/ . Biochem., 71, 447-461 (1972>
Occurrence of Different Types of Cytochrome fe-like
Hemoprotein in Liver Mitochondria and Their
Intramitochondrial Localization
Kazuo FUKUSHIMA,* Akio ITO,t>** Tsuneo OMURA**
and Ryo SATO
Received for publication, August 16, 1971
Hypotonic treatment of rat liver mitochondria caused the solubilization of a fr-type
cytochrome (" IS-cytochrome ") which was spectrally similar to cytochrome b} and
reducible by NADH in the presence of NADH-cytochrome 6S reductase [EC 1.6. 2.2].
Tryptic digestion of hypotonically treated mitochondria resulted in the release of
another 65-like cytochrome (" OM-cytochrome ") into solution.
Cell fractionation studies indicated that IS-cytochrome is a true mitochondrial
constituent. Its solubilization behavior paralleled that of adenylate kinase [EC 2.7.
4.3], suggesting that it is located in the mitochondrial intermembrane space. Hypotonic solubilization of IS-cytochrome was also observed with mitochondria from
rabbit liver, rabbit kidney and pig heart.
The outer membrane purified from hypotonically treated rat liver mitochondria
contained a high concentration of a &5-like cytochrome. This cytochrome could be
solubilized by tryptic digestion of intact mitochondria in an isotonic medium. When
exposed to a hypotonic medium, the trypsin-treated mitochondria could still release
IS-cytochrome, and the outer membrane isolated from these mitochondria was essentially free from 65-like cytochrome. It was thus suggested that OM-cytochrome isfirmly bound to the outer surface of outer mitochondrial membrane.
By Sephadex gel nitration IS-cytochrome was separated into two hemoprotein
components, both having almost the same spectral properties. The large component
(molecular weight, ~ 120,000) was labile and converted gradually to the small one
(molecular weight, ~ 12,000) on incubation of mitochondria in the hypotonic medium.
Although both components could reconstitute an NADH-cytochrome c reductase system when coupled with microsomal NADH-cytochrome bt reductase, their efficiencies
in this system were much lower /than that of cytochrome bt. Neither of the IScytochrome components reacted with an antibody to rat liver microsomal cytochrome 6S.
* Permanent address: Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University,
Matsudo, Chiba. *• Present address: Department of Biology, Faculty of Science, Kyushu University,.
Fukuoka.
•Vol. 71, No. 3, 1972
447
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Institute for Protein Research, Osaka University, Osaka,
and TDepartment of Biochemistry, Kyushu University
School of Medicine, Fukuoka
448
al. (7, 8) and Sottocasa et al. (9, 10) have
later presented evidence that both the btlike cytochrome and the rotenone-insensitive
NADH-cytochrome c reductase activity of
liver mitochondria are associated with the
outer membrane. The localization of the rotenone-insensitive activity in the outer membrane has also been confirmed by Schnaitman
and Greenawalt (11).
On the other hand, in 1967 Siekevitz observed that a 6s-like cytochrome can be released
from liver mitochondria when they are suspended in a hypotonic medium.* Davis and
Kreil (12) have noticed the same phenomenon
and shown that the cytochrome thus released
is similar in molecular weight and spectral
properties to cytochrome bt which has been
enzymatically solubilized from liver microsomes, but differs from the latter in electrophoretic mobility and solubility in ammonium
sulfate solutions. Sottocasa et al. (9, 10) have
also observed the solubilization of a substantial
amount of Vlike cytochrome during the swelling-shrinking and sonication procedure for
mitochondrial subfractionation.
Although these investigators seem to have
believed that the cytochrome released by hypotonic treatment had been derived from the
outer mitochondrial membrane, the observations described above raise a question regarding the relationship between the hypotonically
solubilizable cytochrome and that attached to
* P. Siekevitz, private communication.
the outer membrane. It is also unclear whether or not the cytochrome associated with the
outer membrane is identical with microsomal
cytochrome b-,. The present study was undertaken to clarify these ambiguities.
In this paper, we report that rat liver
mitochondria contain two different types of
i>5-like cytochrome; one is readily solubilizable
by hypotonic treatment and located in the
intermembrane space, whereas the other is
bound to the outer membrane and requires
tryptic digestion for its solubilization. Although the term "hypotonic b" or "hypo b"
has been used in preliminary reports (13—15)
to denote the former hemoprotein, we now
wish to call it "IS-cytochrome" to indicate its
localization in the intermembrane space. Likewise, the latter will be called "OM-cytochrome." We also report that IS-cytochrome
is a mixture of two hemoprotein components
differing in molecular weight and that both
components are different immunologically from
microsomal cytochrome bt. The nature of
OM-cytochrome will be described in a latter
communication.
EXPERIMENTAL PROCEDURE
Preparation of Mitochondria and Microsomes--Male Sprague-Dawley rats, weighing
200-300 g, were fasted for 1 day and killed by
decapitation. The livers were excised, perfused
with cold 0.25 M sucrose, and homogenized
with 9 volumes of cold 0.28M sucrose containing 1 mM Tris-HCl, pH 7.2, and 0.1 mM EDTA
in a Potter homogenizer equipped with a
Teflon pestle. The homogenate was centrifuged
at 900 xg for 15min and the resultant pellet
was discarded. Mitochondria were then sedimented from the supernatant by centrifugation
at 6,000 x g for 20 min. After careful removal
of the fluffy layer, the mitochondrial pellet
was washed twice by suspending it in 1/2 and
1/4 of the initial volume of the medium used
forhomogenization. The washed mitochondria
were suspended in a suitable amount of the
same medium. For isolation of microsomes,
the 6,000xg supernatant was centrifuged at
10,000xg for 20min and the pellet was discarded. Microsomes were then sedimented by
/ . Biochem.
