of micro
these enzyme proteins in rat liver ( 12 ), and
somes was assayed, 1•~10-3 M KCN was included in the
is suitable for a large-scale preparation of reaction mixture to prevent the oxidation of reduced
these enzymes starting from several hundred cytochrome c by contaminating mitochondria.

grams or more of rat liver. It is also the Enzymatic reduction of 2, 6-dichlorophenolindo

purpose of this paper to confirm the homo phenol (DCPI), potassium ferricyanide, and purified
cytochrome b5 by NADPH or by NADH was similar-
geneity of the purified enzyme preparations
obtained by the previous small scale purifica ly measured at 25°C in 0.1 to phosphate buffer (pH

tion method (12 ). 7.5). The concentration of NADPH or NADH in the

reaction mixture was 1•~10-4 M in all cases; the con

centrations of DCPI, potassium ferricyanide and cyto

chrome b, were 4•~10-5 M, 2•~10-4M, and 1•~10-5 M,

249
250 T. OMURA and S . TAKESUE

respectively.
The reductase activities were calculated from the RESULTS

initial rate of reduction of electron acceptor, and ex-

Purification of Cytochrome b5 and NADPH-
pressed by using "unit": one unit of enzyme reduces
Cytochrome c Reductase front
one ƒÊmole of electron acceptor per minute under the
Rat Liner Microsomes
assay condition. The millimolr extinction differences
The following procedure is essentially the
(cm-1 mM-1) between reduced and oxidized electron
acceptors used in calculating enzyme unit were 21.1 same with the previously described small-scale

at 550 mƒÊ for cytochrome c (13), 21.0 at 600 m,u for method (12) except that all steps are modified

DCPI (14 ), 1.02 at 420 mp for potassium ferricyanide to fit for large-scale operations. The elution

(15), and 100 at 424 mƒÊ for cytochrome bs (1). of cytochrome b5 and NADPH-cytochrome c
Assay of Cytochrome b5-The content of cytochrome
reductase from a DEAE-cellulose column was
b5 in microsomes and in trypsinsolubilized superna
effected by a linear concentration gradient of
tant was determined by measuring reduced minus
KCl instead of increasing stepwise the con
oxidized difference spectrum of samples assuming the
centration of KCl as in the previous method
extinction difference of the cytochome between 424

mƒÊ and 409 mƒÊ to be 185 cm-1 mM-1 (16). Micro (121). Unless otherwise stated, all operations

somal suspensions were reduced by NADH ; the trypsin were performed at 0 to 5•Ž.

solubilized supernatant was reduced by Na2S2O4. Step 1. Preparation of " Washed Microsomes "
When interfering colored materials were not
from Rat Liz,er-Frozen rat livers (600-700g)
present in the samples (preparations after Sephadex were quickly thawed, rinsed with ice-chilled
chromatography), the cytochrome was assayed by
0.15M KCl, and then homogenized with 4
measuring the absorption at 423 mp of Na,S.Or-
volumes of 0.15 M KCl using a Waring blendor.
reduced samples using a millimolar extinction of 171
The homogenate was centrifuged at 10,000•~g
cm-1 mM-1 (1).
for 15 min, and the precipitate was discarded.
Other Analytical Methods-Protohernin in purified
cytochrome bs preparations was determined by an The supernatant was further centrifuged at

