- Email: [email protected]
Electron spin resonance of microsomal cytochromes
ARCHIVES
OF
BIOCHEMISTRY
Electron Correlation
AND
121, 742-749 (1967)
BIOPHYSICS
Spin Resonance
of the Amount
Fe, in Microsomes
of Microsomal
of CO-Binding of Normal
Species with So-Called
Tissues and Liver Microsomes
Sudan Ill-Treated YOSHIYUKI Department
ICHIKAWA
of Biochemistry, Iteceived
April
Cytochromes Microsomal of
Animals
AND
Osaka University 7, 1965; accepted
TOSHIO Medical April
YAMANO School, Osaka, Japan
20, 1965
A correlation has been established on intact microsomes of various tissues between the amount of CO-binding pigment (P-450) and microsomal Fe, , which was designated as a component of rabbit liver microsomes with an ESR signal of low-spin type hemoprotein. This correlation also holds with vertebrate microsomes in which the enzyme contents were raised by injection of Sudan III into the animals. The correlat,ion seen with intact microsomes is no longer observed after treatment with deoxycholate or alkaline, which readily destroys P-450. In the microsomes of bovine adrenal medulla a hemoprotein exists with an a-band at 559 rnp but without an ESR signal or CO-binding character.
Whereas cytochrome bS has been established in mammalian liver microsomes by several workers (1, 2)) an NADPH-dependent cytochrome has not yet been well characterized. A CO-binding pigment (P-450) in microsomes was described by Klingenberg (3) and Garfinkel (4), and was proposed by Omura and Sato (5, 6) to be a hemoprotein. The latter authors obtained a hemoprotein other than cytochrome bc from solubilized liver microsomes and named it P-420. On the other hand, Hashimoto et al. (7) have shown by electron spin resonance (ESR) that a certain species of hemoprotein, which they designated as microsomal Fe,, exists in the smooth- and rough-surfaced microsomes of rabbit liver. From the further studies of Mason et al. (8, 9) it appears that this species is an NADPH-dependent hemoprotein. Logically, the next problem is to study the relation between 450 mp absorbing species and microsomal Fe,. In this study t’he correlation between the t’wo species was examined using intact microsomes from
various vertebrate tissues. In addition, Sudan III was found to be effective for inducing microsomal Fe, and P-450 in parallel. A study of the ESR of phenobarbitaltreated rat microsomes was previously reported in a short communication (10). EXPERIMEXTAL
PROCEDURE
The microsomes used in these experiments were prepared from various tissues by a modification of the method of Mitoma et al. (11). Wistar rats (200250 gm), hamsters (150-200 gm), mice (N-70 gm), and rabbits (2.0-2.5 kg) were fed on a standard commercial laboratory diet with water ad Zibitum for more than 2 weeks before Ilse. All operations were conducted in a cold room at 5”, and material from the slaught,er-house was immediately brought t,o t,he laboratory on ice. The tissues were perfused through the main blood vessels with an ice-cold 1.150/d KC1 solrltion to remove as much hemoglobin as possible. Tissues in which this was not possible were carefully washed with isotonic KC1 solution before further preparation procedures. Fat and connective tisslle were removed from the tissues, which were t,hen chopped up with a pair of scissors and homogenized with 5 volumes of 1.15(3& KC1 solution in a Teflon homogenizer of the Potter742
~ITCROSOMAT, Elvehjem type. The homogenate was centrifllged at) 10,OOOgfor 25 minutes in a refrigerated centrifuge, and t.he precipit,ate was discarded. The supernat)ant fluid was filtered through Tetroncotton. The resldting solution was centrifuged at 105,000g for 90 minutes in a refrigerated Martin Christ Omega type 4OP preparative ultracentrifuge. The microsomes were sedimented from the various tisslles, and a Potter-Elvehjem homogenizer was used to carefully resuspend the microsomes in an isotonic KC1 solution. Centrifugation was then repeated to remove as much extraneous material as possible. Contamination of the microsomes of various tissues with mitochondria was found to be less than 47; on a protein basis as judged by succinic oxidase activity. The washed microsomes were stored at -20” under anaerobic conditions and used within 48 hours after preparation. The microsomal preparations were shown to contain no hemoglobin contamination by reducing them with dithionite and then carefully bubbling carbon monoxide through the suspension for about 30 seconds before measuring the spectrum. With the adrenal glands of pig and cow, t,he medldlas were carefully separated from the cortical t,isslle, and medldlary tissrles were homogenized with 0.25 Y sIIcrose. For preparat ioll of adrenal microsomes, 0.25 M sucrose was preferred to 1.157; KU, because their EHR signal was rather low when they were prepared ill KC1 medilun. This was not so with other tissues. The difference spectra of microsome preparations were measlued in a Gary model 14 recording spectrophotoInrter; a l-cm optical path was rlsed escept whell st,ated otherwise ill the text. The amount, of cytochrome bj was measured aerobically by the difference spectrum of the form reduced with KAl>lI minus the oxidized form (4). CO-binding pigment (P-450) was determined by the Omllra and Sato method; for the former, the val\le of 165 cm-l IIIM-’ was assumed for the molar estinction increment between JO9 and 421 rnp, and for the latter the vallle of 91 cm-l nlh~-l ~-as adopted for t.he molar extinrtion incrrrnellt between 450 and -190 mp, based on the difference Ijetween the spectra of the CO-reduced form and the redltced form. All spectrophotometric measrirrments wrre made at room temperatlire. Electron spin resonnlrce spectroscopy was performed with a. \.arian 1. 1500-10 A spectrometer with a 100 Kc field modrdation lulit, and specka were generally obtained at a sample temperatllre of -170”. A silica tribe of 3 mm internal diameter was {Ised on the Yarian variorts temprratnre atachment,. The protein content was determined after addit,ion of sodium cholate to the sample to remove turbidity, by the biuret method of Gornall et al.
