ARCHIVES
OF BIOCHEMISTRY
Vol. 276, No. 2, February
AND
BIOPHYSICS
1, pp. 473-480,199O
Characterization of Three Cytochrome P45Os Purified from Renal Microsomes of Untreated Male Rats and Comparison with Human Renal Cytochrome P450 Susumu
Imaoka,l
Kazuo
Nagashima,*
and Yoshihiko
Funae
Laboratory of Chemistry, Osaka City University Medical School, Abeno-ku, Osaka 545, Japan; and *Department Faculty of Medicine, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060, Japan
of Pathology,
Received ,July 10, 1989, and in revised form October 11,1989
Three renal cytochrome P45Os (P450 K-2, K-4, and K-5) were purified from renal microsomes of untreated male rats. Also, the human renal cytochrome P450 (P450 HK) was partially purified from renal microsomes and its properties were compared with those of the rat renal cytochrome P45Os. The molecular weight of P450 K-2, K-4, and K-5 was 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of P450 K-2, K-4, and K-5 were at 449, 451, and 452 nm, respectively. NH,-terminal sequence analysis of P450 K-2, K-4, and K-5 showed that these forms were different from hepatic cytochrome P45Os purified previously. P450 K-2, K-4, and K-5 catalyzed the 0-dealkylation of ‘7-ethoxycoumarin but were not efficient in the hydroxylation of testosterone. Aminopyrine was metabolized by P450 K-2 and K4 but not by P450 K-5. Laurie acid was metabolized efficiently by all of these forms in the presence of cytochrome bg. The regiospecificity of these forms toward lauric acid was different. P450 K-2 hydroxylated lauric acid only at the (w-1)-position, not at the w-position. P450 K-4 and K-5 hydroxylated lauric acid at both the w- and (o-1)-positions. The ratios of ti/(W-l)-hydroxylation activity of P450 K-4 and K-5 were 2.5 and 7.8, respectively. Human P450 HK was purified 220fold and its specific content was 2.0 nmol/mg of protein. The Soret maxima of P450 HK were at 418 nm for the oxidized form, 416 nm for the reduced form, and 450 nm for the CO-reduced form. P450 HK catalyzed the 0-dealkylation of 7-ethoxycoumarin but was not efficient in aminopyrine N-demethylation or testosterone hydroxylation. P450 HK had high lauric acid W- and (w-l)-hydroxylation activities in the presence of cyto-
’ To whom all correspondence 000%9861/90
should be addressed.
$3.00
Copyright (!I 1990 by Academic Press, Inc. All rights ofreproduction
in any form reserved.
chrome b5, especially w-hydroxylation. These properties resembled those of P450 K-5 most closely. AntiP450 K-5 antibody cross-reacted with P450 HK as well as P450 K-5 and only one band was stained on immunostained Western blotting for partially purified P450 HK. The molecular weight of P450 HK was 52,000 on Western blotting. 0 1990 Academic Press, Inc.