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It is well established that liver microsomes
possess an NADH-cytochrome c reductase
system which is insensitive to respiratory inhibitors such as antimycin A, amytal and
rotenone, and that this system consists of
flavoprotein NADH-cytochrome bt reductase
[EC 1.6.2.2] and cytochrome bt (1, 2). In
1958-1960, Raw, Mahler and coworkers (3-6)
reported that a similar system occurs also in
liver mitochondria. They have shown, among
other things, that a cytochrome closely resembling microsomal cytochrome bt and an
NADH-specific flavoprotein capable of reducing
this cytochrome could be extracted from liver
mitochondria with a Tris buffer containing
10% ethanol (3, 4). Using different methods
for mitochondrial subfractionation, Parsons et
K. FUKUSHIMA, A. ITO, T. OMURA and R. SATO
CYTOCHROME is-LIKE HEMOPROTEINS IN MITOCHONDRIA
Vol. 71, No. 3, 1972
inner membrane-matrix fraction. Centrifugation of this supernatant at 35,000xg for 20
min produced a pellet consisting of an upper
white layer and a lower light-brown layer.
The upper white layer containing the outer
membrane was suspended in a small amount
of 20 mM potassium phosphate buffer, pH 7.2,
by adding the buffer to the tube and gently
swirling. The resultant suspension was layered
over a discontinuous density gradient consisting of 5 ml of 23.7% sucrose (w/v) in 20 mM
potassium phosphate buffer, pH 7.2, and 15 ml
of 37.2% sucrose (w/v) in the same buffer, and
centrifuged at 65,000 X g for 90 min in a RP-30
rotor of a Hitachi 40P centrifuge. The turbid
layer formed at the boundary between the two
sucrose solutions was collected by a bent
Pasteur pipette and used as the purified outer
membrane fraction. The yield was very low
(2-3 mg of protein), indicating that most of
the outer membrane had been lost in other
subfractions, mostly in the inner membranematrix fraction.
An attempt was, therefore, made to recover the outer membrane from the other
subfractions. The inner membrane-matrix
fraction was suspended in 100 ml of the swelling medium, homogenized vigorously in a
Potter homogenizer equipped with a tightly
fitted Teflon pestle (10 passages of the pestle),
and then centrifuged at 1,900xg for 15min.
The resultant pellet was treated as above once
more. The supernatants were combined and
centrifuged at 35,000 x g for 20 min. The pellet
thus obtained was mixed with the light-brown
layer described above and suspended in 0.25M
sucrose. This crude outer membrane fraction
was contaminated by a large amount of inner
membrane fragments, but was useful for estimation of the total content of 6s-like cytochrome associated with the outer membrane.
The recovery of the rotenone-insensitive
NADH-cytochrome c reductase activity of the
starting mitochondrial preparation in the combined purified and crude outer membrane
fractions was about 80%.
In some experiments, mitochondria were
digested with trypsin [EC 3.4.4.4] in an isotonic medium prior to subfractionation. For
this purpose, trypsin (final concentration,
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centrifugation at 77,000 xg for 90min. Mitochondria were also prepared from rabbit livers
and kidneys by essentially the same procedure.
Pig heart mitochondria were isolated as described by Oda and Hayashi (16).
Cell Fractionation—For studies of intracellular enzyme distribution, rat livers were
homogenized with 9 volumes of 0.25 M sucrose
containing 1 mM EDTA (adjusted to pH 7.2),
and the homogenate was fractionatedd as follows. After removal of the cell debris and
nuclei by centrifugation at 1,000 X g for 10 min,
the supernatant was centrifuged successively
at 3,300xg for 10min, at 6,000xg for 10min,
at 11,700xg for 35min, and finally at 77,000
xg for 90 min to sediment the heavy mitochondrial, light mitochondrial, lysosomal, and
microsomal fractions, respectively. Each fraction was washed once by resuspending it in
0.25M sucrose followed by centrifugation.
Hypotonic Treatment of Mitochondria—
Mitochondria freshly prepared or stored at
0°C for less than 24 hr were used. The mitochondrial pellet sedimented from the sucrose
medium by centrifugation at 10,000 xg for 20
min was suspended, with the aid of a Teflon
pestle fitted to the centrifuge tube, in a hypotonic medium, usually 10 mM Tris-phosphate
buffer, pH 7.2, to a protein concentration of
10—20 mg per ml. The suspension was kept
at 0cC for 10 min, unless otherwise stated, and
then subjected to high-speed centrifugation
(usually at 105,000x g for 30 min). High-speed
centrifugation was essential to minimize interferences due to fragmented outer mitochondrial
membrane. The supernatant thus obtained
was referred to as "hypotonic supernatant."
Preparation of Outer Mitochondrial Membrane—This was carried out essentially as described by Parsons et al. ( # ) . Mitochondria
from about 35 g of rat livers were suspended
slowly in 50 ml of 20 mM potassium phosphate
buffer, pH 7.2, containing 0.02% bovine serum
albumin ("swelling medium"). After the suspension had been kept at 0°C for 20 min, it
was centrifuged at 35,000xg for 20min, and
the supernatant was saved ("hypotonic supernatant"). The pellet was resuspended in 100
ml of the swelling medium, and centrifuged
at 1,900xg for 15min to precipitate a swollen
449
450
Protein Determination-Protein was determined by the method of Lowry et al. (18)
with bovine serum albumin as the standard.
In some cases, absorbances at 280 nm were
measured to express relative protein contents.
Enzyme Assays—NADH-Cytochrome c and
NADPH-cytochrome c reductase activities
were measured as described by Takesue and
Omura (19) and Omura and Takesue (20),
respectively. Succinate-cytochrome c reductase, acid phosphatase [EC 3.1.3.2], and urate
oxidase [EC 1.7.3.3] were assayed by the
methods of Stotz (21), Gianetto and de Duve
(22), and Mahler et al. (23), respectively.
In the adenylate kinase [EC 2.7.4.3] assay,
the terminal phosphate group of ATP formed
from ADP was transferred to glucose by hexokinase [EC 2.7.1.1] and the amount of glucose
6-phosphate thus produced was determined by
the use of glucose-6-phosphate dehydrogenase
[EC 1.1.1.49] as described by Schnaitman and
Greenawalt (11). Isocitrate dehydrogenase
[EC 1.1.1.41] was determined also according
to Schnaitman and Greenawalt (11). All the
spectrophotometric measurements were carried
out in a Cary 14 spectrophotometer.