743
CYTOCHROMES
(la), and crystalline bovine plasma albumin was used as standard. The increased absorption in the biuret reaction due to heme in the test samples was avoided by using cupric sulfate-free biuret reagents. NADH and N,4I>PII were obtaitled from the Sigma Chemical Company. Carbon monoxide was prepared from formic acid and concentrated sulfuric acid, and passed through solid potassium 2iydroxide to remove extraneous substances. Ethyl isocyanide was synthesized from silver cyanide and et,hyl iodide (13). The crystalline glucose oxidase used to give anaerobic conditions was purified by the method of Kusai et ccl. (14). The other agents used were obtained commercially. RESULTS
Table I shows the amounts of hemoprotein in the microsomes of various tissues. It is seen that there is a correlation between P-450 and microsomal Fez. The amount of microsomal Fe, is normalized as follows. The double integration of the signal with slope maxima (8,) at g = 2.41 and 1.91, and a g maximum (gm) at g = 2.25, was compared under identical conditions with t>hat of a standard solution of copper containing 2 mnI C&O4 and IO 11111 EDTA. Wit’h rabbit liver microsomes prepared by careful perfusion, an ESR signal was obtained with only a small fluctwkon between gm = 2.25 and s, = 1.91. The estimated amount of microsomal Fe, was found to be of the same order as the amount of P-450 in rabbit liver microsomes, when estimated as described above. Then the height of the ESR signal at gm = 2.25 was measured to estimate the concentrat8ion of microsomal I’e, . As seen from the table, the content of P-450 was highest in the liver tissues of various animals. The contents in kidney microsomes were far less, being about one-tenth of those in liver. The microsomes of pancreas and testis had no P-450 or miclosomal Fe,, but cytochromc 1’5was detected. The result. is in agreement with that reported by CTarfirllrel cxccljt regard to the adrenal gland ( 15). The
with
microsomes of the cort,ex and medulla of bovine adrenal glarlds and pig adrem glands w’crc prepared separately. The ratio of the amount of cyt,ochronle b5 to nlicrosomtll I:eZ varies frorll t,issue to tissue. Pig adrenomedullary
744
ICHIKAWA
AXD
microsomes and cortical microsomes both contain much P-450, t’he amount being second to t’hat in liver microsomes. In contrast with pig adrenomedulla, bovine adrenoTABLE DETERMINATION Sources ofILLmxnes
Rabbit (male) Liver Kidney Small intestine Adrenal gland Rabbit (female) Liver Ovary Rat (male) Liver Rat (female) Liver Kidney Mouse (male) Liver Kidney Guinea pig (female) Liver Kidney Pig (female) Liver Kidney cortex Thyroid Pancreas Adrenal cortex Adrenal medulla Ovary Dog (male) Liver Kidney Carp (female) Liver Gibe1 (female) Liver Bovine Liver Kidney cortex Kidney medulla Cerebellum Pituitary gland Pancreas Adrenal cortex Ovary Testis Calf thymus Hamster (male) Liver Kidney
OF THE
CYTOCHROMES
[21
YAMANO
medullary microsomes contain a cytochrome which shows an a-band at 559 rnp but, no ESR signal. The properties of this cyt.ochrome will be described later. I
IN MICROSOMES
AT VARIOUS
VERTEBRAE
ISI
TISSUES
Cyt. bs
P!%O
0.88 0.10 0.02 0.08
1.72 0.16 0.07 1.20
1.70 0.15 0.08 1.10
1.1 0.9
1.9 1.5 4.0 1.4
0.80 0.21
1.55 0.06
1.60 0.07
1.0 1.2
2.0 0.3
0.64
0.82
0.81
1.0
1.3
0.51 0.08
0.71 0.07
0.70 0.07
1.0 1.0
1.4 0.9
0.40 0.10
0.61 0.06
0.60 0.06
1.0 1.0
1.5 0.6
0.55 0.12
0.79 0.14
0.70 0.13
0.9 0.9
1.3 1.1
0.29 0.17 0.13 0.06 0.24 0.25 0.47
0.42 0.18 0.00 0.00 1.14 0.61 0.17
0.40 0.20 1.00 0.60 0.20
1.0 1.0 0.9 1.0 1.2
1.4 1.1 4.2 1.4 0.4
0.42 0.12
0.75 0.17
0.73 0.18
1.0 1.1
1.7 1.5
0.14
0.38
0.37
1.0
2.6
0.04
0.15
0.15
1.0
3.8
0.79 0.01 0.05 0.04 0.07 0.09 0.13 0.04 0.09 0.05
0.94 Trace 0.00 0.00 0.07 0.00 0.78 0.05 0.00 0.00
1.05 0.07 0.76 Trace -
1.1 1.0 1.0 -
1.3 1.0 5.9 -
0.64 0.08
1.14 0.14
1.10 0.15
1.0 1.1
1.7 1.9
MicI4be
Lz
161
Ratio of 4 to 3 Ratio of 4 to 2
1.0 0.9
MIC:ROSObIAL
CYTOCHROMES
TABLE
745
I-Continued
Cl1
Sources of microsomes
Mic[“i;eI
z
El Ratio of 1 to 3
[61
Ratio of 4 to ?
Hamster (female) Liver Kidney Hen liver Goose (female) Liver Horse (male) Liver Kidney cortex Adrenal cortex Sheep (female) Liver Kidney Adrenal Adrenal
cortex medulla
Orrenius and Ernster (16) have reported the induction of P-450 in liver microsomes by phenobarbital injection into rats. Table II shows the inductive increase of microsomal Fe, in rabbit liver microsomes. Huggins et al. (1’7) reported on the induction of menadione rcductase. In our study, a low dose of Sudan III was found to induce microsomal Fe,. Table II gives these results, and shows t,hat the correlation between P-450 and microsomal Fe, was still present. Sudan III was dissolved in ethanol and injected into a rabbit intraperitoneally. The daily dose was determined by drying a known TABLE
II
INCREASE IN CYTOCHROMES IN PHENOBARBITALAND SUDAN MALE RABBITS
MICROSOMES III-TREATED
OF
Rabbits were injected intraperitoneally with 85 mg of sodium phenobarbital or 4 mg of Sudan III per kg body weight once daily for 4 days. One day after the last inject#ion the animals were killed. The microsomal fraction was isolated from the liver homogenates by a procedure as described in the text. The content,s of cytochrome bg , P-450, and microsomal Fe, are expressed as described in Table I. [II Rabbit liver microsomes
121 cg .