alkali pyridine hemochrome method (16). Flavin of 78,000•~g for 90 min to sediment microsomes.
NADPH-cytochrome c reductase was extracted by the Judging from succinate-cytochrome c reductase
treatment with 10% TCA at 0°C for 5 min, and as- activity, the contamination of the microsomal
sayed by a fluorometric method of BESSEYet at. (17) fraction by mitochondria was usually about
or by a spectrophotometric method (18). Protein was 5% or less.
determined by the method of Lower et al. (19) using
Microsomal pellets were suspended in 700
bovine serum albumin as the standard.
ml of 0.15 ,,1 KCl solution containing 10 mm
Disc gel-electrophoresis was carried out as de
scribed by DAVIS (20) using 7.5%3 acrylamide gel of EDTA (pH 7.0) by gentle homogenization,
and the suspension was centrifuged at 78,000 x g
pH 8.5 as the separation gel. Absorption spectra were
measured with the Cary model 14 recording spectro for 60 min. The pellets were then suspended
photometer. Sedimentation analysis was carried out in 700 ml of 0.1 M potassium phosphate buffer
by using a Hitachi model UCA-1 analytical ultra- (pH 7.5), and sedimented as before. The final
centrifuge. pellets (" washed microsomes ") were suspended
Reagents mid Biochemicats-Crystalline yeast in 350 ml of the same phosphate buffer to give
(Candida krusei) cytochrome c was a generous gift of a protein concentration of 15 to 20 mg per
Sankyo Co.. Tokyo. It was further purified before use ml. The recovery of washed microsomes was
by chromatography on an Amberlite CG-50 column
10-12 mg of protein per g of liver. The con-
(21). Purified cytochrome bs used in the assay of
tents of cytochrome b5 and NADPH-cytochrome
NADPH-cytochrome c reductase was prepared by the
c reductase in washed microsomes are shown
present procedure frorn rat liver microsomes.
NADPH, NADH, and trypsin [BC 3.4.4.4] (twice in Table I.
re-crystallized') were obtained from Sigma Chemical Step 2. Trypsin Digestion-One-tenth vol

Co. DEAF-cellulose and Sephadex G-100 were the ume of a trypsin solution (3 mg/ml) in 2 mm

products of Brown Co., and Pharmacia Co., re HCl was added to the suspension of washed

spectively. Other chemicals used were of reagent grade. microsomes, and the mixture was kept at 0•Ž

for 14 to 16 hr. The trypsin-digested suspension

was then centrifuged at 78,000•~g for 120 min,

 TABLE, I
Summary of /iurilicalion of cytorhromeb, and NADPH-rylochrome c reduetan from rat liner inie10lOins
The purification was carried out as described in text starting rim 700g of' rat liver.

1) The reductase activity of intact microsomes was stimulated by 20 -30;': by trypsin digestion (12 ) . The values listed here are the activ-
ity after trypsin treatment.

FIG
.1. Sephadex G-100 filtration of trypsin FIG.
2 . Chromatography of cytochrome b5 on
e xtract from rat liver microsmes.

DEAE-cellulose.
252 T. OMURA and S. TAKESUE

and clear red supernatant was collected by of the preparation.

decantation. More than 90 of NADPH- To concentrate purified cytochrome b5,
cytochrome ( reductase and 70 to 80% of cyto cytochrome-rich fractions were combined, di
chrome b5 were usually recovered in the super- luted by adding 2 volumes of water, and then
natant (Table I) (12). NADH-cytochrome put on a small column (2 x 3 cm) of DEAE-
b; reductase and cytoehrome P-450 were not cellulose equilibrated with 10 mM phosphate
brought into solution by trypsin digestion, and buffer. The cytochrome formed a narrow red
removed as the sediment (22). band at the top of the column. The column
The supernatant was freeze-dried. The was washed with 20 ml of 10 m.Ni phosphate
freeze-dried crude extract could be stored in buffer, and then the cytochrome was eluted
a freezer for a few months without detectable from the column by 50 mM phosphate buffer
loss of cytochrome b5 and NADPH-cytochrome containing 0.2 M KCl. Solution of concen
c reductase activity. trated cytochrome b5 (5 to 7 ml) eluted from
Step 3. Sephadex Filtration - The freeze- the column was collected, and dialyzed over
dried trypsin extract was dissolved in 100 ml night against 10 mM phosphate buffer of pH 7.5
of 10 mM phosphate buffer (pH 7.5), and the to give a final purified preparation of the cyto
solution was applied to a column (5:•, 80 cm) chrome.
of Sephadex G-100 equilibrated with 10 mM Step 5. Chromatography of NADPH-Cyto
chrome c Reductase on DEAE-Cellulose-Reduetase
phosphate buffer (pH 7.5). The column was
eluted with the same buffer at a flow rate containing fractions of the Sephadex eluate