Immunological
Procedure—The r-globulin
fraction of rabbit antiserum to cytochrome biy
which was purified from rat liver microsomes
after tryptic solubilization, was prepared as
described elsewhere.* The Ouchterlony double
diffusion test in agar gel was carried out according to Kuriyama et al. (24). The test
was run in 1.2% agar ("Special Agar Noble"
from Difco Company) in 0.05M potassium phosphate buffer, pH 7.5, at 4°C for 60 hr. The
well diameter was 7 mm and the center-tocenter distance between the wells was 18 mm.
Enzymes—NADH-Cytochrome h reductase
was solubilized from rat liver microsomes by
lysosomal digestion and purified by the method
of Takesue and Omura (25), and its unit was
expressed in terms of NADH-ferricyanide reductase activity. Cytochrome bt was solubilized
from rat liver microsomes by trypsin and
highly purified (20). Crystalline yeast cytochrome c was supplied from Sankyo Company.
Twice crystallized trypsin and crystalline yeast
glucose-6-phosphate dehydrogenase were purchased from Sigma Chemical Company. Purified yeast hexokinase was a generous gift from
Dr. K. Tagawa of Osaka University School of
Medicine.
* T. Omura and N. Oshino, in preparation.
/ . Biochem.
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0.03%) was added to a mitochondrial suspension in 0.28 M sucrose containing lmM TrisHC1, pH 7.2, and 0.1 mM EDTA (12.5 mg of
protein per ml), and the mixture was incubated at 25°C for 20min. The digest was
cooled and centrifuged at 10,000 X g for 20 min.
The supernatant thus obtained was saved for
determination of trypsin-solubilizable cytochrome, and the residue (trypsin-treated mitochondria) was subfractionated as described
above.
Determination of bs-like Cytochromes-?or
detection and determination of 65-like cytochromes in mitochondrial fractions, NADH-reduced minus air-oxidized difference spectra
were measured in a Cary 14 spectrophotometer. Absolute spectra were not suitable
for this purpose even with clear, soluble preparations, because most of these samples contained hemoproteins such as catalase [EC
1.11.1.6] which interfered with the determination. In the case of membrane preparation, 65-like cytochromes were reduced by 0.1
mM NADH. When necessary, 1 I*M rotenone
was also added to prevent the reduction of
cytochromes associated with contaminating inner membrane. The reduction of 65-like cytochromes in soluble preparations was achieved
by addition of 0.1 mM NADH and an excess
(0.6 unit per ml) of NADH-cytochrome A5 reductase purified from rat liver microsomes.
For examination of hypotonic supernatants,
it was necessary to remove endogenous reductants prior to determination by incubating
the supernatants at 30°C for 60 min followed
by passing them through a small Sephadex
G-25 column (1.5 x 10 cm). Since molar extinction coefficients had not yet been determined
for mitochondrial 65-like cytochromes, it was
assumed arbitrarily that they possessed the
same extinction coefficients as microsomal
cytochrome bs. Thus, their contents were
calculated from the difference spectra by using
a molar extinction coefficient increment of
185 mM-'-cm"1 between 424 and 409 nm (77).
K. FUKUSHIMA, A. ITO, T. OMURA and R. SATO
CYTOCHROME 6 r LIKE HEMOPROTEINS IN MITOCHONDRIA
451
O.O6
DAYS OF STORAGE AT O°C
Fig. 1. Effects of storage and pretreatments of mitochondrial hypotonic supernatant on the amount of
65-like cytochrome detectable by difference spectrophotometry. Rat liver mitochondria were suspended
(12 mg protein/ml) in 10 mM Tris-phosphate buffer, pH
7.2, and incubated at 0°C for lOmin. The hypotonic supernatant was then obtained by centrifugation
at 105,000Xff for 30min, and stored aerobically at
4"C. At indicated time points, 3 ml portions of the
supernatant were withdrawn and assayed for irlike
cytochrome from NADH-reduced minus oxidized difference spectra as described in "EXPERIMENTAL
PROCEDURE" with or without the following pretreatments. Curve 1, no pretreatment; Curve 2 Sephadex G-25 gel filtration ; Curve 3, Sephadex G-25 gel
filtration after aerobic incubation at 30°C for 60 min.
Vol. 71, No. 3, 1972
i?
22
INCUBATION
TIME (HOURS)
Fig. 2. Solubilization of two types of mitochondrial
65-like cytochrome by hypotonic treatment and by
subsequent tryptic digestion. Liver mitochondria
were suspended (12 mg protein/ml) in 10 mM Trisphosphate buffer, pH 7.2, and incubated at 0°C.
After 24 hr of inbubation at 0°C, trypsin was added
to the suspension to a final concentration of 0.1% and
the mixture was further incubated at 30°C. At indicated time points, 3 ml portions of the supernatant
were withdrawn, centrifuged at 105,000 x g for 30 min,
and the supernatants were assayed for is-like cytochrome.
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was partly in the reduced state because of the
presence
of endogenous reductants, which
RESULTS
seemed to be slowly consumed during the
Solubilization of bs-like Cytochromes from storage. The reductants could, however, be
Mitochondria-As reported by Davis and Kreil removed by passing the supernatant through
(12), rat liver mitochondria, when exposed Sephadex G-25 after aerobic incubation at 30°C
briefly to 10 mM Tris-phosphate buffer, pH 7.2, for 60 min (Fig. 1). All the subsequent deterreleased a 65-like cytochrome into the hypotonic minations of 65-like cytochrome in the hyposupernatant. This cytochrome was reducible tonic supernatants were, therefore, conducted
by NADH in the presence of NADH-cytochrome after these pretreatments. The 6s-like cytobs reductase; its reduced minus oxidized differ- chrome solubilized by the hypotonic treatment
ence spectrum was similar to that of cyto- did not seem to have been derived from microchrome bit having absorption peaks at 557, 526 somes contaminating the mitochondrial prepand 424 nm {cf. Fig. 8). The amount of the aration, because no appreciable amount of
cytochrome detectable by reduced minus oxi- cytochrome was released from liver microsomes
dized difference spectrophotometry was, how- under the same conditions.