P-450
131
141 Mic. Fez
Control Phenobarbital Sudan III
0.88 1.50 2.60
1.72 5.40 5.10
1.70 5.10 4.80
R!L of 4 to 3
R?io
1.0 0.9 0.9
1.9 3.4 1.8
of 4 to 2
volume of the ethanol solution and weighing the residue. A daily dose of 5 mg/kg of rabbit caused about a threefold increase in the microsomal Fe, and cytochrome b, cont(ent.8. In view of the observation of Yamano et al. (8) that, the cyt’ochrome which has an a-band at 559 mp is likely to be associated with microsomal Fe,, the increased absorbancy at 559 rnp due to anaerobic XADPH minus the absorption of aerobic NADH should appear more distinct’ly when the difference spectrum is measured on liver microsomes from rabbits after injection of phenobarbital. Figure 1 shows t’he time course of the increase of the absorbancy at 559 rnp after adding NADH or NADPH. The correlation between microsomal Fe, and P-450 was no longer observed after intact microsomes had been treated wit’h cholate or deoxycholate. As seen in Fig. 2, P-450 species decreased at low concentration of deoxycholat’e and almost disappeared at a concentration of 0.5 % deoxycholate, wherea,s the content8 of microsomal Fe, remained nearly unchanged. As the P-450 species disappeared, P-420 species appeared, their concentrat,ions being inversely related to that of P-450. This had previously been observed by Omura and Sato, who reported t’he peak which appears at, 420 mp on dit,hionit,e reduction of the soluble form of P-450 under CO. The P-450 species also disappeared when
746
ICHIKAWA
0.0
0
AFU
I
I
I
I
I
5
IO
15
20
25
TIME
IN MINUTES
FIG. 1. Time course of appearance of increased absorption when KADPH was added to liver microsomes of rabbit, treated with phenobarbital, in the presence of glucose and glucose oxidase (added to give anaerobic conditions). The figure illustrates the autooxidizability of the species which shows an a-band at 559 rnp, as judged from the decrease of the absorption and approach to the absorption level of cytochrome bb after exhaustion of glucose. The mixture contained 2.0 mg of microsomal protein, 1.0 pmole of P\‘ADPH (upper curve) or NADH (lower curve), and 0.7 mg of glucose oxidase in a total volume of 3 ml (0.1 M phosphate buffer, pH 7.0). A cell of l-cm light-path was used. When NADPH was used, 300 wmoles of glucose had previously been added to the mixture.
a microsomal solution of pH 7 was made alkaline. In this case, microsomal Fe, species were decreased in parallel with P-450, and the P-420 species increased in inverse relation. This result is presented in Fig. 3. At pH 6, P-450 species decreased, but the reason for this is unknown. Hemoproteins in rabbit liver microsomes ,qere measured during growth after birth. As presented in Fig. 4, a correlation between microsomal Fe, and P-450 was established. Fouts and Adamson (18) reported that some drug metabolizing activity appeared 2 weeks after birth, and after 4 weeks the act’ivity was about equal to that of the adult. Our observation on the increase in the amount of microsomal Fe, bears some relation to the drug met’abolizing activity of liver microsomes.
YAMANO
Krisch (19) and Spiro and Ball (20) reported t,hat bovine adrenal microsomes, when solubilized with deoxycholate and reduced with dithionite, showed an a-band at 559-560 rnp and a Soret band at 4‘28 rnM. ,4s mentioned above, the medullary microsomes of bovine adrenal glands showed neither an ESR signal nor CO-binding, whereas they showed an a-band at 559 mp. Rledullary microsomes of bovine adrenals were solubilized by adding 0.1% unheated cobra venom to a microsomal suspension in 0.1 M TrisHCl buffer, pH 8.5, and allowing the mixture to stand at 5’ for 15 hours under anaerobic conditions. The cytochrome in the solubilized mixture was reduced by adding ?;ADPH or NADH, as shown m Fig. 5. The rate of reduction was fastest’ with anaerobic NADPH. The cytochrome released from the particles was collected as a reddish pre-
I 0.5
OY 0 DEOXYCHOLATE
CONCENTRATION,
I I 1.0 O %
FIG. 2. Relationship between microsomal CObinding species and microsomal Fe, at various concentrations of deoxycholic acid. The reaction mixture, containing phenobarbital-treated rabbit liver microsomes at a protein concentration of 20 mg/ml, was incubated at 20” for 10 minutes and used for ESR studies. Spectrophotometric measurements were carried out at a protein concentrations of 2.0 mg/ml under the same conditions, in 0.1 M phosphate buffer at pH 7.0 and a cell of l-cm light-path. 0, Microsomal Fe, ; 0, P-450; 0, P-420. The ordinates are the percentages of the residual absorbancy at 450 rnb minus the absorbancy at 490 rnp and of the absorbancy appearing at 420 rnp, when the maximum absorbancy at 420 rnp is assumed to be lo07c. The heights of ESR signals were measured at gm = 2.25, each plot, being expressed as a percentage of the height of the initjR1 ESR signal.
XIICI~OSOMrZL
747
CYTOCIIRONIES DISCUSSIOS
4 2 0” ‘“m 50 4
b s 0
6
7
9
6
IO
II
PH
FIG. 3. Effect of pII on microsomal Fe, and P-450 of phenobarbital-treated rabbit, liver mirrosomes. A mixture containing rabbit liver mirrosomes at a protein coimentration of 20 mg/ml was ttsed for ESR measurements. Spectrophotometric measurements were carried ottt at a protein eon centration of 2.0 mg/ml. The samples were placed in solutions of different pH values. The buffers used are 0.1 M phosphate buffer at below pH 8.3 and 0.1 M glycine-KaOH buffer at above pH 8.5.
The results report,ed here are concerned principally with the hemoprotein in microsomes, which seems to be associated wit,h microsomal Fe, or CDbinding species (P-160). The content of P-450 species was aways present in proportion w&h that of microsomal Fe, in the microsomes of various vertebrate t8issues. However, on treatment of microsomes with deoxycholate or alkali, this parallel relation was lost. In the present experiments, the P-4.50 species were more labile t,han microsomal l‘e, , and on keatment with alkali the former seemed to be degraded to a hemoprot8ein named P-420 by Omura and Sat’0 (5). These three species are not considered to be different hemoprot,eins but to be due to a single hemoprotein, because total-heme analysis showed the existence of only one hemoprotjein besides cytochrome bg. P-450 appears to be microsomal Ipe, in
k
0 WEEKS
FIG. 4. Increase in contents of cytochromes in female rabbit liver microsomes during postnatal growth. Cytochrome contents are expressed as m~moles/nig microsomal protein. 0, Microsomal Fe, ; 0, P-450; A, cytochrome b,
cipitate by adding fksly ;:cwdrr~d anrmonium sulfate to 70% saturation, with stirring. The precipitate was resuspended in 0.1 M phosphate buffer, pH 7.5, and t,he cytochrome was precipitated between 35 and 60 % saturation of ammonium sulfate and collected. Figure 6 shows the absorption spectra of t,his cytochrome isolated from the microsomes.
, IO
1
20
I 30
i
TIME IN MINUTES
FIG. 5. Reduction
of cytorhrome b-559 from microsomes of bovine adrenomedulla by addition of NADPH or KADH to solubilized mixtStue. The microsomes were solubilized by adding O.lyO cobra venom to them and incubating the mixture in 0.1 M Tris-HCl at pH 8.5 for 15 hours at 5” under anaerobic conditions. At zero time 1.0 pmole of SADPH or RADII was added. The mixture contained 3.0 ml of solubilized solutinn of 10 mg protein concentration (0.1 M Tris-HCl, pH 7.5) and 0.7 mg of glttcose oxidase, and 550 rmoles of glricose was added for anerohic experiments. Continuous lines are for SADPH and dotted lines are for NADH. Open marks represent aerobic experiments, and filled marks represent anaerobic experiments.
