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Microsomal monooxygenase system in frog livers
Comp. Biochem. PhysioLVol. 77B, No. 4, pp. 761-767, 1984 Printed in Great Britain
0305-0491/'84 $3.00+0.00 © 1984 Pergamon Press Ltd
MICROSOMAL MONOOXYGENASE SYSTEM IN FROG LIVERS MITSUHIDE NOSHIRO* and TSUNEO OMURA Department of Biology, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812, Japan
(Received 7 September 1983) Abstract--l. Liver microsomes prepared from four species of frog, Rana eatesbeiana, Rana nigromaculata, Bufo bufojaponicus, and Xenopus laevis, contained cytochrome P-450 and showed NAD(P)H-dependent monooxygenase activities to several foreign chemical compounds tested. 2. The oxidations of the chemical compounds by frog liver microsomes showed significant variations among frog species. The oxidation of 7-ethoxycoumarin was much faster than that by rat liver microsomes. The oxidation of benzo(a)pyrene was significantly induced by 3-methylcholanthrene administration. 3. The monooxygenase activities of frog liver microsomes were more sensitive to cyanide inhibition than those of rat liver microsomes.
monooxygenase activity by 3-methylcholanthrene was also studied.
INTRODUCTION Cytochrome P-450-containing monooxygenase system is widespread among various forms of life. In vertebrates, the monooxygenase system of mammalian liver microsomes has been intensively investigated from various viewpoints including the multiple molecular nature of cytochrome P-450. The liver microsomes of birds, reptiles and amphibians have also been shown to oxidize various foreign compounds (Creaven et al., 1965a,b; Creaven et al., 1967). The monooxygenase activities of fish liver preparations have been frequently studied in connection with the biotransformation of water pollutants such as petroleum hydrocarbons and polychlorinated biphenyls (Creaven, et al., 1967; Adamson, 1967; Buhler and Rasmussen, 1968; Pederson et al., 1974; Ahokas et al., 1975; Payne and Penrose, 1975; Ahokas et al., 1977; Bend et al., 1977; Stegeman and Binder, 1979; Stegeman et al., 1979; James and Bend, 1980; Lindstr6m-Sepp~i et al., 1981). However, most of these comparative studies on hepatic monooxygenase activities described only the occurrence of the activities and the metabolites of foreign compounds. In order to compare the liver microsomal monooxygenase systems of various organisms, characterization of the electron transport system of the microsomes is highly desirable. In this study, we examined the electron transport system of the liver microsomes of four species of frog, Rana catesbeiana (bullfrog), Rana nigromaculata, Bufo bufo japonicus (toad) and Xenopus laevis. Hemoprotein contents, reductase activities and oxidations of several chemical compounds were examined and compared. The multiple nature of microsomal cytochrome P-450 was studied by the titration with cyanide. The induction of the *Present address, Department of Biochemistry, Hiroshima University School of Dentistry, Kasuni 1-2-3, Minamiku, Hiroshima, Hiroshima 734 Japan.
MATERIALS AND METHODS
Animals Rana catesbeiana (270-380g body wt), Rana nigromaculata (30-60 g) and Bufo bufojaponicus (200-360 g) were obtained from a commercial source. Xenopus laevis (20-70 g) was generously supplied by Dr. Misumi and Dr. Yamana of Kyushu University. Male rats of SpragueDawley strain weighing about 200 g were also used for comparison. When Xenopus was treated with 3-methylcholanthrene, 1% solution of the chemical in olive oil was injected once intraperitoneally in a dose of 25 mg per kg body wt 5 days before killing. Preparation of microsomes from frog livers Animals were killed by a blow on the head. The abdominal cavity was opened and the liver was thoroughly perfused in situ from the hepatic vein with chilled 0.9% NaCI. The perfused liver was excised, finely chopped with scissors and homogenized with 9 vol of 0.25 M sucrose solution containing 10mM Tris-HCl buffer (pH 7.5) and 1 mM EDTA in a Potter-type glass homogenizer equipped with a Teflon pestle. The homogenate was centrifuged at 10,000g for 15 min and the precipitate was discarded. The supernatant was centrifuged at 105,000g for 60min, and the packed pellets formed two layers. The lower colourless jelly-like layer of glycogen granules was discarded. The upper layer of microsomes was suspended in 0.15 M KCI containing 1 mM EDTA and centrifuged again as above. The washed microsomes were finally suspended in 0.25 M sucrose containing 10mM Tris-HC1 (pH 7.5) and t mM EDTA. All assays with microsomal preparations were performed within the same day. Analytical methods The contents of cytochrome P-450 and cytochrome b5 in microsomes were determined according to Omura and Sato (1964) using extinction coefficients of 91 and 185/cm/mM- i, respectively. NADPH- and NADH-cytochrome c reductase activities were determined as described previously (Omura and Takesue, 1970). The content of protoheme in microsomes was measured spectrophotometrically after con761
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MITSUHIDE NOSHIRO and TSUNEO OMURA Table 1. Chemical composition of frog liver microsomes Recovery of Contents of Ms protein Phospholipid RNA Protoheme Flavin Animal species (mg/g liver) (#g/mg Ms protein) (nmol/mg Ms protein) Rana catesbeiana 7.4 580 140 1.12 0.141 Bufo bufo japonicus 5.4 600 350 1.56 0.131 Xenopus laevis 4.2 490 210 1.04 0.199 Rat 12.7 590 120 1.17 0.336 All data are mean values of two or three preparations. Ms: microsomes.
verting the heme into pyridine hemochromogen in the presence of 0.1 M NaOH and 20~o pyridine (Omura and Sato, 1964). The content of total flavin in microsomes was determined by the method of Bessey et al. (1949). Phospholipid and RNA were extracted according to Schneider (1957) and determined by the methods of Lees (1957) and Schneider (1957), respectively. Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Cyanide titration of cytochrome P-450 in microsomes Spectrophotometric titration of liver microsomes with potassium cyanide was performed using a Shimadzu Double-40-DF spectrophotometer according to the method of Comai and Gaylor (1973). Suspensions of microsomes (1 mg/ml) in 0.l M potassium phosphate buffer (pH 7.5) were titrated with freshly prepared potassium cyanide solution which had been adjusted to neutral pH with HCI. The titrations were performed over range of 0.1 to 32mM cyanide. The pH of the titration mixtures did not change during the titration. Assays of drug oxidations Assays of aniline hydroxylase, aminopyrine Ndemethylase, 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase were performed as described by Imai et al. (1966), Orrenius (1965), Ullrich and Weber (1972), and Nebert and Gelboin (1968), respectively. In the assays of the oxidations of aniline and aminopyrine, an NADPH-generating system consisting of 0.5 mM NADP +, 5 mM isocitrate, 5 mM MgC12 and 0.36 unit of isocitrate dehydrogenase or 2 mM NADH were used. In the assays of 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase, 0.1 mM NADPH or 0.1 mM NADH and 0.4 mM NADPH or 1 mM NADH, respectively, were used. Other assay conditions were the same as described previously (Noshiro and Omura, 1978). Reagents and biochemicals Yeast cytochrome c was a generous gift of Sankyo Co., Tokyo. 7-Ethoxycoumarin was kindly provided by Dr. V. Ullrich. All other chemicals were of the highest purity commercially available. RESULTS
Chemical composition o f f r o g liver microsomes Table 1 shows the chemical compositions of liver