ever, variable, and it increased gradually when
Figure 2 shows that the solubilization of
the supernatant was stored aerobically at 4°C &s-like cytochrome by the hypotonic treatment
(Fig. 1). This suggested that the cytochrome took place in a short time, and no further
liberation was observed on prolonged incubation at 0°C. However, the addition of trypsin
(final concentration, 0.1%) to the mitochondrial
suspension followed by incubation at 30°C resulted in further liberation of a substantial
K. FUKUSHIMA, A. ITO, T. OMURA and R. SATO
452
considerable portion of the cytochrome released
by trypsin as depicted in Fig. 2 was of the
mitochondrial origin. That the trypsin-solubilized fraction contained a mitochondrial 6s-like
pigment in addition to microsomal cytochrome
bt could further be confirmed by the finding,
as will be reported in a later communication,
that a bs-tike cytochrome other than cytochrome
bt could be purified from tryptic digest of
mitochondria.
It may, therefore, be concluded from the
results shown in Fig. 2 that rat liver mitochondria contain two types of 65-like cytochrome differing in solubilization behavior. In
what follows the cytochrome readily solubilizable by hypotonic treatment will be called
"IS-cytochrome" (preliminarily called "hypotonic b" or "hypo b" (13-15)), whereas the
hemoprotein liberated from mitochondria by
tryptic digestion will be termed "OM-cytochrome." The amounts of both cytochromes
solubilized by the above treatments were
proportionally dependent on the amount of
mitochondria employed.
In the above experiments, the hypotonic
TABLE I. Solubilization offtj-Iikecytochrome by hypotonic treatment in different media and by subsequent
tryptic digestion. For Experiments A and B, mitochondria were suspended (10 mg protein/ml) in 10 mM
Tris-phosphate buffer, pH7.2 (A), or in 20 mM potassium phosphate buffer, pH7.2, containing 0.02% bovine
serum albumin (BSA) (B). The suspension was kept at 0°C for lOmin and then centrifuged at 105,000X0
for 30min to obtain the "first hypotonic supernatant." The pellet was again treated as above to obtain
the "second hypotonic supernatant." The residue was suspended in the original volume of 10mM Trisphosphate buffer, pH7.2, digested with trypsin (final concentration, 0.1%) at 30°C for 60 min, and centrifuged
as above to obtain the "trypsin supernatant." For Experiment C, mitochondria were suspended in 10mM
Tris-HCl buffer, pH 7.4, containing 10% ethanol (v/v). The suspension was rapidly frozen with dry iceacetone, thawed, heated to 40°C for 10 min, frozen and thawed again, and centrifuged (first supernatant).
The pellet was again subjected to the same treatment (second supernatant). The residue was finally
suspended in 10 mM Tris-phosphate buffer, pH7.2, and digested with trypsin (trypsin supernatant). The
contents of 65-like cytochrome in the supernatants were determined as described in " EXPERIMENTAL
PROCEDURE."
B
Fraction
10 mM Tris20 mM phosphate
10 mM Tris-HCl
phosphate
+0.02% BSA
+10% ethanol
(nmole of cyt./lOOmg of mitochondrial protein) .
First hypotonic supernatant
2.32
2.47
2.72
Second hypotonic supernatant
0.02
0.02
0.01
Trypsin supernatant
1.16
1.13
0.67
/ . Biochem.
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amount of 65-like cytochrome. Since trypsin
can solubilize cytochrome h from microsomes
(20, 26), it is probable that this cytochrome
was derived from contaminating microsomes.
With the NADPH-cytochrome c reductase activity as a microsomal marker (27), the ex- .
tent of microsomal contamination in the mitochondrial preparation employed in the experiment shown in Fig. 2 was found to be 3.0%
on a protein basis. On the other hand, the
cytochrome bs content in microsomes from the
same source was determined to be 0.40 nmole
per mg of protein. From these data it was
concluded that cytochrome bs from contaminating microsomes could account for about
80% of the trypsin-solubilized cytochrome. As
will be described in detail in a later communication, however, it was found that microsomal
bound cytochrome bt was more resistant to
proteolytic solubilization than the 65-like cyto-.
chrome associated with hypotonically treated
mitochondria. This fact, together with our
observation that tryptic digestion of microsomes under comparable conditions only partially solubilized cytochrome bit suggested that a
CYTOCHROME ftj-LIKE HEMOPROTEINS IN MITOCHONDRIA
cytochrome. However, the amount of
the solubilized cytochrome was somewhat
larger than those obtained in usual hypotonic
treatments, and the yield of trypsin-solubilized
cytochrome was correspondingly lower. It
therefore seemed that this rather drastic procedure partially solubilized OM-cytochrome in
addition to IS-cytochrome.
Figure 3 shows that Mike cytochrome
(IS-cytochrome) was liberated by the hypotonic
treatment not only from mitochondria prepared
from rat liver but also from those prepared
from rabbit liver, rabbit kidney and pig heart.
IS-CYTOCHROME
SUCCINATE-CYT. c
REDUCTASE
URATE OXIDASE
ACID rMUSFrfATASE
PHOSPHATASF NADPH-CYT. "c
Agio
REDUCTASE
J
30
60
90
120
INCUBATION TIME (MIN)
Fig. 3. Solubilization of 65-Iike cytochrome on hypotonic treatment of mitochondria from different
sources. Mitochondria obtained from rat liver (12
mg protein/ml), pig heart (11 mg protein/ml), rabbit
liver (12.5 mg protein/ml) and rabbit kindney (9 mg
protein/ml) were suspended in 10 mM Tris-phosphate
buffer, pH 7.2, and incubated at 0°C. At indicated
time points, portions of the suspensions were centrifuged, and the supematants were assayed for 65like cytochrome as described in Fig. 2. Curve 1,
rat liver mitochondria; Curve 2, pig heart mitochondria ; Curve 3, rabbit liver mitochondria; Curve 4,
rabbit kidney mitochondria.