30 to 40 ml per hour, and the eluate was col were combined, and put on a column (2•~30

lected in 20 ml fractions. Fig. I shows a typ cm) of well-packed DEAE-cellulose equilibrat

ical elution pattern indicating a clear separa ed with 10 mM phosphate buffer (pH 7.5). The

tion of cytochrome b; and NADPH-cytochrome reductase formed a yellow broad band at the

c reductase at this step. Fractions rich in cyto top of the column. The column was washed
with 50 ml of 50 mm phosphate buffer, and
chrome bs and fractions containing N ADPH-
cytochrome c reductase were separately col then eluted with an increasing linear concen

lected. tration gradient of KC1 from 0 to 0.35 M in

50 mM phosphate buffer prepared with 200 ml
Step 4. Chromatography of Cytochrome b5 on
of 50 mM phosphate and 200 ml of 50 mM phos
DEAE-Cellulose-Cytochrome b5-rich fractions
of the Sephadex eluate (120-150 ml) were com phate buffer containing 0.35M KC1. Thus
the condition for elution of the reductase is
bined, and applied to a column (2 x 25 cm) of
very similar with that for cytochrome b5. The
DEAE-cellulose, which had been well packed
flow rate was 20 to 30 ml per hr, and the
by applying pressure with a rubber bulb and
eluate was collected in 10 ml fractions
equilibrated with 10 mM phosphate buffer (pH . The
reductase came out of the column forming a
7.5). Cytochrome b5 formed a deep red band
single peak (Fig. 3), but the shape of the peak
at the top of the column. The column was
washed with 50 ml of 50 mM phosphate buffer
of the same pH, and then eluted with an in-
creasing linear concentration gradient of KC1
from 0 to 0.35 NI in 50 m n phosphate buffer
prepared with 175 ml of 50 mM phosphate buffer
and 175 ml of 50 mM phosphate buffer contain-
ing 0.35 ,,1 KCI. The flow rate was 20 to 30 ml
per hr, and the eluate was collected in 10 ml
fractions. Most of the adsorbed cytochrome
b5 came out of the column forming a single
peak (Fig. 2+, and the content of the cyto
chrome per mg of protein in fractions around FIG. 3. Chromatography of NADPH-cvto-
the peak was constant indicating high purity chrome c reductase on DEAE-cellulose.
 Cyt. b5 and NADPH-cyt. c Reductase 253

was always asymmetric showing a considerable cytochrome b5 gave a single band when ex
tailing of reductase activity in the higher KCl amined by disc electrophoresis (Fig. 4). Ul
concentration range. The specific activity of tracentrifugal analysis of purified preparations
the reductase in fractions around the peak also showed a single peak (Fig. 5) ; the sedi
was, however, almost constant. mentation coefficient (520) was 1.49 S in 50 mm
Reductase-rich fractions were combined, potassium phosphate buffer (pH 7.5) at a pro
and concentrated to a few milliliters by ultra- tein concentration of 0.5%.
filtration using a collodion bag (Sartorius Spectral properties of purified cytochrome
hlembranfilter Co.). The concentrated solu b5 were identical with those described by pre
tion was dialyzed against 10 mitt phosphate vious workers for the cytochrome from other
buffer (pH 7.5) overnight to give the purified animals (1-3,5). Reduced form showed ab
preparation of the reductase. sorption peaks at 423, 526, and 555 m t with
Table I shows the results of a typical puri a shoulder at 559 mu. Oxidized form had
fication experiment. The contents of the en absorption maxima at 414, 530, and 560 mg.
zymes in purified preparations were 78 to 82 The ratio of the optical density at a-peak of
mƒÊmoles per mg of protein for cytochrome reduced cytochrome to that at 280 my of the
b5 and 35 to 44 units per mg of protein for oxidized form was 1.28 to 1.32 (Fig. 6). The
the reductase. The purification ratios of the reduced cytochrome did not combine with
cytochrome and the reductase were usually carbon monoxide.
about 150 and 200 over microsomes, respec The content of protohemin in purified

tively, and the recovery of these enzymes cytochrome b5 was 80 to 81 mƒÊmoles per mg