748
ICHIKAWA
AND
YAMANO
WAVELENGTH
(my 1
FIG. 6. Absorption spectra of the oxidized and reduced forms of the cytochrome isolated from medullary microsomes of bovine adrenal gland. The cytochrome was prepared by ammonium sulfate fractionation of a mixture solubilized by treatment with O.l’$& cobra venom. The spectra were taken in a Beckman model DK-2 spectrophotometer with a mixture containing 1.2 mg of protein in a total volume of 3 ml (0.1 M Tris-HCI buffer, pH 7.5), and reduction was carried out by adding a small amount of solid dithionite to the mixture in a cell of l-cm light-path.
TABLE COMPARISON Source:
Absorption bands (mp) oxidized reduced Carbon monoxide EtNC ESR signal (gm = 2.25) Reduction-velocity by NADPH or NADH Prosthetic group Stabilitya
OF PROPERTIES P-420 liver
414 ) 535
427, 530, 559 Combination Combination f NADPH > NADH Protoheme Labile
11Stability against solubilization b Microsomal Fe, .
-
III
OF CYTOCHROMES
IN MAMMALIAN
Mic. Fez6 liver
414, 427, 530, 559 Combination Combination + NADPH > NADH Protoheme Stable
by 1.0% deoxycholate
interaction with other components. The fact that the decrease of absorbancy at 450 rnp of dithionite-reduced microsomes under CO is followed by an increase of absorbancy at 420 rnp when the mixture is rendered alkaline, does not mean that dissociation of the hemoprotein takes place. This change of absorbancy is not reversed by neutralization
MICROSOMES
-
b-559 bovine adrenal medulla
Cyt.
Cyt. bs liver
360, 413, 530,561 424, 526, 556 No combination No combination -
415, 525, 565 428, 530, 559 No combination No combination
NADH> NADPH Protoheme Stable
NADPH > NADH Protoheme Stable
in 0.1
M
phosphate
buffer
at pH 7.0.
but is accompanied by loss of the capacity to be reduced by NADPH. Administration of Sudan III to a rabbit caused a marked increase in the content of microsomal Fe, as well as of P-450. Both Sudan III and phenobarbital appear to induce a substantial absolute increase in new endoplasmic reticulum, after a high content
MICROSO&L~L
of microsomal Fe, has been reached on the basis of microsomal protein. A dramatic hypertrophy was caused by injection of phenobarbital plus SKF 525 A (p-diethyla~minoetlhyldiphcnylpropylacetate-HCl), and this was much greater than that following trcnt,ment, with phenobarbital alone, while the miarosomal E’e, content’ per milligram of microsomal protein mairkained at, the only phenobarbital-treated level. We have studied the cytochromes of adrenal microsomcs to see whether t,hey are identical with t,hose of liver microsomes. Our observations on the absorption maxima of t,he cytochromes from adrenal microsomes are consist’ent with those of Krisch (19) and Estabrook et al. (21). In this paper, the cytochromes of the medullary and of the cortical microsomes of bovine and pig adrenal glands were studied separately. In pig adrenal gland, cytochrome 6s and microsoma, E’e, could be detected both in the cortex and in the medulla. In bovine adrenal glands, they were detected only in cortex. Information was obtained about the cytochrome from bovine medullary microsomes in relat,ion to the ESR signal, CO, or ethylisocyanide (EtXC) binding and enzymic reactivity in SADPH and XADH oxidizing systems. These arc summarized in Table III, where the properties of microsomal Fe, and cyt,ochrome 65 are listed for comparison. REFERENCES 1. YOSHIICAWA, H., J. B&hem. 38, 1 (1951). 2, STRITTMATTER,~. F., AND BALL, E. G., Proc. Xafl. Acad. Sci. L1.S. 38, 19 (1952). 3. KLINGENRERG, M., drch. Biochem. Biophys. 75, 376 (1958).
I CYTO(‘HROi\Zb’S
il
749
4. CLRFINKEI., I)., .I (1-h. Biochcnc. Biophys. 77, 4Q3 (1958). 5. Ounn.4, T., AND SATO, R., J. Biol. Chcm. 237, PC 1375 (1962). 0. OMURA, T., AND Sn~o, R., J. niol. Chcm. 233, 2370 (1964). 7. HASHIMOTO, y., 1'.4MASo, T., AND ~~IAso?;, II. S., J. Biol. Chwt. 237, PC 3843 (1962). y., AND hISSON, 8. ~AMANO, T., &SHIMOTO, 11. S., Federation Proc. 22, 5% (1903). 9. X~SOS, 1-I. S., Y-4xAs0, T., XORTH, J. C., HASHIMOTO, Y., END SAKAGISHI, P., “Oxidases and Related Redox System,” ed. by T. E. King et al., Vol. 2, p. 879, Wiley, New York (1965). 10. WADA, II., IIIGASHI, T., ICHIK.~WA, Y., TADA, K., AND SAI~AMOTO, T., I&chim. Bioph ys. Ada 88, 654 (1964). 11. Rh~onI.4, C., ~OSNER, 11. d., REITZ, IT. C., ~ZSD IJDENFRIEND, S., rlrch. Uioch,em. Biophys. 61, 431 (1956). 12. GORN~LL, A. G., ASI) BARDAWILL, c. J., J. Biol. Chem. 177, 751 (1949). 13. JACKSON, 11. L., AXD &KUSICK, B. C., in “Organic Syntheses” (T. L. Cairns, cd.), Vol. 35, p. G2. Wiley, Xew York (1955). 14. KUSAI, K., .-inn. Repf. Sci. IVor~s, Fat. Sci. Osaka Unir. 8, 13 (1960). 15. GARFINKEL, I)., (‘amp. Wiochem. Phgsiol. 8, 367 (1963). 16. ORRENIUS, S., AND ERNSTER, L., Hiochem. Biophys. Res. Convmzrn. 16, GO (1964). 17. HUGGINS, C., BND PATAKI, T., Proc. Lvatl. Acad. Sci. U.S. 53, 791 (1965). 18. F~IJTS, J. R., AND .~DAMSON, R. H., Science 129, 897 (1959). 19. KRISCH, K., Mature 193, 982 (1962). 20. SPIRO, RI. J., .~XD BALI., E. G., J. Bio/. Chem. 236, 225 (1961). 21. ESTABROOK, R. w., COOPER, n. Y., 4ND ROSENTHAL, O., Biochem. %. 338, 741 (1963).