Vol. 71, No. 3, 1972
HM
LMLMi
HM
J
LM L Ms
Fig. 4. Distribution of IS-cytochrome and some
marker enzymes in the heavy mitochondrial (HM),
light mitochondrial (LM), lysosomal (L), and microsomal (Ms) fractions of rat liver homogenate. The
subcellular fractions were isolated and enzyme activities determined as described in " EXPERIMENTAL PROCEDURE." The content of IS-cytochrome
was obtained by measuring the £>5-like cytochrome
solubilized by hypotonic treatment (0°C, 10 min) in
10 mM Tris-phosphate buffer, pH7.2. The ordi nates
indicate the relative specific content or activities per
protein of the isolated subcellular fractions. The
fractions are represented in the abscissas by their
relative protein contents in the order of this isolation
(from left to right).
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treatment was performed in 10 mM Tris-phosphate buffer, pH 7.2, according to Davis and
Kreil (12). It was, however, found that 20
mM potassium phosphate buffer, pH 7.2, containing 0.02% bovine serum albumin, which
was used by Parsons et al. (7, 8) as the
swelling medium for the isolation of outer
mitochondrial membrane, was as effective as
the Tris-phosphate buffer in solubilizing IScytochrome from mitochondria, and the residue
obtained in this medium could release OMcytochrome upon subsequent tryptic digestion
(Table I). It was also shown that solubilization of IS-cytochrome was almost complete by
only one hypotonic treatment. Raw et al. {3)
have reported that a Mike cytochrome could
be extracted from liver mitochondria when
they were suspended in 10 mM Tris-HCl buffer,
pH 7.4, containing 10% ethanol, rapidly frozen
and thawed, heated to 40°C for 10 min, and
finally frozen and thawed again. As shown
in Table I, this treatment actually solubilized
453
454
To obtain information concerning the intramitochondrial localization of IS-cytochrome,
its solubilization behavior was compared with
that of two mitochondrial enzymes, i.e. adenylate kinase which has been shown' to be
located in the intermembrane space (the compartment between the outer and inner mitochondrial membranes) (11, 30) and isocitrate
dehydrogenase which is a typical matrix enzyme (31). As shown in Fig. 5, both IScytochrome and adenylate kinase were completely solubilized upon exposure of mitochondria to 10 mM Tris-phosphate buffer, pH 7.2,
for lOmin, and.no further release took place
on prolonged incubation. On the other hand,
only about 30% of the total isocitrate dehydrogenase activity present in mitochondrial preparation was solubilized under the same conditions. This release of isocitrate dehydrogenase
seemed to have been caused by partial breakage of the inner membrane. A similar parallelism between the solubilization of IS-cytochrome and adenylate kinase could also be
10 20 30
40
50
INCUBATION TIME (MIN)
60
Fig. 5. Solubilization behavior of mitochondrial IScytochrome, adenylate kinase, and isocitrate dehydrogenase on hypotonic treatment. A mitochondrial
suspension (12 mg protein/ml) in 10 mM Tris-phosphate buffer, pH 7.2, was incubated at 0°C for indicated periods of time, and centrifuged at 105,000 xg
for 30min. IS-Cytochrome (is-like cytochrome), adenylate kinase, and isocitrate dehydrogenase released
into the supernatant were assayed as described in
"EXPERIMENTAL PROCEDURE." The amounts
of IS-cytochrome and adenylate kinase relased after
60min incubation were asssumed to be 100%. The
total mitochondrial content of isocitrate dehydrogenase was determined with intact mitochondria and
expressed as 100%. Curve 1, IS-cytochrome; Curve2, adenylate kinase; Curve 3, isocitrate dehydrogenase.
TABLE II. Parallel solubilization of IS-cytochromeand adenylate kinase from liver mitochondria at
various osmolarities. The experimental conditionswere the same as described in the legend for Fig. 5,
except that indicated concentration of sucrose wasadded to the hypotonic medium and incubation of
mitochondrial suspension was performed for lOmin.
The amounts of IS cytochrome and adenylate kinase
solubilized in the sucrose-free medium were takea
as 100%.
% Solubilization
Sucrose
concentration
(M)
IS-Cytochrome
Adenylate kinase
0.00
100
100
0.10
38.3
32.0
0.175
29.0
26.6
0.25
14.2
14.0
/ . Biochem.
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Localization of IS-Cytochrome in Intermembrane Space—As mentioned above, it was
clear that IS-cytochrome solubilized from mitochondrial fraction had not been derived from
contaminating microsomes. However, the possibility still existed that it had been solubilized
from lysosomes, peroxisomes or other subcellular structures that are present in considerable quantities in usual mitochondrial preparations. To check this point, rat liver homogenate was fractionated into several fractions
by differential centrifugation, and the amounts
of IS-cytochrome released from these fractions
were compared with the distribution patterns
of several marker enzymes. For this purpose,
succinate-cytochrome c reductase (28), acid
phosphatase (28), urate oxidase (29), and
NADPH-cytochrome c reductase' (27) were
chosen as markers for mitochondria, lysosomes,
peroxisomes, and microsomes, respectively. It
was found that the distribution pattern of IScytochrome was quite similar to that of succinate-cytochrome c reductase, but differed
significantly from those of non-mitochondrial
markers (Fig. 4). It could, therefore, be concluded that IS-cytochrome is a true mitochondrial constituent.
K. FUKUSHIMA, A. ITO, T. OMURA and R. SATO
CYTOCHROME ij-LIKE HEMOPROTEINS IN MITOCHONDRIA
455
TABLE III. Intramitochondrial distribution of 6$-like cytochromes. Mitochondria from 35 g of rat livers
were subfractionated as described in "EXPERIMENTAL PROCEDURE." Microsomal contamination in the
outer membrane preparations was estimated from NADPH-cytochrome c reductase activities.
6s-Iike cytochrome
Submitochondrial
Fraction
Mitochondria
Hypotonic supernatant
Purified outer membrane
Crude outer membrane
(mg)
Total content
(nmole)
420
49.2
1.87
56.0
9.9
0.98 (0.88) n
7.06" (6.40)°.