microsomes from three species of flog. A l t h o u g h the recovery of m i c r o s o m a l protein was lower than that of rat, the contents of phospholipids, p r o t o h e m e and flavin of frog liver microsomes were not m u c h different from those of rat liver microsomes. Hemoprotein contents and reductase activities q f frog liver microsomes Table 2 shows the h e m o p r o t e i n contents and reductase activities of liver microsomes prepared from four species o f frog. The c o r r e s p o n d i n g values of rat liver microsomes are also shown for comparison, B o t h c y t o c h r o m e P-450 and c y t o c h r o m e b s were present in all microsomal samples but their contents varied significantly from one species of frog to another. In the case of Xenopus laevis which showed especially low c o n t e n t of c y t o c h r o m e P-450, 3-methylcholanthrene-treated animals were also examined. The sum of the contents of c y t o c h r o m e P-450 a n d c y t o c h r o m e bs was always lower t h a n the p r o t o h e m e content of frog liver microsomes. C a r b o n monoxide-difference spectra of the microsomal prepa r a t i o n s showed a significant a m o u n t of cont a m i n a t i n g h e m o g l o b i n in Xenopus microsomes, but the a m o u n t s of h e m o g l o b i n in the microsomes of other three species of frog were too small to account for the excess protoheme. The excess p r o t o h e m e in microsomes was p r o b a b l y due to some h e m o p r o t e i n s o f other c o n t a m i n a t i n g cell o r g a n d i e s including catalase of peroxisomes. Judging from succinatec y t o c h r o m e c reductase activity of the microsomal p r e p a r a t i o n s (data not shown), m i t o c h o n d r i a l cont a m i n a t i o n s to the microsomal fractions were a b o u t 2 - 6 % on the protein basis. N A D ( P ) H - s u p p o r t e d c y t o c h r o m e c reductase activities of frog liver microsomes were lower t h a n those of rat liver microsomes. 3 - M e t h y l c h o l a n t h r e n e - t r e a t m e n t of Xenopus laevis resulted in a two-fold increase of c y t o c h r o m e P-450 in liver microsomes, but the t r e a t m e n t did not affect the content of c y t o c h r o m e b5 n o r the activities of the reductases. It was not possible to detect a shift of Soret peak in the CO-difference spectrum of induced microsomes (Alvares et al., 1967), since the presence
Table 2. Hemoprotein contents and reductase activities of frog liver microsomes NADPH-cyt. c NADH-cyt.c Cyt. P-450 Cyt. bs reductase reductase (nmol/mg Ms protein) (gmol reduced/min/mg Ms protein) Animal species 0.43 0.16 0.074 0.372 Rana catesbeiana 0.77 0.32 0.116 0.524 Rana nigromaculata 0.73 0.16 0.048 0.268 Bufo bufo japonicus 0.10 0.072 0.082 0.469 Xenopus laevis Xenopus laevis 0.23 0.078 0.093 0.375 (3-MC-treated) 0.67 0.39 0.239 1.05 Rat Assay procedures are described in Materials and Methods. All data are mean values of more than three preparations. Ms and 3-MC: microsomes and 3-methylcholanthrene, respectively.
Microsomal monooxygenase system in frog livers Table
3.
NADPH-supported oxidation of aniline, aminopyrine, benzo(a)pyrene by frog liver microsomes
763
7-ethoxycoumarin and
Substrates Aniline Animal species
Rana catesbeiana Rana nigromaculata Bufo bufo japonicus Xenopus laevis Xenopus laevis (3-MC-treated) Rat
Aminopyrine 7-ethoxycoumarin Benzo(a)pyrene nmol product formed/min/mg Ms protein (nmol product formed/min/nmol P-450)
0.463 (1.07) 0.797 (1.03) 0.632 (0.880) 0.062 (0.61) 0.089 (0.387) 1.02 (1.04)
h29 (2.99) 8.05 (10.4) 3.65 (5.09) 0.422 (4.18) 0.487 (2.10) 3.23 (3.28)
2.00 (4.63) 3.94 (5.09) 1.32 (1.84) 0.163 (1.6t) 0.486 (2.10) 0.331 (0.338)
0.390 (0.90) 0.150 (0.239) 0.840 (1.17) 0.013 (0.13) 0.094 (0.407) 1.05 (1.07)
Oxidation activities were assayed as described in Materials and Methods. The data are mean values of more than three microsomal preparations. Ms and 3-MC, microsomes and 3-methylcholanthrene, respectively.
of contaminating hemoglobin interferred with the spectral observation of cytochrome P-450.
Oxidation of chemical compounds by frog liver microsomes Table 3 shows the NADPH-supported oxidations of four chemical compounds catalyzed by frog liver microsomes. Aniline hydroxylation activities of frog liver microsomes were lower on the protein basis than rat liver microsomes. However, when the hydroxylation activities were expressed on the basis of cytochrome P-450, the activities of the frog microsomes were almost identical with that of rat microsomes. Aminopyrine N-demethylation activity of Rana nigromaculata microsomes was much higher than that of rat liver microsomes on the basis of both protein and cytochrome P-450. The activities of other frogs were not much different from that of rat on the basis of cytochrome P-450. 7-Ethoxycoumarin O-deethylation activities of frog liver microsomes were much higher than that of rat liver microsomes on the basis of cytochrome P-450. The activities of bullfrog and Rana nigromaculata microsomes were about fifteen times higher than that of rat and the activities of toad and Xenopus microsomes were about five times higher than rat. Benzo(a)pyrene hydroxylation activities of the liver microsomes of bullfrog and toad were about the same as that of rat liver microsomes on the basis of cytochrome P-450, whereas the activities of Rana
nigromaculata and Xenopus microsomes were much lower than the other frogs. NADPH-supported oxidations of aminopyrine and 7-ethoxycoumarin by bullfrog liver microsomes were inhibited by 40 and 90~o, respectively, in a gas mixture of 80~ CO and 20~o 02, confirming the participation of cytochrome P-450 in these oxidation reactions. The liver microsomes of Xenopus laevis showed a very low content of cytochrome P-450 (Table 2) and a weak activity of benzo(a)pyrene hydroxylation compared with other frogs. However, when Xenopus was treated with 3-methylcholanthrene, benzo(a)pyrene hydroxylation activity of the liver microsomes was markedly increased on the basis of both protein and cytochrome P-450. O-Deethylation of 7-ethoxycoumarin was also stimulated by the treatment, whereas the oxidations of aniline and aminopyrine did not change on the basis of protein and rather decreased on the basis of cytochrome P-450. Although N A D P H is the preferred source of reducing equivalents for the monooxygenase reactions catalyzed by microsomal cytochrome P-450, N A D H is also capable of sustaining the oxidation reactions although at lower rates than the corresponding NADPH-supported reactions. In some cases, addition of NADPH-supported microsomal oxidation systems enhanced the rates of reactions (Cohen and Estabrook, 1971a,b), which is called " N A D H synergism". We investigated the effect of N A D H on the monooxygenase reactions catalyzed by frog liver
Table 4. Effects of NADH on the oxidations of aniline, aminopyrine and 7-ethoxycoumarin by frog liver microsomes Substrates Frog species
Electron donors
Aniline Aminopyrine 7-Ethoxycoumarin nmol product formed/min/nmol P-450 (%)
Rana catesbeiana
NADPH NADH NADPH + NADH
1.07 (100) 0.13 (12) 1.04 (97)
3.15 (100) 2.22 (70) 3.79 (120)
4.89 (100) 0.29 (6) 4.94 (101)
Rana nigromaculata
NADPH NADH NADPH ÷ NADH
0.98 (100) 0.11 ( 11) 0.97 (97)
6.34 (100) h 57 (24) 8.43 (131)
5.09 (100) 0.10 (2) 8.44 (166)
Bufo bufo japonicus
NADPH NADH NADPH + NADH
0.83 (100) 0.056 (7) 0.85 (102)
3.95 (100) 0.79 (20) 3.87 (98)
3.06 (100) 0.37 (12) 3.12 (102)
Assay procedures are described in Materials and Methods. Concentrations of NADH were 2 mM for the assays of aniline and aminopyrine oxidations and 0.1 mM for 7-ethoxycoumarin oxidation.
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MITSUHIDE NOSHIRO and TSUNEO OMURA
Table 5. Effects of cyanide on NADPH-supported oxidations of aminopyrine and 7-ethoxycoumarin by frog and rat liver microsomes Substrates KCN Aminopyrine 7-Ethoxycoumarin Animal species (raM) nmol product formed/min/nmol P-450 (%) 0 1.75 (100) 5.78 (100) Rana catesbeiana 0.5 1.20 (69) 0.65 (I 1) 0 8.85 (100) 5.09 (100) Rana nigromaculata 0.5 5.76 (65) 2.57 (50) 0 3.19 (100) 0.5 1.74 (55) 0 1.88 (100) Rat 0.5 1.85 (98) Assay procedures are described in Materials and Methods.