OF
BIOCHEMISTRY
Electron Correlation
AND
121, 742-749 (1967)
BIOPHYSICS
Spin Resonance
of the Amount
Fe, in Microsomes
of Microsomal
of CO-Binding of Normal
Species with So-Called
Tissues and Liver Microsomes
Sudan Ill-Treated YOSHIYUKI Department
ICHIKAWA
of Biochemistry, Iteceived
April
Cytochromes Microsomal of
Animals
AND
Osaka University 7, 1965; accepted
TOSHIO Medical April
YAMANO School, Osaka, Japan
20, 1965
A correlation has been established on intact microsomes of various tissues between the amount of CO-binding pigment (P-450) and microsomal Fe, , which was designated as a component of rabbit liver microsomes with an ESR signal of low-spin type hemoprotein. This correlation also holds with vertebrate microsomes in which the enzyme contents were raised by injection of Sudan III into the animals. The correlat,ion seen with intact microsomes is no longer observed after treatment with deoxycholate or alkaline, which readily destroys P-450. In the microsomes of bovine adrenal medulla a hemoprotein exists with an a-band at 559 rnp but without an ESR signal or CO-binding character.
Whereas cytochrome bS has been established in mammalian liver microsomes by several workers (1, 2)) an NADPH-dependent cytochrome has not yet been well characterized. A CO-binding pigment (P-450) in microsomes was described by Klingenberg (3) and Garfinkel (4), and was proposed by Omura and Sato (5, 6) to be a hemoprotein. The latter authors obtained a hemoprotein other than cytochrome bc from solubilized liver microsomes and named it P-420. On the other hand, Hashimoto et al. (7) have shown by electron spin resonance (ESR) that a certain species of hemoprotein, which they designated as microsomal Fe,, exists in the smooth- and rough-surfaced microsomes of rabbit liver. From the further studies of Mason et al. (8, 9) it appears that this species is an NADPH-dependent hemoprotein. Logically, the next problem is to study the relation between 450 mp absorbing species and microsomal Fe,. In this study t’he correlation between the t’wo species was examined using intact microsomes from
various vertebrate tissues. In addition, Sudan III was found to be effective for inducing microsomal Fe, and P-450 in parallel. A study of the ESR of phenobarbitaltreated rat microsomes was previously reported in a short communication (10). EXPERIMEXTAL
PROCEDURE
The microsomes used in these experiments were prepared from various tissues by a modification of the method of Mitoma et al. (11). Wistar rats (200250 gm), hamsters (150-200 gm), mice (N-70 gm), and rabbits (2.0-2.5 kg) were fed on a standard commercial laboratory diet with water ad Zibitum for more than 2 weeks before Ilse. All operations were conducted in a cold room at 5”, and material from the slaught,er-house was immediately brought t,o t,he laboratory on ice. The tissues were perfused through the main blood vessels with an ice-cold 1.150/d KC1 solrltion to remove as much hemoglobin as possible. Tissues in which this was not possible were carefully washed with isotonic KC1 solution before further preparation procedures. Fat and connective tisslle were removed from the tissues, which were t,hen chopped up with a pair of scissors and homogenized with 5 volumes of 1.15(3& KC1 solution in a Teflon homogenizer of the Potter742
~ITCROSOMAT, Elvehjem type. The homogenate was centrifllged at) 10,OOOgfor 25 minutes in a refrigerated centrifuge, and t.he precipit,ate was discarded. The supernat)ant fluid was filtered through Tetroncotton. The resldting solution was centrifuged at 105,000g for 90 minutes in a refrigerated Martin Christ Omega type 4OP preparative ultracentrifuge. The microsomes were sedimented from the various tisslles, and a Potter-Elvehjem homogenizer was used to carefully resuspend the microsomes in an isotonic KC1 solution. Centrifugation was then repeated to remove as much extraneous material as possible. Contamination of the microsomes of various tissues with mitochondria was found to be less than 47; on a protein basis as judged by succinic oxidase activity. The washed microsomes were stored at -20” under anaerobic conditions and used within 48 hours after preparation. The microsomal preparations were shown to contain no hemoglobin contamination by reducing them with dithionite and then carefully bubbling carbon monoxide through the suspension for about 30 seconds before measuring the spectrum. With the adrenal glands of pig and cow, t,he medldlas were carefully separated from the cortical t,isslle, and medldlary tissrles were homogenized with 0.25 Y sIIcrose. For preparat ioll of adrenal microsomes, 0.25 M sucrose was preferred to 1.157; KU, because their EHR signal was rather low when they were prepared ill KC1 medilun. This was not so with other tissues. The difference spectra of microsome preparations were measlued in a Gary model 14 recording spectrophotoInrter; a l-cm optical path was rlsed escept whell st,ated otherwise ill the text. The amount, of cytochrome bj was measured aerobically by the difference spectrum of the form reduced with KAl>lI minus the oxidized form (4). CO-binding pigment (P-450) was determined by the Omllra and Sato method; for the former, the val\le of 165 cm-l IIIM-’ was assumed for the molar estinction increment between JO9 and 421 rnp, and for the latter the vallle of 91 cm-l nlh~-l ~-as adopted for t.he molar extinrtion incrrrnellt between 450 and -190 mp, based on the difference Ijetween the spectra of the CO-reduced form and the redltced form. All spectrophotometric measrirrments wrre made at room temperatlire. Electron spin resonnlrce spectroscopy was performed with a. \.arian 1. 1500-10 A spectrometer with a 100 Kc field modrdation lulit, and specka were generally obtained at a sample temperatllre of -170”. A silica tribe of 3 mm internal diameter was {Ised on the Yarian variorts temprratnre atachment,. The protein content was determined after addit,ion of sodium cholate to the sample to remove turbidity, by the biuret method of Gornall et al.