from microsomes was 35 to 45%. of protein. The molecular weight calculated

Properties of Purified 6),tochrome b5-Purified from the heme content was about 12,500,
which agrees well with the value for cyto
chrome b5 of liver microsomes of other animals

reported by previous workers (4,23,6). In

this experiment, protein was determined by
LOWRY's method (17). The content of tyrosine

in cytochrome b5 is 3 moles per 90 amino acid

residues ( 24,6), which is almost identical with

FIG. 4. Disc electrophoresis of purified cyto

chrome b5 (right) and NADPH-cytochrome c reduc
tase (left). Fin. 5. Sedimentation patterns of cytochrome
Purified cytochrome b5 and NADPH-cyto b5 (right) and NADPH-cytochrome c reductase
chrome c reductase were separately analyzed by (left) in 50 mm potassium phosphate buffer of pH
the disc electrophoresis using columns (5.<70 mm) 7.5.
of 7.5% polyacrylamide gel of pH 8.5 as the Protein concentration, 0.50%. Rotor speed,

separation gel. 100 ƒÊg of the enzyme proteins 54,600 rpm (right) and 54,200 rpm (left). Rotor

were applied on each column, and the electro temperature, 15.0•Ž (right) and 15.9•Ž (left).

Time of centrifugation, 195 min (right) and 50

phoresis was carried out at 4°C with a current
of 3 mA per column. min (left) after attaining the top speed.
254 T. OMURA and S. TAKESUE

FIG. 6. Absorption spectra of purified cyto

chrome e b; .
Absorption spectra of a solution of purified Fm. 7. Absorption spectra of purified-
cytochrome b5 (0.059 mg protein per ml) in 0.1 M NADPH-cytochrome c reductase.

phosphate buffer (pH 7.5). Optical path, 1 cm. Absorption spectra of a solution of purified
Temperature, 25"C. NADPH-cytochrome c reductase (0,466 mg protein
Oxidized, per ml) in 0.1 M phosphate buffer (pH 7.5). Optical
---- Reduced with Na2S2O4 . path, 1 cm. Temperature, 25°C.
- Oxidized
,
---- Reduced by the addition of 0 .1 m+,t NADH
that of bovine serum albumin (20 moles of
to the air-saturated sample,
tyrosine per 602 amino acid residues) (25)
•c•c Reduced with Na.S,O,.
used in the present study as the standard for
protein determination.
Purified cytochrome b5 was stable. Storage in a few preparations, which could be elimi
of frozen solutions or freeze-drying did not nated by repeating Step 5 (DEAE-cellulose
affect the spectral properties of the cytochrome. chromatography).
The cytochrome was fully reducible by NADH Judging from the fluorometric assay ( 17)
and purified NADH-cytochrome b5 reductase, of the extracted flavin, the purified reductase

which was prepared from rat liver microsomes contained FAD. The content of the flavin

by the procedure described in one of the fol was 21 to 24 mƒÊmoles per mg of protein giv

lowing papers (2(;). ing a minimal molecular weight of 42,000 to

Properties of Purified NADPH-Cytochrorne c 48,000 per mole of the flavin. The oxidized

Redurtase-Analytical ultracentrifuation of puri enzyme had absorption peaks at 455, 380, and

fied reductase preparations showed a single 275 mp. The peak at 455 m,u disappeared by

reducing the enzyme with NADPH or Na2S2O4.

peak with a sedimentation coefficient (S20)of
4.6 S in 50 mu potassium phosphate buffer The purified enzyme was also reducible by

(pH 7.5) at a protein concentration of 0.5% NADH even in air-saturated solution (Fig. 7).

(Fig. 5). Examination by disc electrophoresis The ratio of the optical density at 455 mp to

also usually gave a single band (Fig. 4). How- that at 275 mƒÊ of the oxidized enzyme was

ever, disc electrophoresis revealed the presence 6.3. Judging from the oxidized and reduced

of trace amounts of contaminating proteins spectra (Fig. 7), purified reductase is completely
 Cyt. b5 and NADPH-cyt. c Reductase 255

TABLE II ity was noticed.