4.86 (100) 3.10 (63) 0.39 (100) 0.28 (72)
Bufo bufojaponicus
microsomes and we found that N A D H - s u p p o r t e d oxidation activities were usually about 10-20% of the corresponding N A D P H - s u p p o r t e d activities (Table 4). However, bullfrog microsomes showed exceptionally high N A D H - s u p p o r t e d aminopyrine Ndemethylation activity, which was as high as 70% of the corresponding N A D P H - s u p p o r t e d activity. A significant NADH-synergism was observed only with the 7-ethoxycoumarin O-deethylation catalyzed by Rana nigromaculata microsomes, although the microsomal preparation showed a very low N A D H supported oxidation of the same substrate.
Effects of cyanide on the monooxygenase reactions catalyzed by frog liver microsomes Table 5 shows the effects of cyanide on the oxidations of aminopyrine and 7-ethoxycoumarin catalyzed by frog and rat liver microsomes. At a concen-
tration of 0.5 mM, cyanide inhibited the aminopyrine N-demethylation activities of frog liver microsomes by 30-45%, whereas the corresponding activity of rat liver microsomes was not affected at all. 7-Ethoxycoumarin O-deethylation activities by frog liver microsomes were also significantly inhibited by cyanide, and the activity of bullfrog microsomes was especially sensitive to cyanide. The O-deethylation activity of rat liver microsomes was also slightly inhibited by cyanide. Since 7-ethoxycoumarin O-deethylation by bullfrog microsomes was very sensitive to cyanide, effects of various concentrations of cyanide on the N A D P H supported O-deethylation by bullfrog microsomes were examined at various concentrations of the substrate. Figure 1 showed the double reciprocal plot of the O-deethylation activities in the presence and absence of cyanide. Two distinct phases having
5 mM
/
0.1 mM
I---E 4 +
/
/
/
005
mM
E
0.02 mM
J KCN
-1
0
1 [ 7 - ethoxycoumorln} -1
2 1 {pMl-
0 mM
3
Fig. l. Double reciprocal plots of 7-ethoxycoumarin O-deethylation reaction catalyzed by bullfrog liver microsomes. Experimental procedures are described in Materials and Methods. The incubation mixture contained 0.1 mg of microsomal protein and various concentrations of 7-ethoxycoumarin and cyanide as indicated in the figure in 2 ml of 0.1 M Tris-HC1 buffer (pH 7.5). The assays were started by the addition of 0.1 mM NADPH and carried out at 30°C for 5min.
Microsomal monooxygenase system in frog livers
0.05
0,04
E E m o
0.03
~
765
8
i ~t
•,a" 0 . 0 2 <1
0.01
o
I
i
I
I
0.02
0.04
o.o6
o.os
, A/[CN-}
I
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Fig. 2. Hoffstee plots of the cyanide titration and bullfrog liver microsomes. Both sample and reference cuvettes contained the same microsomal suspension (1 mg/ml) in 0.1 M potassium phosphate buffer (pH 7.5). After recording the base line, the sample cuvette was titrated with cyanide solution and reference cuvette was also added with same volumes of water and the difference spectrum was recorded between 370 and 500 nm. When the titration was carried out in the presence of 7-ethoxycoumarin, the substrate was added to both the sample and the reference cuvette at a final concentration of 10 #M. O, in the absence of 7-ethoxycoumarin; O, in the presence of 7-ethoxycoumarin. different Km values for 7-ethoxycoumarin (2.8 × 10 6 M and 4.7 × 10 -7 M) were observed for all concentrations of cyanide examined, which indicated the existence of at least two types of cytochrome P-450 catalyzing the oxidation of 7-ethoxycoumarin in bullfrog liver microsomes. These two phases were both significantly inhibited by cyanide and showed a mixed-type inhibition. Two inhibition constants of 3.4 × 10 -5 M and 2.1 × 10-5 M for cyanide were obtained from the first and second phases of Fig. 1, respectively. Since the cytochrome P-450 of frog liver microsomes was more sensitive to cyanide than the cytochrome P-450 of rat liver microsomes, we performed spectrophotometric titrations of microsomal cytochrome P-450 with cyanide. Figure 2 shows the Hoffstee plots of the cyanide titration of bullfrog liver microsomes. At least three dissociation constants (0.16, 0.79 and 3.1 mM) were obtained from the titration curves (Table 6). The liver microsomes of Rana nigromaculata also gave almost same dissociation constants (0.18, 1.04 and 4.7mM) of cyanide, although the oxidation of 7-ethoxycoumarin by Rana nigromaculata liver microsomes was less sensitive to cyanide than bullfrog liver microsomes. Under the same experimental conditions, three dissociation constants of 0.54, 2.4 and 8.5 mM were obtained from the cyanide titration of rat liver micro-
somes, which were slightly larger than the data of Comai and Gaylor (1973). The lower dissociation constants of frog liver microsomes than those of rat liver microsomes explain the higher sensitivity to cyanide of the oxidation activities of the former. It was noticed, however, that the inhibition constants of cyanide obtained from the inhibition experiments (Fig. 1) were much lower than the dissociation constants obtained from the cyanide titration. Since the same dissociation constants were obtained from the titration experiments in the presence and absence of 7-ethoxycoumarin, the binding of the substrate to cytochrome P-450 does not seem to alter the affinity of this hemoprotein to cyanide. DISCUSSION The liver microsomes prepared from four species of frog, Rana catesbeiana, Rana nigromaculata, Bufo bufo japonicus and Xenopus laevis, contained cytochrome P-450, cytochrome b5. NADPH-cytochrome c and NADH-cytochrome c reductase activities suggesting the existence of an electron transfer system similar to that of mammalian liver microsomes. Several chemical compounds examined (aniline, aminopyrine, 7-ethoxycoumarin and benzo(a)pyrene) were all oxidized by frog liver microsomes in the presence of NADPH. When expressed on cyto-
Table 6. The dissociation constants for cyanide obtained from the titration of microsome-bound cytochrome P-450 with KCN
Rana catesbeiana Rana catesbeiana ( + 10pM 7-ethoxycoumarin) Rana nigromaculata Rat
Number of experiments
K~
K2
K3
6
0.16 + 0.08
0.79 _+0.15
3.1 + 0.8
3 3 2
0.16_+0.11 0.18 + 0.04 0.54
0.81+0.20 1.04 _+0.06 2.4
2.9_+1.0 4.7 ___0.4 8.5
Experimental procedure is the same as the experiment shown in Fig. 2.