743
CYTOCHROMES
(la), and crystalline bovine plasma albumin was used as standard. The increased absorption in the biuret reaction due to heme in the test samples was avoided by using cupric sulfate-free biuret reagents. NADH and N,4I>PII were obtaitled from the Sigma Chemical Company. Carbon monoxide was prepared from formic acid and concentrated sulfuric acid, and passed through solid potassium 2iydroxide to remove extraneous substances. Ethyl isocyanide was synthesized from silver cyanide and et,hyl iodide (13). The crystalline glucose oxidase used to give anaerobic conditions was purified by the method of Kusai et ccl. (14). The other agents used were obtained commercially. RESULTS
Table I shows the amounts of hemoprotein in the microsomes of various tissues. It is seen that there is a correlation between P-450 and microsomal Fez. The amount of microsomal Fe, is normalized as follows. The double integration of the signal with slope maxima (8,) at g = 2.41 and 1.91, and a g maximum (gm) at g = 2.25, was compared under identical conditions with t>hat of a standard solution of copper containing 2 mnI C&O4 and IO 11111 EDTA. Wit’h rabbit liver microsomes prepared by careful perfusion, an ESR signal was obtained with only a small fluctwkon between gm = 2.25 and s, = 1.91. The estimated amount of microsomal Fe, was found to be of the same order as the amount of P-450 in rabbit liver microsomes, when estimated as described above. Then the height of the ESR signal at gm = 2.25 was measured to estimate the concentrat8ion of microsomal I’e, . As seen from the table, the content of P-450 was highest in the liver tissues of various animals. The contents in kidney microsomes were far less, being about one-tenth of those in liver. The microsomes of pancreas and testis had no P-450 or miclosomal Fe,, but cytochromc 1’5was detected. The result. is in agreement with that reported by CTarfirllrel cxccljt regard to the adrenal gland ( 15). The
with
microsomes of the cort,ex and medulla of bovine adrenal glarlds and pig adrem glands w’crc prepared separately. The ratio of the amount of cyt,ochronle b5 to nlicrosomtll I:eZ varies frorll t,issue to tissue. Pig adrenomedullary
744
ICHIKAWA
AXD
microsomes and cortical microsomes both contain much P-450, t’he amount being second to t’hat in liver microsomes. In contrast with pig adrenomedulla, bovine adrenoTABLE DETERMINATION Sources ofILLmxnes
Rabbit (male) Liver Kidney Small intestine Adrenal gland Rabbit (female) Liver Ovary Rat (male) Liver Rat (female) Liver Kidney Mouse (male) Liver Kidney Guinea pig (female) Liver Kidney Pig (female) Liver Kidney cortex Thyroid Pancreas Adrenal cortex Adrenal medulla Ovary Dog (male) Liver Kidney Carp (female) Liver Gibe1 (female) Liver Bovine Liver Kidney cortex Kidney medulla Cerebellum Pituitary gland Pancreas Adrenal cortex Ovary Testis Calf thymus Hamster (male) Liver Kidney
OF THE
CYTOCHROMES
[21
YAMANO
medullary microsomes contain a cytochrome which shows an a-band at 559 rnp but, no ESR signal. The properties of this cyt.ochrome will be described later. I
IN MICROSOMES
AT VARIOUS
VERTEBRAE
ISI
TISSUES
Cyt. bs
P!%O
0.88 0.10 0.02 0.08
1.72 0.16 0.07 1.20
1.70 0.15 0.08 1.10
1.1 0.9
1.9 1.5 4.0 1.4
0.80 0.21
1.55 0.06
1.60 0.07
1.0 1.2
2.0 0.3
0.64
0.82
0.81
1.0
1.3
0.51 0.08
0.71 0.07
0.70 0.07
1.0 1.0
1.4 0.9
0.40 0.10
0.61 0.06
0.60 0.06
1.0 1.0
1.5 0.6
0.55 0.12
0.79 0.14
0.70 0.13
0.9 0.9
1.3 1.1
0.29 0.17 0.13 0.06 0.24 0.25 0.47
0.42 0.18 0.00 0.00 1.14 0.61 0.17
0.40 0.20 1.00 0.60 0.20
1.0 1.0 0.9 1.0 1.2
1.4 1.1 4.2 1.4 0.4
0.42 0.12
0.75 0.17
0.73 0.18
1.0 1.1
1.7 1.5
0.14
0.38
0.37
1.0
2.6
0.04
0.15
0.15
1.0
3.8
0.79 0.01 0.05 0.04 0.07 0.09 0.13 0.04 0.09 0.05
0.94 Trace 0.00 0.00 0.07 0.00 0.78 0.05 0.00 0.00
1.05 0.07 0.76 Trace -
1.1 1.0 1.0 -
1.3 1.0 5.9 -
0.64 0.08
1.14 0.14
1.10 0.15
1.0 1.1
1.7 1.9
MicI4be
Lz
161
Ratio of 4 to 3 Ratio of 4 to 2
1.0 0.9
MIC:ROSObIAL
CYTOCHROMES
TABLE
745
I-Continued
Cl1
Sources of microsomes
Mic[“i;eI
z
El Ratio of 1 to 3
[61
Ratio of 4 to ?
Hamster (female) Liver Kidney Hen liver Goose (female) Liver Horse (male) Liver Kidney cortex Adrenal cortex Sheep (female) Liver Kidney Adrenal Adrenal
cortex medulla
Orrenius and Ernster (16) have reported the induction of P-450 in liver microsomes by phenobarbital injection into rats. Table II shows the inductive increase of microsomal Fe, in rabbit liver microsomes. Huggins et al. (1’7) reported on the induction of menadione rcductase. In our study, a low dose of Sudan III was found to induce microsomal Fe,. Table II gives these results, and shows t,hat the correlation between P-450 and microsomal Fe, was still present. Sudan III was dissolved in ethanol and injected into a rabbit intraperitoneally. The daily dose was determined by drying a known TABLE
II
INCREASE IN CYTOCHROMES IN PHENOBARBITALAND SUDAN MALE RABBITS
MICROSOMES III-TREATED
OF
Rabbits were injected intraperitoneally with 85 mg of sodium phenobarbital or 4 mg of Sudan III per kg body weight once daily for 4 days. One day after the last inject#ion the animals were killed. The microsomal fraction was isolated from the liver homogenates by a procedure as described in the text. The content,s of cytochrome bg , P-450, and microsomal Fe, are expressed as described in Table I. [II Rabbit liver microsomes
121 cg .