Specific content
(nmole/mg protein
0.20
0.52
0.13
(0.54)2'
observed when mitochondria were suspended
in the Tris-phosphate buffer containing various
concentrations of sucrose (Table II). These
findings suggested strongly that the localization
site of IS-cytochrome is the intermembrane
space of mitochondria.
Association of OM-Cytochrome with Outer
Mitochondrial Membrane-Since it was likely
that OM-cytochrome corresponded to the outer
membrane-bound 6s-like cytochrome described
by Parsons et al. {7,8) and Sottocasa et al.
(9, 10), an attempt was made to confirm
this by subfractionating liver mitochondria.
As mentioned above, exposure of mitochondria
to the swelling medium of Parsons et al. ( # ) ,
i.e. 20 mM potassium phosphate buffer, pH 7.2,
containing 0.02% bovine serum albumin, resulted in the release of a considerable quantity
of IS-cytochrome; its yield was 9.9 nmoles
from 420 mg of mitochondrial protein, corresponding to an IS-cytochrome content of 0.024
nmole per mg of mitochondrial protein (Table
III). The pellet of swollen mitochondria, to
which OM-cytochrome was still attached, was
then subjected to subfractionation as described
"EXPERIMENTAL PROCEDURE." The purified
outer membrane fraction thus isolated was
found to exhibit an NADH-reduced minus oxidized difference spectrum resembling that of
cytochrome bt (Fig. 6). As shown in Table
III, the content of 65-like cytochrome in the
purified outer membrane fraction was 0.54
nmole per mg of protein when corrected for
Vol. 71, No. 3, 1972
4OO
5OO
600
WAVELENGTH(nm)
Fig. 6. NADH-Reduced minus oxidized difference
spectrum of purified outer mitochondrial membrane
fraction. The outer membrane preparation containing 0.52 nmole of 6s-like cytochrome per mg of protein was suspended in 10 mM potassium phosphate
buffer, pH7.2, at a concentration of 0.6 mg of protein per ml. Reduction of the cytochrome was
achieved by adding 0.1 mM NADH.
the contribution of cytochrome bt due to contaminating microsomes. This value was even
higher than the cytochrome h content in microsomes from the same source (0.38 nmole per
mg of protein). Since the yield of purified
outer membrane was low! the outer membrane
was recovered from the outer submitochondrial
fractions as complete as possible; its total recovery was about 80% as judged from that of
the rotenone-insehsitive NADH-cytochrome c
reductase activity. The recovery of 65-like
cytochrome both in this crude outer membrane
Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
15
This value was determined in the presence of 1/<M rotenone. :> These values represent the net contents
of mitochondrial 65-like cytochrome, obtained after correction for the contribution of cytochrome 65 in contaminating microsomes.
456
K. FUKUSHIMA, A. ITO, T. OMURA and R. SATO
TABLE IV. Selective solubilization of OM-cytochrome by trypsin treatment of intact mitochondria in an
isotonic sucrose medium. Mitochondria from 21 g of rat livers were treated with trypsin in an isotonic
medium and the trypsin-treated mitochondria were then subfractionated as described in " EXPERIMENTAL
PROCEDURE." The ftrKke cytochrome contents of the subtractions were determined.
Submitochondrial
(nig)
Mitochondria
Trypsin supernatant
Hypotonic supernatant
Purified outer membrane
Crude outer membrane
Total content
(nmole)
Specific content
(nmole/mg protein)
7.90
6.00
0.62
0.12
0.04
0.02
313
12.7
48.8
3.0
40.2
0.13
0.68"
This value was determined in the presence of lfiM rotenone.
fraction and in the purified fraction was 7.28
nmoles from 420 mg of mitochondrial protein
after correction for contaminating microsomal
cytochrome bt was made. When corrected for
the recovery of outer membrane, this value
corresponded to a content of outer membranebound Vlike cytochrome of about 0.021 nmole
per mg of mitochondrial protein. Since this
content was roughly in agreement with the
amount of OM-cytochrome expected from the
data of Fig. 2, it could be concluded that OMcytochrome is the ftj-like cytochrome firmly ]
bound to the outer mitochondrial membrane.
In another experiment, mitochondria were
first digested with trypsin in an isotonic medium, in which no swelling of mitochondria
occurred, and then subfractionated. As shown
in Table IV, the trypsin treatment solubilized
a considerable amount of &5-like cytochrome,
and subsequent exposure of mitochondria to
the swelling (hypotonic) medium resulted in a
second solubilization of £5-like cytochrome.
This latter cytochrome seemed to correspond
to IS-cytochrome from its solubilization behavior. It was further found that both the
purified and crude outer membrane fractions
isolated from the mitochondria thus treated
contained only very small amounts of 6j-like
cytochrome. Its content in the purified outer
membrane fraction thus obtained was only
0.04 nmole per mg of protein, as compared
. with the value of 0.54 nmole for the outer
membrane purified from untreated mitochon-
dria (c/. Table III). Furthermore, it seemed
likely that the small amount of 65-like cytochrome remaining in the outer membrane
fractions was mostly cytochrome 65 bound to
contaminating microsomes in view of the
aforementioned resistance of microsomal cytochrome bi toward tryptic solubilization (only
9% of the total cytochrome 65 was solubilized
from liver microsomes under the tryptic digestion conditions employed in Table IV).
These observations indicated clearly that
OM-cytochrome bound to the outer membrane
could be solubilized quantitatively when intact
mitochondria were treated with trypsin in the
isotonic medium. This digestion, however,
must have caused practically no rupture of the
outer membrane since IS-cytochrome was still
retained in the intermembrane space even after
the treatment and was solubilized only by
subsequent hypotonic treatment. This fact,
together with the consideration that trypsin
does not seem to penetrate into the intermembrane space under the isotonic condition, suggested that OM-cytochrome is situated at the
outer surface of the outer mitochondrial membrane.