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MITSUHIDE NOSHIRO and TsUNEo OMURA
chrome P-450 basis, frog liver microsomes catalyzed microsomes, and found that the reactions were oxidations of aniline, aminopyrine and ben- significantly inhibited by cyanide even at a concenzo(a)pyrene at similar rates as with rat liver micro- tration of 0.5mM. The O-deethylation of somes. The oxidation of 7-ethoxycoumarin by frog 7-ethoxycoumarin catalyzed by the liver microsomes liver microsomes was significantly faster, however, of bullfrog was particularly sensitive to cyanide, and than the corresponding activity of rat liver micro- gave an inhibition constant of 2-3 x 10 5M for somes. It is likely that frog liver microsomes contain cyanide, which was much smaller than those obtained a molecular species of cytochrome P-450 which is for the drug oxidation reactions by rat liver microhighly active in the oxidation of 7-ethoxycoumarin, somes (Matsubara and Tochino, 1975; Kitada et al., although we do not know the natural substrate for 1977). Miura et al. (1978) described an inhibitory effect of cyanide on the co-hydroxylation of laurate that particular type of cytochrome P-450. Out of the four species of frog examined in this by the liver microsomes of bullfrog, but the Ostudy, two Rana species are typical amphibians, deethylation of 7-ethoxycoumarin was much more whereas the toad is terrestrial except for the breeding sensitive to cyanide than the laurate hydroxylation. season, and Xenopus lives always in an aquatic envi- Spectrophotometric titration of frog liver microronment. The examination of the oxidations of for- somes by cyanide was also carried out, and gave three eign chemical compounds by the liver microsomes dissociation constants of cytochrome P-450-cyanide indicated no essential difference between the two complex, which were significantly smaller than the Rana species and toad in spite of a significant corresponding values of rat liver microsomal cytodifference in their life styles, whereas Xenopus liver chrome P-450 (Comai and Gaylor, 1973). Three dissociation constants for cyanide of frog microsomes showed much lower oxidation activities. However, we can not ascribe the low microsomal liver microsomal cytochrome P-450 and multiple Km monooxygenase activity of Xenopus liver to the values for the substrate in 7-ethoxycoumarin Oaquatic life style of this particular frog species, since deethylation catalyzed by bullfrog liver microsomes other three species were obtained commercially, indicated the existence of multiple forms of cytowhereas Xenopus was bred and raised in the labora- chrome P-450 in frog liver microsomes as in the case tory. Commercially obtained frogs might have been of mammalian liver microsomes. Miura (1982) examined the effects of inhibitions on exposed to some environmental chemical pollutants which could have induced the monooxygenase activ- co- and (co - 1)-hydroxylation of laurate by the liver microsomes of bullfrog, and he also suggested the ity of their liver microsomes. Aryl hydrocarbon hydroxylase of mammalian liver contributions of different cytochrome P-450 species microsomes is inducible by the treatment of the to the co- and (co - l)-hydroxylation reactions. The animals with certain aromatic hydrocarbons includ- isolation and characterization of those multiple forms ing 3-methylcholanthrene (Conney, 1967). Ben- of cytochrome P-450 from frog liver microsomes and zo(a)pyrene hydroxylation and 7-ethoxycoumarin O- the elucidation of their physiological functions are deethylation activities of Xenopus liver microsomes the subjects of future studies. were also induced by the treatment of the frog with 3-methylcholanthrene and the content of cytochrome SUMMARY P-450 in the liver microsomes was increased about Liver microsomes were prepared from four species two-fold. However, it was not possible to detect a shift of the soret peak of the CO compound of of frog, Rana catesbeiana, Rana n(~romaculata, BuJb cytochrome P-450 with the liver microsomes of bufo japonicus and Xenopus laevis, and the contents 3-methylcholanthrene-treated Xenopus, since the ab- &electron transfer components and the oxidations of sorption of contaminating hemoglobin affected the several chemical compounds were investigated. Cytoshape of the CO-difference spectrum of cytochrome chrome P-450, cytochrome bs, NADPH-cytochrome c reductase and NADH-cytochrome c reductase acP-450. As already observed with mammalian liver micro- tivities were detected with all frog species but the somes, frog liver microsomes catalyzed the oxidation cytochrome contents and the reductase activities were of foreign chemical compounds in the presence of lower than those of rat liver microsomes. The oxidations of aniline, aminopyrine, N A D H as well as NADPH. The NADH-supported oxidation activities were generally lower than the 7-ethoxycoumarin and benzo(a)pyrene were catacorresponding NADPH-supported activities, but a lyzed by frog liver microsomes in the presence of significant N A D H synergism (Cohen and Estabrook, NADPH or NADH. The oxidation activities were not much different from those of rat liver micro1971a,b) was observed with some substrates. The oxidized form of microsomal cytochrome P- somes, but 7-ethoxycoumarin O-deethylation activity 450 can combine with cyanide (Jefcoate et al., 1969), of some frog species was much higher than the but the monooxygenase activities of rat liver micro- corresponding activity of rat liver microsomes. The somes are not sensitive to low concentrations of liver microsomes of Xenopus showed a very low cyanide (Oshino et al., 1966) suggesting a large content of cytochrome P-450 and low oxidation dissociation constant of cytochrome P-450~zyanide activities, but the treatment of Xenopus with complex. Matsubara and Tochino (1975) examined 3-methylcholanthrene resulted in a significant inthe inhibitory effect of cyanide on the hydroxylation crease in the content of cytochrome P-450 and of aniline by rat liver microsomes and obtained an benzo(a)pyrene hydroxylation activity. The monooxygenase activities of frog liver microinhibition constant of 7 x 10 3 M. We also examined the effects of cyanide on the oxidations of somes were more sensitive to cyanide than rat liver 7-ethoxycoumarin and aminopyrine by frog liver microsomes. Cyanide titration of cytochrome P-450
Microsomal monooxygenase system in frog livers in frog liver microsomes gave three dissociation constants, which were significantly lower t h a n the corres p o n d i n g c o n s t a n t s of rat liver m i c r o s o m a l cytoc h r o m e P-450. These observations indicate the existence o f multiple forms o f c y t o c h r o m e P-450 in frog liver microsomes as in the case of m a m m a l i a n liver microsomes.
REFERENCES Adamson R. H. (1967) Drug metabolism in marine vertebrates. Fed. Proc. Fed. Am. Socs. exp. Biol. 26, 1047-1055. Ahokas J. T., Pelkonen O. and Karki N. T. (1975) Metabolism of polycyclic hydrocarbon by a highly active aryl hydrocarbon hydroxylase system in the liver of a trout species. Biochem. biophys. Res. Commun. 63, 635-641. Ahokas J. T., Pelkonen O. and Karki K. T. (1977) Characterization of benzo(a)pyrene hydroxylase of trout liver. Cancer Res. 37, 3737 3743. Alvares A. P., Schilling G., Levin W. and Kuntzman R. (1967) Studies on the induction of CO-binding pigments in liver microsomes by phenobarbital and 3-methylcholanthrene. Biochem. biophys. Res. Commun. 29, 521 526. Bend J. R., James M. O. and Dansette P. M. (1977) In vitro metabolism of xenobiotics in some marine animals. Ann. N.Y. Acad. Sci. 298, 505-521. Bessey O. A., Lowry O. H. and Love R. H. (1949) The fluorometric measurement of the nucleotides of riboflavin and their concentration in tissues. J. biol. Chem. 180, 755-769. Buhler D. R. and Rasmussen M. E. (1968) The oxidation of drugs by fishes. Comp. Biochem. Physiol. 25, 223-239. Cohen B. S. and Estabrook R. W. (1971a) Microsomal electron transport reactions. II. The use of reduced triphosphopyridine nucleotide and/or diphosphopyridine nucleotide for the oxidative N-demethylation of aminopyrine and other drug substrates. Archs Biochem. Biophys. 143, 46-53. Cohen B. S. and Estabrook R. W. (1971b) Microsomal electron transport reactions. III. Cooperative interactions between reduced diphosphopyridine nucleotide and reduced triphosphopyridine nucleotide linked reactions. Archs Biochem. Biophys. 143, 54-65. Comai K. and Gaylor J. L. (1973) Existence and separation of three forms of cytochrome P-450 from rat liver microsomes. J. biol. Chem. 248, 49474955. Conney A. H. (1967) Pharmacological implications of microsomal enzyme induction. Pharmac. Rev. 19, 317-366. Creaven P. J., Davies W. H. and Williams R. T. (1967) Dealkylation of alkoxybiphenyls by trout and frog liver preparations. Life Sci. 6, 105 111. Creaven P. J., Parke D. V. and Williams R. T. (1965a) A spectrofluorimetric study of the 7-hydroxylation of coumarin by liver microsomes. Biochem. J. 96, 390-398. Creaven P. J., Parke D. V. and Williams R. T. (1965b) A fluorimetric study of the hydroxylation of biphenyl in vitro by liver preparations of various species. Biochem. J. 96, 879-885. Imai Y., Ito A. and Sato R. (1966) Evidence for biochemically different types of vesicles in the hepatic microsomal fraction. J. Biochem. 60, 417428. James M. O. and Bend J. R. (1980) Polycyclic aromatic hydrocarbon induction of cytochrome P-450-dependent