Cytochrome P450 and its associated monooxygenase activities are predominantly found in the liver, but are also present in many other tissues, although usually at lower levels (1). There are species, sex, age, and tissue differences in cytochrome P45O.s (2-4). The balance of metabolic activation and detoxification of drugs and other chemicals by individual forms of cytochrome P450 in different tissues or species is an important factor in explaining organ- or species-specific toxicity. Cytochrome P450 also has organ-specific roles involving, for example, the synthesis of steroids by adrenal cytochrome P450 (5) and the synthesis of thromboxane by platelet cytochrome P450 (6). Most studies of cytochrome P450 have focused on hepatic forms in rats (713) and humans (14-20). Much less is known about of renal cytochrome P45Os in rats and humans. Renal cytochrome P450 efficiently catalyzes the hydroxylation of prostaglandins and fatty acids such as lauric acid but metabolizes testosterone and drugs such as aminopyrine and 7-ethoxycoumarin little if at all (21~ 24). The substrate specificity of renal cytochrome P450 is different from that of hepatic cytochrome P45Os. Arachidonic acid can be metabolized by renal cytochrome P450, being converted to w- or (w-1)-hydroxyarachidonic acids and epoxyeicosatrienoic acids (25,26). We have investigated arachidonic acid metabolism by a cytochrome P450 purified from the rat renal microsomes, finding that purified renal cytochrome P450 produces epoxyei473
474
IMAOKA,
NAGASHIMA,
cosatrienoic acid and w- and (w-1)-hydroxyarachidonic acid (27). However, the contribution to renal function of arachidonic acid or prostaglandin metabolites arising from cytochrome P450-dependent monooxygenase is unknown. Further characterization of renal cytochrome P450 and a study of its regulation are needed. The characterization of cytochrome P450 in humans has been limited by the difficulty in obtaining organs in good condition. Hepatic cytochrome P450 of humans has been purified and studied in detail (14-20). It has been compared with rat hepatic cytochrome P450, which has also been studied extensively. The study of extrahepatic cytochrome P450 in humans is only in microsomes, because the specific content of cytochrome P450 in extrahepatic organs of humans is low (28-30) and the purification of cytochrome P450 is difficult. Because of the species differences in cytochrome P450, renal cytochrome P450 in humans must be purified for characterization. In this study, we characterized P450” K-2, K-4, and K5 purified to homogeneity from rat kidney by their NH2terminal sequence, spectral properties, and catalytic activity. Furthermore, we partially purified renal cytochrome P450 of humans and compared it with rat renal cytochrome P45Os. MATERIALS
AND
METHODS
Chemicals. Testosterone, sodium laurate, and w-hydroxylauric acid were obtained from Sigma Chemical Co. (St. Louis, MO). (w-l)Hydroxylauric acid was the kind gift of Dr. Y. Imai of Osaka University. 2n-, 6/j-, 7a-, 16a-, and 16fi-hydroxytestosterone were the kind gift of Dr. Y. Nakamura (Shionogi Research Laboratory, Osaka, Japan). Both 6n- and 15u-hydroxytestosterone were provided by Prof. D. N. Kirk (Queen Mary University of London, UK). Dilauroylphosphatidylcholine was obtained from Sigma. NADPH was obtained from Oriental Yeast Co. (Tokyo, Japan). Emulgen 911 was a gift of the Kao Corp. (Tokyo, Japan). tu-Chymotrypsin was obtained from Miles Diagnostics (Kankakee, IL). 9-Anthryldiazomethane was obtained from Funakoshi Chemical Industry (Tokyo, Japan). Coomassie brilliant blue R-250 was obtained from Bio-Rad Laboratories (Richmond, CA). A Vectastain ABC kit containing the reagent consisting of avidin and biotinylated horseradish peroxidase was obtained from Vector Laboratories (Burlingame, CA). A gradient poiyacrylamide gel (10~20%) for SDS-polyacrylamide gel electrophoresis was obtained from Daiichi Pure Chemicals Co. (Tokyo, Japan). Other reagents and organic solvents were obtained from Wako Pure Chemical Industries (Tokyo, Japan). Purification of renal cytochrome P4.50. Male Sprague-Dawley rats weighing 200-250 g were obtained from
OF BIOCHEMISTRY
Vol. 276, No. 2, February
AND
BIOPHYSICS
1, pp. 473-480,199O
Characterization of Three Cytochrome P45Os Purified from Renal Microsomes of Untreated Male Rats and Comparison with Human Renal Cytochrome P450 Susumu