Characterization of IS-Cytochrome—As an
attempt to purity IS-cytochrome, the hypotonic
supernatant.(in 10 mM Tris-phosphate buffer,
pH 7.2) of rat liver mitochondria was concentrated with a collodion bag and subjected to
gel filtration through a Sephadex G-200 column
equilibrated with 10 mM Tris-phosphate buffer,
/ . Biochem.
Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
s>
brlike cytochrome
Protein
CYTOCHROME 6 r LIKE HEMOPROTEINS IN MITOCHONDRIA
400
500
600
WAVELENGTH ( nm )
5
10
15
20
25
30
FRACTION NUMBER
35
Fig. 7. Separation of IS-cytochrome into two hemoprotein components by Sephadex G-200 gel filtration.
A suspension (15 ml) of liver mitochondria in 10 mM
Tris-phosphate buffer, pH 7.2 (12 mg protein/ml), was
incubated at 0°C for 15min (A), 3hr (B), or 20 hr
(C), and then centrifuged at 20,000 x.g for 60min.
The supernatant was concentrated to about 5 ml by
means of a collodion bag (Sartorius Membranfilter
Co.), and applied to a Sephadex G-200 column (1.5x
47 cm) equilibrated with 10 mM Tris-phosphate buffer,
pH 7.5. Elution was conducted with the same buffer,
and 2 ml fractions were colleted. IS-Cytochrome
(fts-like cytochrome) in the eluates was determined as
described in " EXPERIMENTAL PROCEDURE " and
protein by measuring the absorbance at 280 nm.
,
IS-cytochrome;
, protein.
pH 7.5. It was thus revealed that IS-cytochrome was actually a mixture of two bt-like
hemoproteins differing in molecular weight
(Fig. 7). Although detailed determinations are
yet to be made, a molecular weight of about
12,000 was assigned to the small component,
because this component was eluted from the
Sephadex column at the same position as microsomal cytochrome b% purified after tryptic
solubilization (cf Ref. 20). On the other hand,
the molecular weight of the large component
was estimated to be about 120,000 from its
elution position. Figure 7 shows further that
the large component was labile and converted
Vol. 71, No. 3, 1972
Fig. 8. NADH-Reduced minus oxidized difference
spectra of the large and small components of IS-cytochrome. The two components were separated from
a mitochondrial hypotonic supernatant essentially as
described in the legend for Fig. 7, except that a
Sephadex G-100 column was used and the hypotonic
supernatant was prepared after lOmin incubation of
mitochondria at 0°C. The cytochrome in the sample
cuvette was reduced by NADH in the presence of
NADH-cytochrome 65 reductase, and the control
cuvette contained the untreated cytochrome. A,
large component; B, small component.
gradually to the small one upon prolonged incubation of mitochondria at 0°C in 10 mM Trisphosphate buffer, pH 7.2. This conversion
could be accelerated and brought to completion
when the large component was treated with
trypsin.
Figure 8 shows the reduced minus oxidized
difference spectra of the large and small components of IS-cytochrome. Both spectra are
practically identical with each other and resemble that of cytochrome bt.
Like cytochrome bit both components of
IS-cytochrome could catalyze the reduction of
cytochrome c by NADH when supplemented
with an excess of microsomal NADH-cytochrome bs reductase. However, as shown in
Fig. 9, the reactivity of the small component
in this reconstructed system was less than
50% of that of trypsin-solubilized cytochrome
bt, and the reactivity of the large component
Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
0.02
457
458
K. FUKUSHIMA, A. ITO, T. OMURA and R. SATO
Fig. 10. Immunological reactivities of cytochrome
bt and two components of IS-cytochrome with an
antibody to cytochrome bs. Ouchterlony double difFig. 9. Comparison of reactivities of cytochrome b> fusion test in an agar gel was carried out as deand of two components of IS-cytochrome in recon- scribed in " EXPERIMENTAL PROCEDURE." Well
stituted NADH-cytochrome c reductase system. The 1, 3 mg of y-globulin fraction of rabbit antiserum to
two components of IS-cytochrome used were the same purified rat liver microsomal cytochrome b}. Well
as in Fig. 8. The NADH-cytochrome c reductase assay 2, a trypsin-digested supernatant of rat liver microsystem contained 0.1 M potassium phosphate buffer, some containing 0.4 nmole of cytochrome 6S. Well
pH7.5, 0.1 mM NADH, 3 units of NADH-cytochrome 3, unfractionated hypotonic supernatant of rat liver
bs reductase, 20 fiM cytochrome c, and indicated mitochondria (prepared as in Fig. 8) containing 0.6
amount of either trypsin-solubilized cytochrome 45 nmole of IS-cytochrome. Well 4, 0.4 nmole of the
(Curve 1), the small IS-cytochrome component large IS-cytochrome component (prepared as in Fig.
(Curve 2), or the large IS-cytochrome component 8). Well 5, 0.4 nmole of the small IS-cytochrome
(Curve 3) in a total volume of 2.0 ml.
component (prepared as in Fig. 8).
02
0.4
O£
OS
was only about 7%. Such low reactivities of
the IS-cytochrome components were not due
to the presence of inhibitors in their preparations, because the NADH-cytochrome c reductase activity of the system reconstructed from
purified cytochrome bt and its reductase was
not influenced by the addition of these preparations (the additions resulted in additive increases in the reductase activity). These
findings indicated that the components of IScytochrome, though spectrally similar to cytochrome bi, are different in reactivity from the
microsomal hemoprotein.
The molecular differences between cytochrome bi and the two components were more
clearly demonstrated in an experiment in
which a rabbit antibody to trypsin-solubilized
rat liver microsomal cytochrome bt was reacted
with these components by the agar gel double
diffusion technique of Ouchterlony. As shown
in Fig. 10, purified rat liver microsomal cytochrome bt formed a clear precipitation line
with the antibody, as expected. However,
corresponding amounts of the large component,
small component and the hypotonic supernatant
of mitochondria showed no sign of interaction
with the antibody. It was thus clear that
both the large and small components of IScytochrome are different immunologically from
microsomal cytochrome bt.