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mixed-function oxidases in marine fish. Toxic. appl. Pharmac. 54, 117-133. Jefcoate C. R. E., Gaylor J. L. and Calabrese R. L. (1969) Ligand interactions with cytochrome P-450. I. Binding of primary amines. Biochemistry 8, 3455-3463. Kitada M., Chiba K., Kamataki T. and Kitagawa H. (1977) Inhibition by cyanide of drug oxidations in rat liver microsomes. Japan J. Pharmac. 27, 601-608. Lees M. B. (1957) Preparation and analysis of phosphatides. In Methods in Enzymology, Vol. 3, pp. 328-345. Academic Press, New York. Linstr6m-Sepp/i P., Koivusaari U. and Hanninen O. (1981) Metabolism of xenobiotics by vendace (Coregonus albula). Comp. Biochem. Physiol. 68e, 121-126. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Matsubara T. and Tochino T. (1975) Inhibitory action of cyanide on aniline hydroxylase system. FEBS Lett. 52, 77-80. Miura Y. (1982) Effect of inhibitors on o~- and (~o - l)-hydroxylation of lauric acid by frog liver microsomes. Lipids 17, 864-869. Miura Y., Hisaki H. and Ueta N. (1978) ~o- and (oJ - 1)-hydroxylation of fatty acids by frog liver microsomes. Substrate specificity and other properties. Biochim. biophys. Acta 531, 149 157. Nebert D. W. and Gelboin H. V. (1968) Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. J. biol. Chem. 243, 6242-6249. Noshiro M. and Omura T. (1978) Immunochemical study on the electron pathway from NADH to cytochrome P-450 of liver microsomes. J. Biochem. 83, 61-77. Omura T. and Sato R. (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. biol. Chem. 239, 2370-2378. Omura T. and Takesue S. (1970) A new method for sinmltaneous purification of cytochrome b 5 and NADPHcytochrome c reductase from rat liver microsomes. J. Biochem. 67, 249-257. Orrenius S. (1965) On the mechanism of drug hydroxylation in rat liver microsomes. J. Cell Biol. 26, 713-723. Oshino N., Imai Y. and Sato R. (1966) Electron-transfer mechanism associated with fatty acid desaturation catalyzed by liver microsomes. Biochim. biophys. Acta 128, 13-28. Payne J. F. and Penrose W. R. (1975) Induction of aryl hydrocarbon (benzo(a)pyrene) hydroxylase in fish by petroleum. Bull. Environ. Contain. Toxic. 14, 112-116. Pedersen M. G., Hershberger W. K. and Juchau M. R. (1974) Metabolism of 3,4-benzopyrene in rainbow trout (Salmo gairdneri). Bull. Environ. Contain. Toxic. 12, 481486. Schneider W. C. (1957) Determination of nuleic acids in tissues by pentose analysis. In Methods in Enzymology, Vol. 3, pp. 680-684. Academic Press, New York. Stegeman J. J. and Binder R. L. (1979) High benzopyrene hydroxylase activity in the marine fish Stenotomus versicolor. Biochem. Pharmac. 28, 1686-1688. Stegeman J. J., Binder R. L. and Orren A. (1979) Hepatic and extrahepatic microsomal electron transport components and mixed-function oxygenases in the marine fish Stenotomus versicolor. Biochem. Pharmac. 28, 3431-3439. Ullrich V. and Weber P. (1972) The O-dealkylation of 7-ethoxycoumarin by liver microsomes. Hoppe-Seyler's Z. physiol. Chem. 353, 1171-1177.
0305-0491/'84 $3.00+0.00 © 1984 Pergamon Press Ltd
MICROSOMAL MONOOXYGENASE SYSTEM IN FROG LIVERS MITSUHIDE NOSHIRO* and TSUNEO OMURA Department of Biology, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812, Japan
(Received 7 September 1983) Abstract--l. Liver microsomes prepared from four species of frog, Rana eatesbeiana, Rana nigromaculata, Bufo bufojaponicus, and Xenopus laevis, contained cytochrome P-450 and showed NAD(P)H-dependent monooxygenase activities to several foreign chemical compounds tested. 2. The oxidations of the chemical compounds by frog liver microsomes showed significant variations among frog species. The oxidation of 7-ethoxycoumarin was much faster than that by rat liver microsomes. The oxidation of benzo(a)pyrene was significantly induced by 3-methylcholanthrene administration. 3. The monooxygenase activities of frog liver microsomes were more sensitive to cyanide inhibition than those of rat liver microsomes.
monooxygenase activity by 3-methylcholanthrene was also studied.
INTRODUCTION Cytochrome P-450-containing monooxygenase system is widespread among various forms of life. In vertebrates, the monooxygenase system of mammalian liver microsomes has been intensively investigated from various viewpoints including the multiple molecular nature of cytochrome P-450. The liver microsomes of birds, reptiles and amphibians have also been shown to oxidize various foreign compounds (Creaven et al., 1965a,b; Creaven et al., 1967). The monooxygenase activities of fish liver preparations have been frequently studied in connection with the biotransformation of water pollutants such as petroleum hydrocarbons and polychlorinated biphenyls (Creaven, et al., 1967; Adamson, 1967; Buhler and Rasmussen, 1968; Pederson et al., 1974; Ahokas et al., 1975; Payne and Penrose, 1975; Ahokas et al., 1977; Bend et al., 1977; Stegeman and Binder, 1979; Stegeman et al., 1979; James and Bend, 1980; Lindstr6m-Sepp~i et al., 1981). However, most of these comparative studies on hepatic monooxygenase activities described only the occurrence of the activities and the metabolites of foreign compounds. In order to compare the liver microsomal monooxygenase systems of various organisms, characterization of the electron transport system of the microsomes is highly desirable. In this study, we examined the electron transport system of the liver microsomes of four species of frog, Rana catesbeiana (bullfrog), Rana nigromaculata, Bufo bufo japonicus (toad) and Xenopus laevis. Hemoprotein contents, reductase activities and oxidations of several chemical compounds were examined and compared. The multiple nature of microsomal cytochrome P-450 was studied by the titration with cyanide. The induction of the *Present address, Department of Biochemistry, Hiroshima University School of Dentistry, Kasuni 1-2-3, Minamiku, Hiroshima, Hiroshima 734 Japan.
MATERIALS AND METHODS
Animals Rana catesbeiana (270-380g body wt), Rana nigromaculata (30-60 g) and Bufo bufojaponicus (200-360 g) were obtained from a commercial source. Xenopus laevis (20-70 g) was generously supplied by Dr. Misumi and Dr. Yamana of Kyushu University. Male rats of SpragueDawley strain weighing about 200 g were also used for comparison. When Xenopus was treated with 3-methylcholanthrene, 1% solution of the chemical in olive oil was injected once intraperitoneally in a dose of 25 mg per kg body wt 5 days before killing. Preparation of microsomes from frog livers Animals were killed by a blow on the head. The abdominal cavity was opened and the liver was thoroughly perfused in situ from the hepatic vein with chilled 0.9% NaCI. The perfused liver was excised, finely chopped with scissors and homogenized with 9 vol of 0.25 M sucrose solution containing 10mM Tris-HCl buffer (pH 7.5) and 1 mM EDTA in a Potter-type glass homogenizer equipped with a Teflon pestle. The homogenate was centrifuged at 10,000g for 15 min and the precipitate was discarded. The supernatant was centrifuged at 105,000g for 60min, and the packed pellets formed two layers. The lower colourless jelly-like layer of glycogen granules was discarded. The upper layer of microsomes was suspended in 0.15 M KCI containing 1 mM EDTA and centrifuged again as above. The washed microsomes were finally suspended in 0.25 M sucrose containing 10mM Tris-HC1 (pH 7.5) and t mM EDTA. All assays with microsomal preparations were performed within the same day. Analytical methods The contents of cytochrome P-450 and cytochrome b5 in microsomes were determined according to Omura and Sato (1964) using extinction coefficients of 91 and 185/cm/mM- i, respectively. NADPH- and NADH-cytochrome c reductase activities were determined as described previously (Omura and Takesue, 1970). The content of protoheme in microsomes was measured spectrophotometrically after con761
762
MITSUHIDE NOSHIRO and TSUNEO OMURA Table 1. Chemical composition of frog liver microsomes Recovery of Contents of Ms protein Phospholipid RNA Protoheme Flavin Animal species (mg/g liver) (#g/mg Ms protein) (nmol/mg Ms protein) Rana catesbeiana 7.4 580 140 1.12 0.141 Bufo bufo japonicus 5.4 600 350 1.56 0.131 Xenopus laevis 4.2 490 210 1.04 0.199 Rat 12.7 590 120 1.17 0.336 All data are mean values of two or three preparations. Ms: microsomes.