P-450
131
141 Mic. Fez
Control Phenobarbital Sudan III
0.88 1.50 2.60
1.72 5.40 5.10
1.70 5.10 4.80
R!L of 4 to 3
R?io
1.0 0.9 0.9
1.9 3.4 1.8
of 4 to 2
volume of the ethanol solution and weighing the residue. A daily dose of 5 mg/kg of rabbit caused about a threefold increase in the microsomal Fe, and cytochrome b, cont(ent.8. In view of the observation of Yamano et al. (8) that, the cyt’ochrome which has an a-band at 559 mp is likely to be associated with microsomal Fe,, the increased absorbancy at 559 rnp due to anaerobic XADPH minus the absorption of aerobic NADH should appear more distinct’ly when the difference spectrum is measured on liver microsomes from rabbits after injection of phenobarbital. Figure 1 shows t’he time course of the increase of the absorbancy at 559 rnp after adding NADH or NADPH. The correlation between microsomal Fe, and P-450 was no longer observed after intact microsomes had been treated wit’h cholate or deoxycholate. As seen in Fig. 2, P-450 species decreased at low concentration of deoxycholat’e and almost disappeared at a concentration of 0.5 % deoxycholate, wherea,s the content8 of microsomal Fe, remained nearly unchanged. As the P-450 species disappeared, P-420 species appeared, their concentrat,ions being inversely related to that of P-450. This had previously been observed by Omura and Sato, who reported t’he peak which appears at, 420 mp on dit,hionit,e reduction of the soluble form of P-450 under CO. The P-450 species also disappeared when
746
ICHIKAWA
0.0
0
AFU
I
I
I
I
I
5
IO
15
20
25
TIME
IN MINUTES
FIG. 1. Time course of appearance of increased absorption when KADPH was added to liver microsomes of rabbit, treated with phenobarbital, in the presence of glucose and glucose oxidase (added to give anaerobic conditions). The figure illustrates the autooxidizability of the species which shows an a-band at 559 rnp, as judged from the decrease of the absorption and approach to the absorption level of cytochrome bb after exhaustion of glucose. The mixture contained 2.0 mg of microsomal protein, 1.0 pmole of P\‘ADPH (upper curve) or NADH (lower curve), and 0.7 mg of glucose oxidase in a total volume of 3 ml (0.1 M phosphate buffer, pH 7.0). A cell of l-cm light-path was used. When NADPH was used, 300 wmoles of glucose had previously been added to the mixture.
a microsomal solution of pH 7 was made alkaline. In this case, microsomal Fe, species were decreased in parallel with P-450, and the P-420 species increased in inverse relation. This result is presented in Fig. 3. At pH 6, P-450 species decreased, but the reason for this is unknown. Hemoproteins in rabbit liver microsomes ,qere measured during growth after birth. As presented in Fig. 4, a correlation between microsomal Fe, and P-450 was established. Fouts and Adamson (18) reported that some drug metabolizing activity appeared 2 weeks after birth, and after 4 weeks the act’ivity was about equal to that of the adult. Our observation on the increase in the amount of microsomal Fe, bears some relation to the drug met’abolizing activity of liver microsomes.
YAMANO
Krisch (19) and Spiro and Ball (20) reported t,hat bovine adrenal microsomes, when solubilized with deoxycholate and reduced with dithionite, showed an a-band at 559-560 rnp and a Soret band at 4‘28 rnM. ,4s mentioned above, the medullary microsomes of bovine adrenal glands showed neither an ESR signal nor CO-binding, whereas they showed an a-band at 559 mp. Rledullary microsomes of bovine adrenals were solubilized by adding 0.1% unheated cobra venom to a microsomal suspension in 0.1 M TrisHCl buffer, pH 8.5, and allowing the mixture to stand at 5’ for 15 hours under anaerobic conditions. The cytochrome in the solubilized mixture was reduced by adding ?;ADPH or NADH, as shown m Fig. 5. The rate of reduction was fastest’ with anaerobic NADPH. The cytochrome released from the particles was collected as a reddish pre-
I 0.5
OY 0 DEOXYCHOLATE
CONCENTRATION,
I I 1.0 O %
FIG. 2. Relationship between microsomal CObinding species and microsomal Fe, at various concentrations of deoxycholic acid. The reaction mixture, containing phenobarbital-treated rabbit liver microsomes at a protein concentration of 20 mg/ml, was incubated at 20” for 10 minutes and used for ESR studies. Spectrophotometric measurements were carried out at a protein concentrations of 2.0 mg/ml under the same conditions, in 0.1 M phosphate buffer at pH 7.0 and a cell of l-cm light-path. 0, Microsomal Fe, ; 0, P-450; 0, P-420. The ordinates are the percentages of the residual absorbancy at 450 rnb minus the absorbancy at 490 rnp and of the absorbancy appearing at 420 rnp, when the maximum absorbancy at 420 rnp is assumed to be lo07c. The heights of ESR signals were measured at gm = 2.25, each plot, being expressed as a percentage of the height of the initjR1 ESR signal.
XIICI~OSOMrZL
747
CYTOCIIRONIES DISCUSSIOS
4 2 0” ‘“m 50 4
b s 0
6
7
9
6
IO
II
PH
FIG. 3. Effect of pII on microsomal Fe, and P-450 of phenobarbital-treated rabbit, liver mirrosomes. A mixture containing rabbit liver mirrosomes at a protein coimentration of 20 mg/ml was ttsed for ESR measurements. Spectrophotometric measurements were carried ottt at a protein eon centration of 2.0 mg/ml. The samples were placed in solutions of different pH values. The buffers used are 0.1 M phosphate buffer at below pH 8.3 and 0.1 M glycine-KaOH buffer at above pH 8.5.
The results report,ed here are concerned principally with the hemoprotein in microsomes, which seems to be associated wit,h microsomal Fe, or CDbinding species (P-160). The content of P-450 species was aways present in proportion w&h that of microsomal Fe, in the microsomes of various vertebrate t8issues. However, on treatment of microsomes with deoxycholate or alkali, this parallel relation was lost. In the present experiments, the P-4.50 species were more labile t,han microsomal l‘e, , and on keatment with alkali the former seemed to be degraded to a hemoprot8ein named P-420 by Omura and Sat’0 (5). These three species are not considered to be different hemoprot,eins but to be due to a single hemoprotein, because total-heme analysis showed the existence of only one hemoprotjein besides cytochrome bg. P-450 appears to be microsomal Ipe, in
k
0 WEEKS
FIG. 4. Increase in contents of cytochromes in female rabbit liver microsomes during postnatal growth. Cytochrome contents are expressed as m~moles/nig microsomal protein. 0, Microsomal Fe, ; 0, P-450; A, cytochrome b,
cipitate by adding fksly ;:cwdrr~d anrmonium sulfate to 70% saturation, with stirring. The precipitate was resuspended in 0.1 M phosphate buffer, pH 7.5, and t,he cytochrome was precipitated between 35 and 60 % saturation of ammonium sulfate and collected. Figure 6 shows the absorption spectra of t,his cytochrome isolated from the microsomes.
, IO
1
20
I 30
i
TIME IN MINUTES
FIG. 5. Reduction
of cytorhrome b-559 from microsomes of bovine adrenomedulla by addition of NADPH or KADH to solubilized mixtStue. The microsomes were solubilized by adding O.lyO cobra venom to them and incubating the mixture in 0.1 M Tris-HCl at pH 8.5 for 15 hours at 5” under anaerobic conditions. At zero time 1.0 pmole of SADPH or RADII was added. The mixture contained 3.0 ml of solubilized solutinn of 10 mg protein concentration (0.1 M Tris-HCl, pH 7.5) and 0.7 mg of glttcose oxidase, and 550 rmoles of glricose was added for anerohic experiments. Continuous lines are for SADPH and dotted lines are for NADH. Open marks represent aerobic experiments, and filled marks represent anaerobic experiments.