DISCUSSION
The results reported in this paper seem to
leave little doubt that two different types of
cytochrome £5-like hemoprotein occur in liver
mitochondria; one, called IS-cytochrome, is
easily solubilizable by hypotonic treatment and
is situated between the two mitochondrial
membranes, whereas the other, called OMcytochrome, is bound to the outer membrane
and requires tryptic digestion for its solubilization. Our data also show that IS-cytochrome
is a mixture of two hemoprotein components
differing in molecular weight and reactivity.
/ . Biochem.
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CYTOCHROME ADDED (nmole)
CYTOCHROME *5-LIKE HEMOEROTEINS IN MITOCHONDRIA
Vol. 7i, No. 3, 1972
purpose would be the OM-cytochrome which
can be selectively solubilized from mitochondria
by trypsin treatment in an isotonic medium
(Table IV). The half life of 4.4 days determined by Druyan et al. (32) for the decay in
vivo of what they believed to be the outermembrane hemoprotein should, therefore, be
regarded as reflecting the average metabolic
stability of a mixture of two components of
IS-cytochrome and a small fraction of OMcytochrome.
Although both of the two components of
IS-cytochrome are spectrally similar to microsomal cytochrome bt and reducible by NADH
in the presence of microsomal NADH-cytochrome bt reductase, they are in fact different
from the microsomal hemoprotein in immunological properties (Fig. 10) and in reactivity
in the reconstructed NADH-cytochrome c reductase system (Fig. 9). Differences from
microsomal cytochrome £>5 have also been
noticed with two bt-like cytochrome preparations which appear to be identical with the
small component of IS-cytochrome. Davis and
Kreil (12) have reported that the cytochrome
they solubilized from mitochondria by hypotonic treatment differs from cytochrome bt in
electrophoretic mobility and solubility in ammonium sulfate solutions. According to Raw
et al. (3), on the other hand, the hemoprotein
purified from the Tris-ethanol extract of mitochondria is not reducible by cysteine, which
however reduces cytochrome 65 partly. It can,
therefore, be concluded that the two IS-cytochrome components are entities which are
entirely different from microsomal cytochrome
b>.
The large component of IS-cytochrome is
labile as evidenced by its slow conversion in
hypotonic media to a smaller hemoprotein
(Fig. 7). Raw et al. (3) have also noticed the
presence of a labile hemoprotein, in addition
to the one they purified, in their mitochondrial
extract, but they did not study this labile
component in detail. The mechanism of conversion of the large component is not yet
clear, though the observation that trypsin accelerates the transformation suggests that it
is induced by an endogenous or contaminating
protease. Nor is clear the relationship between
Downloaded from http://jb.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 2016
As a matter of fact, previous workers had already observed both of these two types of
mitochondrial 65-like cytochrome, but no one
has so far recognized the distinction between
them. Instead, the view has been prevailing
in recent years that the hemoprotein associated
with the outer membrane is the only 65-like
cytochrome of mitochondria, and it has been
believed that hypotonic treatment solubilizes
this cytochrome partially because of its loose
binding to the membrane (9, 10, 12). If this
interpretation were correct, it would be expected that all the Mike cytochrome of mitochondria could be solubilized quantitatively on
prolonged or repeated hypotonic treatments.
The present work, however, indicates that
this is not the case (Fig. 2 and Table I).
In the light of the present results, it is
now certain that the hemoprotein detected by
Parsons et al. {7,8) and by Sottocasa et al.
(9, 10) in their outer membrane preparations
corresponds to our OM-cytochrome. It can
also be concluded that the cytochrome solubilized hypotonically by Davis and Kreil (12)
is equivalent to the small component of IScytochrome, although they were unable to
detect the large component. The observation
of Sottocasa et al. (9, 10) that a large part
of £>5-like cytochrome was released into solution
during their swelling-shrinking and sonication
procedure of mitochondrial subfractionation
may be interpreted to mean that the linkage
of OM-cytochrome to the membrane is not
very tight, though sufficiently resistant to
prolonged or repeated hypotonic treatments,
and is partially broken by their rather drastic
procedure. Similarly, the extraction method
of Raw et al. (3) using an ethanol-containing
Tris buffer also seems to cause partial solubilization of OM-cytochrome, as mentioned
earlier. It is, however, evident that the hemoprotein present in such extracts is mostly IScytochrome (Table I). Assuming that it is
originated from the outer membrane, Druyan
et al. (32) have employed the extractable
hemoprotein as a marker of. the outer membrane in a study of assembly and degradation
of mitochondrial membranes. It is, however,
certain that this is not a true marker of the
outer membrane. A suitable marker for this
459
460
Coninck and Wattiaux (39). It is, therefore,
certain that the large component of IS-cytochrome functions as sulfite oxidase. The
physiological significance of the small component is yet to be elucidated. It is to be reminded in this connection that IS-cytochrome
is distributed not only in liver mitochondria
but also in those from kidney and heart (Fig.
3).
The finding that trypsin treatment of
mitochondria in an isotonic medium solubilizes
OM-cytochrome without breaking the outer
membrane (Table IV) suggests that this hemoprotein is bound to the outer surface of the
outer membrane. This conclusion is in line
with the observations by Kuylenstierna et al.
(40) that the rotenone-insensitive NADHcytochrome c reductase system of the outer
membrane is more sensitive to trypsin than
the microsomal counterpart and is readily inactivated by brief tryptic digestion of mitochondria in an isotonic medium. They have
actually suggested that the reductase system
is located at the outer surface of the membrane. It is now certain that our OM-cytochrome is a component of the rotenone-insensitive reductase system of the outer mitochondrial membrane, though the role of this system
in cell physiology is still obscure.
The clarification of the identity of OMcytochrome with microsomal cytochrome bs is
important in studying the biogenetic interrelationship between the endoplasmic reticulum
and outer mitochondrial membrane, but this
iproblem has not yet been solved, although
Parsons et al. (8) have noticed a small difference in the low-temperature spectrum bej^tween the two cytochromes. We have recently solubilized and purified OM-cytochrome and
obtained evidence that it is actually different
from the microsomal cytochrome. These findings will constitute the subject of a later
communication.
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