verting the heme into pyridine hemochromogen in the presence of 0.1 M NaOH and 20~o pyridine (Omura and Sato, 1964). The content of total flavin in microsomes was determined by the method of Bessey et al. (1949). Phospholipid and RNA were extracted according to Schneider (1957) and determined by the methods of Lees (1957) and Schneider (1957), respectively. Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Cyanide titration of cytochrome P-450 in microsomes Spectrophotometric titration of liver microsomes with potassium cyanide was performed using a Shimadzu Double-40-DF spectrophotometer according to the method of Comai and Gaylor (1973). Suspensions of microsomes (1 mg/ml) in 0.l M potassium phosphate buffer (pH 7.5) were titrated with freshly prepared potassium cyanide solution which had been adjusted to neutral pH with HCI. The titrations were performed over range of 0.1 to 32mM cyanide. The pH of the titration mixtures did not change during the titration. Assays of drug oxidations Assays of aniline hydroxylase, aminopyrine Ndemethylase, 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase were performed as described by Imai et al. (1966), Orrenius (1965), Ullrich and Weber (1972), and Nebert and Gelboin (1968), respectively. In the assays of the oxidations of aniline and aminopyrine, an NADPH-generating system consisting of 0.5 mM NADP +, 5 mM isocitrate, 5 mM MgC12 and 0.36 unit of isocitrate dehydrogenase or 2 mM NADH were used. In the assays of 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase, 0.1 mM NADPH or 0.1 mM NADH and 0.4 mM NADPH or 1 mM NADH, respectively, were used. Other assay conditions were the same as described previously (Noshiro and Omura, 1978). Reagents and biochemicals Yeast cytochrome c was a generous gift of Sankyo Co., Tokyo. 7-Ethoxycoumarin was kindly provided by Dr. V. Ullrich. All other chemicals were of the highest purity commercially available. RESULTS
Chemical composition o f f r o g liver microsomes Table 1 shows the chemical compositions of liver
microsomes from three species of flog. A l t h o u g h the recovery of m i c r o s o m a l protein was lower than that of rat, the contents of phospholipids, p r o t o h e m e and flavin of frog liver microsomes were not m u c h different from those of rat liver microsomes. Hemoprotein contents and reductase activities q f frog liver microsomes Table 2 shows the h e m o p r o t e i n contents and reductase activities of liver microsomes prepared from four species o f frog. The c o r r e s p o n d i n g values of rat liver microsomes are also shown for comparison, B o t h c y t o c h r o m e P-450 and c y t o c h r o m e b s were present in all microsomal samples but their contents varied significantly from one species of frog to another. In the case of Xenopus laevis which showed especially low c o n t e n t of c y t o c h r o m e P-450, 3-methylcholanthrene-treated animals were also examined. The sum of the contents of c y t o c h r o m e P-450 a n d c y t o c h r o m e bs was always lower t h a n the p r o t o h e m e content of frog liver microsomes. C a r b o n monoxide-difference spectra of the microsomal prepa r a t i o n s showed a significant a m o u n t of cont a m i n a t i n g h e m o g l o b i n in Xenopus microsomes, but the a m o u n t s of h e m o g l o b i n in the microsomes of other three species of frog were too small to account for the excess protoheme. The excess p r o t o h e m e in microsomes was p r o b a b l y due to some h e m o p r o t e i n s o f other c o n t a m i n a t i n g cell o r g a n d i e s including catalase of peroxisomes. Judging from succinatec y t o c h r o m e c reductase activity of the microsomal p r e p a r a t i o n s (data not shown), m i t o c h o n d r i a l cont a m i n a t i o n s to the microsomal fractions were a b o u t 2 - 6 % on the protein basis. N A D ( P ) H - s u p p o r t e d c y t o c h r o m e c reductase activities of frog liver microsomes were lower t h a n those of rat liver microsomes. 3 - M e t h y l c h o l a n t h r e n e - t r e a t m e n t of Xenopus laevis resulted in a two-fold increase of c y t o c h r o m e P-450 in liver microsomes, but the t r e a t m e n t did not affect the content of c y t o c h r o m e b5 n o r the activities of the reductases. It was not possible to detect a shift of Soret peak in the CO-difference spectrum of induced microsomes (Alvares et al., 1967), since the presence
Table 2. Hemoprotein contents and reductase activities of frog liver microsomes NADPH-cyt. c NADH-cyt.c Cyt. P-450 Cyt. bs reductase reductase (nmol/mg Ms protein) (gmol reduced/min/mg Ms protein) Animal species 0.43 0.16 0.074 0.372 Rana catesbeiana 0.77 0.32 0.116 0.524 Rana nigromaculata 0.73 0.16 0.048 0.268 Bufo bufo japonicus 0.10 0.072 0.082 0.469 Xenopus laevis Xenopus laevis 0.23 0.078 0.093 0.375 (3-MC-treated) 0.67 0.39 0.239 1.05 Rat Assay procedures are described in Materials and Methods. All data are mean values of more than three preparations. Ms and 3-MC: microsomes and 3-methylcholanthrene, respectively.
Microsomal monooxygenase system in frog livers Table
3.
NADPH-supported oxidation of aniline, aminopyrine, benzo(a)pyrene by frog liver microsomes
763
7-ethoxycoumarin and
Substrates Aniline Animal species
Rana catesbeiana Rana nigromaculata Bufo bufo japonicus Xenopus laevis Xenopus laevis (3-MC-treated) Rat
Aminopyrine 7-ethoxycoumarin Benzo(a)pyrene nmol product formed/min/mg Ms protein (nmol product formed/min/nmol P-450)
0.463 (1.07) 0.797 (1.03) 0.632 (0.880) 0.062 (0.61) 0.089 (0.387) 1.02 (1.04)
h29 (2.99) 8.05 (10.4) 3.65 (5.09) 0.422 (4.18) 0.487 (2.10) 3.23 (3.28)
2.00 (4.63) 3.94 (5.09) 1.32 (1.84) 0.163 (1.6t) 0.486 (2.10) 0.331 (0.338)
0.390 (0.90) 0.150 (0.239) 0.840 (1.17) 0.013 (0.13) 0.094 (0.407) 1.05 (1.07)
Oxidation activities were assayed as described in Materials and Methods. The data are mean values of more than three microsomal preparations. Ms and 3-MC, microsomes and 3-methylcholanthrene, respectively.
of contaminating hemoglobin interferred with the spectral observation of cytochrome P-450.