748
ICHIKAWA
AND
YAMANO
WAVELENGTH
(my 1
FIG. 6. Absorption spectra of the oxidized and reduced forms of the cytochrome isolated from medullary microsomes of bovine adrenal gland. The cytochrome was prepared by ammonium sulfate fractionation of a mixture solubilized by treatment with O.l’$& cobra venom. The spectra were taken in a Beckman model DK-2 spectrophotometer with a mixture containing 1.2 mg of protein in a total volume of 3 ml (0.1 M Tris-HCI buffer, pH 7.5), and reduction was carried out by adding a small amount of solid dithionite to the mixture in a cell of l-cm light-path.
TABLE COMPARISON Source:
Absorption bands (mp) oxidized reduced Carbon monoxide EtNC ESR signal (gm = 2.25) Reduction-velocity by NADPH or NADH Prosthetic group Stabilitya
OF PROPERTIES P-420 liver
414 ) 535
427, 530, 559 Combination Combination f NADPH > NADH Protoheme Labile
11Stability against solubilization b Microsomal Fe, .
-
III
OF CYTOCHROMES
IN MAMMALIAN
Mic. Fez6 liver
414, 427, 530, 559 Combination Combination + NADPH > NADH Protoheme Stable
by 1.0% deoxycholate
interaction with other components. The fact that the decrease of absorbancy at 450 rnp of dithionite-reduced microsomes under CO is followed by an increase of absorbancy at 420 rnp when the mixture is rendered alkaline, does not mean that dissociation of the hemoprotein takes place. This change of absorbancy is not reversed by neutralization
MICROSOMES
-
b-559 bovine adrenal medulla
Cyt.
Cyt. bs liver
360, 413, 530,561 424, 526, 556 No combination No combination -
415, 525, 565 428, 530, 559 No combination No combination
NADH> NADPH Protoheme Stable
NADPH > NADH Protoheme Stable
in 0.1
M
phosphate
buffer
at pH 7.0.
but is accompanied by loss of the capacity to be reduced by NADPH. Administration of Sudan III to a rabbit caused a marked increase in the content of microsomal Fe, as well as of P-450. Both Sudan III and phenobarbital appear to induce a substantial absolute increase in new endoplasmic reticulum, after a high content
MICROSO&L~L
of microsomal Fe, has been reached on the basis of microsomal protein. A dramatic hypertrophy was caused by injection of phenobarbital plus SKF 525 A (p-diethyla~minoetlhyldiphcnylpropylacetate-HCl), and this was much greater than that following trcnt,ment, with phenobarbital alone, while the miarosomal E’e, content’ per milligram of microsomal protein mairkained at, the only phenobarbital-treated level. We have studied the cytochromes of adrenal microsomcs to see whether t,hey are identical with t,hose of liver microsomes. Our observations on the absorption maxima of t,he cytochromes from adrenal microsomes are consist’ent with those of Krisch (19) and Estabrook et al. (21). In this paper, the cytochromes of the medullary and of the cortical microsomes of bovine and pig adrenal glands were studied separately. In pig adrenal gland, cytochrome 6s and microsoma, E’e, could be detected both in the cortex and in the medulla. In bovine adrenal glands, they were detected only in cortex. Information was obtained about the cytochrome from bovine medullary microsomes in relat,ion to the ESR signal, CO, or ethylisocyanide (EtXC) binding and enzymic reactivity in SADPH and XADH oxidizing systems. These arc summarized in Table III, where the properties of microsomal Fe, and cyt,ochrome 65 are listed for comparison. REFERENCES 1. YOSHIICAWA, H., J. B&hem. 38, 1 (1951). 2, STRITTMATTER,~. F., AND BALL, E. G., Proc. Xafl. Acad. Sci. L1.S. 38, 19 (1952). 3. KLINGENRERG, M., drch. Biochem. Biophys. 75, 376 (1958).
I CYTO(‘HROi\Zb’S
il
749
4. CLRFINKEI., I)., .I (1-h. Biochcnc. Biophys. 77, 4Q3 (1958). 5. Ounn.4, T., AND SATO, R., J. Biol. Chcm. 237, PC 1375 (1962). 0. OMURA, T., AND Sn~o, R., J. niol. Chcm. 233, 2370 (1964). 7. HASHIMOTO, y., 1'.4MASo, T., AND ~~IAso?;, II. S., J. Biol. Chwt. 237, PC 3843 (1962). y., AND hISSON, 8. ~AMANO, T., &SHIMOTO, 11. S., Federation Proc. 22, 5% (1903). 9. X~SOS, 1-I. S., Y-4xAs0, T., XORTH, J. C., HASHIMOTO, Y., END SAKAGISHI, P., “Oxidases and Related Redox System,” ed. by T. E. King et al., Vol. 2, p. 879, Wiley, New York (1965). 10. WADA, II., IIIGASHI, T., ICHIK.~WA, Y., TADA, K., AND SAI~AMOTO, T., I&chim. Bioph ys. Ada 88, 654 (1964). 11. Rh~onI.4, C., ~OSNER, 11. d., REITZ, IT. C., ~ZSD IJDENFRIEND, S., rlrch. Uioch,em. Biophys. 61, 431 (1956). 12. GORN~LL, A. G., ASI) BARDAWILL, c. J., J. Biol. Chem. 177, 751 (1949). 13. JACKSON, 11. L., AXD &KUSICK, B. C., in “Organic Syntheses” (T. L. Cairns, cd.), Vol. 35, p. G2. Wiley, Xew York (1955). 14. KUSAI, K., .-inn. Repf. Sci. IVor~s, Fat. Sci. Osaka Unir. 8, 13 (1960). 15. GARFINKEL, I)., (‘amp. Wiochem. Phgsiol. 8, 367 (1963). 16. ORRENIUS, S., AND ERNSTER, L., Hiochem. Biophys. Res. Convmzrn. 16, GO (1964). 17. HUGGINS, C., BND PATAKI, T., Proc. Lvatl. Acad. Sci. U.S. 53, 791 (1965). 18. F~IJTS, J. R., AND .~DAMSON, R. H., Science 129, 897 (1959). 19. KRISCH, K., Mature 193, 982 (1962). 20. SPIRO, RI. J., .~XD BALI., E. G., J. Bio/. Chem. 236, 225 (1961). 21. ESTABROOK, R. w., COOPER, n. Y., 4ND ROSENTHAL, O., Biochem. %. 338, 741 (1963).