Rdductase aetioities of Purified NADPH-
cvtochromec reductase DISCUSSION

Conditions for the assay of reductase activ The purification procedure for microsomal
ities are described in text. The correction for cytochrome b, and NADPH-cytochrome c re
non-enzymatic reduction of electron-acceptors by
ductase described here is a preparative applica
NADH or NADPH is made in all assays. The
tion of the small-scale method reported earlier
alues listed are the mean values of three separate
preparations, all of which were homogeneous in
( 12 ). Starting from several hundred grams
sedimentation analysis and disc electrophoresis. of rat liver, both enzymes were easily purified
with good recovery, and the final preparations
of these enzymes were homogeneous when ex
amined by sedimentation analysis and by disc
electrophoresis in polyacrylamide gel. The
present procedure gave reproducible results,
and has already been employed in preparing
the reductase from rat liver microsomes (27 ),
and also in the preparation of trypsin-solu
bilized cytochrome b5 from rabbit liver micro
somes (28). A preliminary experiment showed
that the procedure was applicable to bovine
liver microsomes with very similar results.
Since the flavin prosthetic group of micro
somal NADPH-cytochrome c, reductase is easily
free from contaminating hemoproteins. dissociated from the enzyme protein in a
The activities of the reductase with differ mildly acidic solution containing a high con
ent electron donors and acceptors are shown centration of ammonium sulfate (29), frac
in Table II. As has already been described tionation by ammonium sulfate was purposely
by previous workers, the reductase is virtually avoided in the present method. The content
specific for NADPH (7-9). However, the of FAD in our purified preparations of the
purified enzyme catalyzed a slow NADH-linked reductase was fairly constant (21 to 24 mpmoles
reduction of cytochrome c. Since the purified per mg of protein), and the minimal molecular
preparation had practically no NADH-cyto weight of the enzyme per mole of flavin was
chrome b5 reductase activity, this weak NADH- about 42,000 to 48,000 which is considerably
linked reduction of cytochrome c was not caused lower than the value obtained by previous
by the contamination of microsomal NADH- workers (8, 9 ).
cytochrome b reductase. The association of When solubilized from microsomes by
a weak NADH-linked reductase activity and trypsin treatment, cytochrome b5 and NADPH
a strong NADPH-cytochrome c reductase ac cytochrome c reductase behave similarly toward
tivity with the same enzyme molecule was ion-exchange chromatography on DEAE-cel
also suggested by the reduction of the flavin lulose (9 ). The difficulty to eliminate con
of purified enzyme by NADH. taminating hemoproteins from NADPH-cyto
The purified reductase was stable. When chrome c reductase preparations was described
kept frozen at -15 to -20°C, solutions of the by previous workers (30), and the absorption
reductase in 10 mM phosphate buffer (pH 7.5) spectra of most of their preparations showed
did not lose its reductase activity for months. the presence of trace amounts of hemin-com
The sclutions could also be kept at 0°C for a
pounds (8, 9 ). In the present method, cyto
few days without measurable decrease in ac chrome b5 was clearly separated from the re
tivity. However, when a very dilute solution ductase by a gel-filtration technique using
of the reductase in a neutral buffer was kept Sephadex G-100. Small amount of hemoglobin,
at 25•Ž or 30•Ž, gradual loss of enzyme activ- which was present in the reductase-rich frac-
256 T. OMURA and S. TAKESUE

tions of Sephadex eluate (Step 3), was then the contents of enzymes per mg of protein in

removed by chromatography on DEAE-cel final preparations are usually 75 to 80 mƒÊmoles

lulose. Our final preparations of the reductase of cytochrome b5 and 32 to 40 units of NADPH-
were always free from contaminating hemin cytochrome c reductase. These values are al-

compounds. most the same with those of purified enzymes

Some previous workers noticed the pres obtained by the present method, and thus

ence of two separable cytochrome b5 species confirm the high purity of final preparations

in their purified preparations of the cyto prepared by the small-scale procedure which
chrome ( 21, 6, 31). These two forms of the has already been used in studying the rate of

cytochrome have identical spectral properties synthesis and degradation of these microsomal

and very similar molecular size, and can be components in vivo (12, 27).

separated by electrophoresis or by chromato

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