Oxidation of chemical compounds by frog liver microsomes Table 3 shows the NADPH-supported oxidations of four chemical compounds catalyzed by frog liver microsomes. Aniline hydroxylation activities of frog liver microsomes were lower on the protein basis than rat liver microsomes. However, when the hydroxylation activities were expressed on the basis of cytochrome P-450, the activities of the frog microsomes were almost identical with that of rat microsomes. Aminopyrine N-demethylation activity of Rana nigromaculata microsomes was much higher than that of rat liver microsomes on the basis of both protein and cytochrome P-450. The activities of other frogs were not much different from that of rat on the basis of cytochrome P-450. 7-Ethoxycoumarin O-deethylation activities of frog liver microsomes were much higher than that of rat liver microsomes on the basis of cytochrome P-450. The activities of bullfrog and Rana nigromaculata microsomes were about fifteen times higher than that of rat and the activities of toad and Xenopus microsomes were about five times higher than rat. Benzo(a)pyrene hydroxylation activities of the liver microsomes of bullfrog and toad were about the same as that of rat liver microsomes on the basis of cytochrome P-450, whereas the activities of Rana
nigromaculata and Xenopus microsomes were much lower than the other frogs. NADPH-supported oxidations of aminopyrine and 7-ethoxycoumarin by bullfrog liver microsomes were inhibited by 40 and 90~o, respectively, in a gas mixture of 80~ CO and 20~o 02, confirming the participation of cytochrome P-450 in these oxidation reactions. The liver microsomes of Xenopus laevis showed a very low content of cytochrome P-450 (Table 2) and a weak activity of benzo(a)pyrene hydroxylation compared with other frogs. However, when Xenopus was treated with 3-methylcholanthrene, benzo(a)pyrene hydroxylation activity of the liver microsomes was markedly increased on the basis of both protein and cytochrome P-450. O-Deethylation of 7-ethoxycoumarin was also stimulated by the treatment, whereas the oxidations of aniline and aminopyrine did not change on the basis of protein and rather decreased on the basis of cytochrome P-450. Although N A D P H is the preferred source of reducing equivalents for the monooxygenase reactions catalyzed by microsomal cytochrome P-450, N A D H is also capable of sustaining the oxidation reactions although at lower rates than the corresponding NADPH-supported reactions. In some cases, addition of NADPH-supported microsomal oxidation systems enhanced the rates of reactions (Cohen and Estabrook, 1971a,b), which is called " N A D H synergism". We investigated the effect of N A D H on the monooxygenase reactions catalyzed by frog liver
Table 4. Effects of NADH on the oxidations of aniline, aminopyrine and 7-ethoxycoumarin by frog liver microsomes Substrates Frog species
Electron donors
Aniline Aminopyrine 7-Ethoxycoumarin nmol product formed/min/nmol P-450 (%)
Rana catesbeiana
NADPH NADH NADPH + NADH
1.07 (100) 0.13 (12) 1.04 (97)
3.15 (100) 2.22 (70) 3.79 (120)
4.89 (100) 0.29 (6) 4.94 (101)
Rana nigromaculata
NADPH NADH NADPH ÷ NADH
0.98 (100) 0.11 ( 11) 0.97 (97)
6.34 (100) h 57 (24) 8.43 (131)
5.09 (100) 0.10 (2) 8.44 (166)
Bufo bufo japonicus
NADPH NADH NADPH + NADH
0.83 (100) 0.056 (7) 0.85 (102)
3.95 (100) 0.79 (20) 3.87 (98)
3.06 (100) 0.37 (12) 3.12 (102)
Assay procedures are described in Materials and Methods. Concentrations of NADH were 2 mM for the assays of aniline and aminopyrine oxidations and 0.1 mM for 7-ethoxycoumarin oxidation.
764
MITSUHIDE NOSHIRO and TSUNEO OMURA
Table 5. Effects of cyanide on NADPH-supported oxidations of aminopyrine and 7-ethoxycoumarin by frog and rat liver microsomes Substrates KCN Aminopyrine 7-Ethoxycoumarin Animal species (raM) nmol product formed/min/nmol P-450 (%) 0 1.75 (100) 5.78 (100) Rana catesbeiana 0.5 1.20 (69) 0.65 (I 1) 0 8.85 (100) 5.09 (100) Rana nigromaculata 0.5 5.76 (65) 2.57 (50) 0 3.19 (100) 0.5 1.74 (55) 0 1.88 (100) Rat 0.5 1.85 (98) Assay procedures are described in Materials and Methods.
4.86 (100) 3.10 (63) 0.39 (100) 0.28 (72)
Bufo bufojaponicus
microsomes and we found that N A D H - s u p p o r t e d oxidation activities were usually about 10-20% of the corresponding N A D P H - s u p p o r t e d activities (Table 4). However, bullfrog microsomes showed exceptionally high N A D H - s u p p o r t e d aminopyrine Ndemethylation activity, which was as high as 70% of the corresponding N A D P H - s u p p o r t e d activity. A significant NADH-synergism was observed only with the 7-ethoxycoumarin O-deethylation catalyzed by Rana nigromaculata microsomes, although the microsomal preparation showed a very low N A D H supported oxidation of the same substrate.
Effects of cyanide on the monooxygenase reactions catalyzed by frog liver microsomes Table 5 shows the effects of cyanide on the oxidations of aminopyrine and 7-ethoxycoumarin catalyzed by frog and rat liver microsomes. At a concen-
tration of 0.5 mM, cyanide inhibited the aminopyrine N-demethylation activities of frog liver microsomes by 30-45%, whereas the corresponding activity of rat liver microsomes was not affected at all. 7-Ethoxycoumarin O-deethylation activities by frog liver microsomes were also significantly inhibited by cyanide, and the activity of bullfrog microsomes was especially sensitive to cyanide. The O-deethylation activity of rat liver microsomes was also slightly inhibited by cyanide. Since 7-ethoxycoumarin O-deethylation by bullfrog microsomes was very sensitive to cyanide, effects of various concentrations of cyanide on the N A D P H supported O-deethylation by bullfrog microsomes were examined at various concentrations of the substrate. Figure 1 showed the double reciprocal plot of the O-deethylation activities in the presence and absence of cyanide. Two distinct phases having
5 mM
/
0.1 mM
I---E 4 +
/
/
/
005
mM
E
0.02 mM
J KCN
-1
0
1 [ 7 - ethoxycoumorln} -1
2 1 {pMl-
0 mM
3
Fig. l. Double reciprocal plots of 7-ethoxycoumarin O-deethylation reaction catalyzed by bullfrog liver microsomes. Experimental procedures are described in Materials and Methods. The incubation mixture contained 0.1 mg of microsomal protein and various concentrations of 7-ethoxycoumarin and cyanide as indicated in the figure in 2 ml of 0.1 M Tris-HC1 buffer (pH 7.5). The assays were started by the addition of 0.1 mM NADPH and carried out at 30°C for 5min.
Microsomal monooxygenase system in frog livers
0.05
0,04
E E m o
0.03
~
765
8
i ~t
•,a" 0 . 0 2 <1
0.01
o
I
i
I
I
0.02
0.04
o.o6
o.os
, A/[CN-}
I
o.lo
mM -1
Fig. 2. Hoffstee plots of the cyanide titration and bullfrog liver microsomes. Both sample and reference cuvettes contained the same microsomal suspension (1 mg/ml) in 0.1 M potassium phosphate buffer (pH 7.5). After recording the base line, the sample cuvette was titrated with cyanide solution and reference cuvette was also added with same volumes of water and the difference spectrum was recorded between 370 and 500 nm. When the titration was carried out in the presence of 7-ethoxycoumarin, the substrate was added to both the sample and the reference cuvette at a final concentration of 10 #M. O, in the absence of 7-ethoxycoumarin; O, in the presence of 7-ethoxycoumarin. different Km values for 7-ethoxycoumarin (2.8 × 10 6 M and 4.7 × 10 -7 M) were observed for all concentrations of cyanide examined, which indicated the existence of at least two types of cytochrome P-450 catalyzing the oxidation of 7-ethoxycoumarin in bullfrog liver microsomes. These two phases were both significantly inhibited by cyanide and showed a mixed-type inhibition. Two inhibition constants of 3.4 × 10 -5 M and 2.1 × 10-5 M for cyanide were obtained from the first and second phases of Fig. 1, respectively. Since the cytochrome P-450 of frog liver microsomes was more sensitive to cyanide than the cytochrome P-450 of rat liver microsomes, we performed spectrophotometric titrations of microsomal cytochrome P-450 with cyanide. Figure 2 shows the Hoffstee plots of the cyanide titration of bullfrog liver microsomes. At least three dissociation constants (0.16, 0.79 and 3.1 mM) were obtained from the titration curves (Table 6). The liver microsomes of Rana nigromaculata also gave almost same dissociation constants (0.18, 1.04 and 4.7mM) of cyanide, although the oxidation of 7-ethoxycoumarin by Rana nigromaculata liver microsomes was less sensitive to cyanide than bullfrog liver microsomes. Under the same experimental conditions, three dissociation constants of 0.54, 2.4 and 8.5 mM were obtained from the cyanide titration of rat liver micro-
somes, which were slightly larger than the data of Comai and Gaylor (1973). The lower dissociation constants of frog liver microsomes than those of rat liver microsomes explain the higher sensitivity to cyanide of the oxidation activities of the former. It was noticed, however, that the inhibition constants of cyanide obtained from the inhibition experiments (Fig. 1) were much lower than the dissociation constants obtained from the cyanide titration. Since the same dissociation constants were obtained from the titration experiments in the presence and absence of 7-ethoxycoumarin, the binding of the substrate to cytochrome P-450 does not seem to alter the affinity of this hemoprotein to cyanide. DISCUSSION The liver microsomes prepared from four species of frog, Rana catesbeiana, Rana nigromaculata, Bufo bufo japonicus and Xenopus laevis, contained cytochrome P-450, cytochrome b5. NADPH-cytochrome c and NADH-cytochrome c reductase activities suggesting the existence of an electron transfer system similar to that of mammalian liver microsomes. Several chemical compounds examined (aniline, aminopyrine, 7-ethoxycoumarin and benzo(a)pyrene) were all oxidized by frog liver microsomes in the presence of NADPH. When expressed on cyto-
Table 6. The dissociation constants for cyanide obtained from the titration of microsome-bound cytochrome P-450 with KCN
Rana catesbeiana Rana catesbeiana ( + 10pM 7-ethoxycoumarin) Rana nigromaculata Rat
Number of experiments
K~
K2
K3
6
0.16 + 0.08
0.79 _+0.15
3.1 + 0.8
3 3 2
0.16_+0.11 0.18 + 0.04 0.54
0.81+0.20 1.04 _+0.06 2.4
2.9_+1.0 4.7 ___0.4 8.5
Experimental procedure is the same as the experiment shown in Fig. 2.
766
MITSUHIDE NOSHIRO and TsUNEo OMURA
chrome P-450 basis, frog liver microsomes catalyzed microsomes, and found that the reactions were oxidations of aniline, aminopyrine and ben- significantly inhibited by cyanide even at a concenzo(a)pyrene at similar rates as with rat liver micro- tration of 0.5mM. The O-deethylation of somes. The oxidation of 7-ethoxycoumarin by frog 7-ethoxycoumarin catalyzed by the liver microsomes liver microsomes was significantly faster, however, of bullfrog was particularly sensitive to cyanide, and than the corresponding activity of rat liver micro- gave an inhibition constant of 2-3 x 10 5M for somes. It is likely that frog liver microsomes contain cyanide, which was much smaller than those obtained a molecular species of cytochrome P-450 which is for the drug oxidation reactions by rat liver microhighly active in the oxidation of 7-ethoxycoumarin, somes (Matsubara and Tochino, 1975; Kitada et al., although we do not know the natural substrate for 1977). Miura et al. (1978) described an inhibitory effect of cyanide on the co-hydroxylation of laurate that particular type of cytochrome P-450. Out of the four species of frog examined in this by the liver microsomes of bullfrog, but the Ostudy, two Rana species are typical amphibians, deethylation of 7-ethoxycoumarin was much more whereas the toad is terrestrial except for the breeding sensitive to cyanide than the laurate hydroxylation. season, and Xenopus lives always in an aquatic envi- Spectrophotometric titration of frog liver microronment. The examination of the oxidations of for- somes by cyanide was also carried out, and gave three eign chemical compounds by the liver microsomes dissociation constants of cytochrome P-450-cyanide indicated no essential difference between the two complex, which were significantly smaller than the Rana species and toad in spite of a significant corresponding values of rat liver microsomal cytodifference in their life styles, whereas Xenopus liver chrome P-450 (Comai and Gaylor, 1973). Three dissociation constants for cyanide of frog microsomes showed much lower oxidation activities. However, we can not ascribe the low microsomal liver microsomal cytochrome P-450 and multiple Km monooxygenase activity of Xenopus liver to the values for the substrate in 7-ethoxycoumarin Oaquatic life style of this particular frog species, since deethylation catalyzed by bullfrog liver microsomes other three species were obtained commercially, indicated the existence of multiple forms of cytowhereas Xenopus was bred and raised in the labora- chrome P-450 in frog liver microsomes as in the case tory. Commercially obtained frogs might have been of mammalian liver microsomes. Miura (1982) examined the effects of inhibitions on exposed to some environmental chemical pollutants which could have induced the monooxygenase activ- co- and (co - 1)-hydroxylation of laurate by the liver microsomes of bullfrog, and he also suggested the ity of their liver microsomes. Aryl hydrocarbon hydroxylase of mammalian liver contributions of different cytochrome P-450 species microsomes is inducible by the treatment of the to the co- and (co - l)-hydroxylation reactions. The animals with certain aromatic hydrocarbons includ- isolation and characterization of those multiple forms ing 3-methylcholanthrene (Conney, 1967). Ben- of cytochrome P-450 from frog liver microsomes and zo(a)pyrene hydroxylation and 7-ethoxycoumarin O- the elucidation of their physiological functions are deethylation activities of Xenopus liver microsomes the subjects of future studies. were also induced by the treatment of the frog with 3-methylcholanthrene and the content of cytochrome SUMMARY P-450 in the liver microsomes was increased about Liver microsomes were prepared from four species two-fold. However, it was not possible to detect a shift of the soret peak of the CO compound of of frog, Rana catesbeiana, Rana n(~romaculata, BuJb cytochrome P-450 with the liver microsomes of bufo japonicus and Xenopus laevis, and the contents 3-methylcholanthrene-treated Xenopus, since the ab- &electron transfer components and the oxidations of sorption of contaminating hemoglobin affected the several chemical compounds were investigated. Cytoshape of the CO-difference spectrum of cytochrome chrome P-450, cytochrome bs, NADPH-cytochrome c reductase and NADH-cytochrome c reductase acP-450. As already observed with mammalian liver micro- tivities were detected with all frog species but the somes, frog liver microsomes catalyzed the oxidation cytochrome contents and the reductase activities were of foreign chemical compounds in the presence of lower than those of rat liver microsomes. The oxidations of aniline, aminopyrine, N A D H as well as NADPH. The NADH-supported oxidation activities were generally lower than the 7-ethoxycoumarin and benzo(a)pyrene were catacorresponding NADPH-supported activities, but a lyzed by frog liver microsomes in the presence of significant N A D H synergism (Cohen and Estabrook, NADPH or NADH. The oxidation activities were not much different from those of rat liver micro1971a,b) was observed with some substrates. The oxidized form of microsomal cytochrome P- somes, but 7-ethoxycoumarin O-deethylation activity 450 can combine with cyanide (Jefcoate et al., 1969), of some frog species was much higher than the but the monooxygenase activities of rat liver micro- corresponding activity of rat liver microsomes. The somes are not sensitive to low concentrations of liver microsomes of Xenopus showed a very low cyanide (Oshino et al., 1966) suggesting a large content of cytochrome P-450 and low oxidation dissociation constant of cytochrome P-450~zyanide activities, but the treatment of Xenopus with complex. Matsubara and Tochino (1975) examined 3-methylcholanthrene resulted in a significant inthe inhibitory effect of cyanide on the hydroxylation crease in the content of cytochrome P-450 and of aniline by rat liver microsomes and obtained an benzo(a)pyrene hydroxylation activity. The monooxygenase activities of frog liver microinhibition constant of 7 x 10 3 M. We also examined the effects of cyanide on the oxidations of somes were more sensitive to cyanide than rat liver 7-ethoxycoumarin and aminopyrine by frog liver microsomes. Cyanide titration of cytochrome P-450
Microsomal monooxygenase system in frog livers in frog liver microsomes gave three dissociation constants, which were significantly lower t h a n the corres p o n d i n g c o n s t a n t s of rat liver m i c r o s o m a l cytoc h r o m e P-450. These observations indicate the existence o f multiple forms o f c y t o c h r o m e P-450 in frog liver microsomes as in the case of m a m m a l i a n liver microsomes.
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