Imaoka,l
Kazuo
Nagashima,*
and Yoshihiko
Funae
Laboratory of Chemistry, Osaka City University Medical School, Abeno-ku, Osaka 545, Japan; and *Department Faculty of Medicine, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060, Japan
of Pathology,
Received ,July 10, 1989, and in revised form October 11,1989
Three renal cytochrome P45Os (P450 K-2, K-4, and K-5) were purified from renal microsomes of untreated male rats. Also, the human renal cytochrome P450 (P450 HK) was partially purified from renal microsomes and its properties were compared with those of the rat renal cytochrome P45Os. The molecular weight of P450 K-2, K-4, and K-5 was 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of P450 K-2, K-4, and K-5 were at 449, 451, and 452 nm, respectively. NH,-terminal sequence analysis of P450 K-2, K-4, and K-5 showed that these forms were different from hepatic cytochrome P45Os purified previously. P450 K-2, K-4, and K-5 catalyzed the 0-dealkylation of ‘7-ethoxycoumarin but were not efficient in the hydroxylation of testosterone. Aminopyrine was metabolized by P450 K-2 and K4 but not by P450 K-5. Laurie acid was metabolized efficiently by all of these forms in the presence of cytochrome bg. The regiospecificity of these forms toward lauric acid was different. P450 K-2 hydroxylated lauric acid only at the (w-1)-position, not at the w-position. P450 K-4 and K-5 hydroxylated lauric acid at both the w- and (o-1)-positions. The ratios of ti/(W-l)-hydroxylation activity of P450 K-4 and K-5 were 2.5 and 7.8, respectively. Human P450 HK was purified 220fold and its specific content was 2.0 nmol/mg of protein. The Soret maxima of P450 HK were at 418 nm for the oxidized form, 416 nm for the reduced form, and 450 nm for the CO-reduced form. P450 HK catalyzed the 0-dealkylation of 7-ethoxycoumarin but was not efficient in aminopyrine N-demethylation or testosterone hydroxylation. P450 HK had high lauric acid W- and (w-l)-hydroxylation activities in the presence of cyto-
’ To whom all correspondence 000%9861/90
should be addressed.
$3.00
Copyright (!I 1990 by Academic Press, Inc. All rights ofreproduction
in any form reserved.
chrome b5, especially w-hydroxylation. These properties resembled those of P450 K-5 most closely. AntiP450 K-5 antibody cross-reacted with P450 HK as well as P450 K-5 and only one band was stained on immunostained Western blotting for partially purified P450 HK. The molecular weight of P450 HK was 52,000 on Western blotting. 0 1990 Academic Press, Inc.
Cytochrome P450 and its associated monooxygenase activities are predominantly found in the liver, but are also present in many other tissues, although usually at lower levels (1). There are species, sex, age, and tissue differences in cytochrome P45O.s (2-4). The balance of metabolic activation and detoxification of drugs and other chemicals by individual forms of cytochrome P450 in different tissues or species is an important factor in explaining organ- or species-specific toxicity. Cytochrome P450 also has organ-specific roles involving, for example, the synthesis of steroids by adrenal cytochrome P450 (5) and the synthesis of thromboxane by platelet cytochrome P450 (6). Most studies of cytochrome P450 have focused on hepatic forms in rats (713) and humans (14-20). Much less is known about of renal cytochrome P45Os in rats and humans. Renal cytochrome P450 efficiently catalyzes the hydroxylation of prostaglandins and fatty acids such as lauric acid but metabolizes testosterone and drugs such as aminopyrine and 7-ethoxycoumarin little if at all (21~ 24). The substrate specificity of renal cytochrome P450 is different from that of hepatic cytochrome P45Os. Arachidonic acid can be metabolized by renal cytochrome P450, being converted to w- or (w-1)-hydroxyarachidonic acids and epoxyeicosatrienoic acids (25,26). We have investigated arachidonic acid metabolism by a cytochrome P450 purified from the rat renal microsomes, finding that purified renal cytochrome P450 produces epoxyei473
474
IMAOKA,
NAGASHIMA,
cosatrienoic acid and w- and (w-1)-hydroxyarachidonic acid (27). However, the contribution to renal function of arachidonic acid or prostaglandin metabolites arising from cytochrome P450-dependent monooxygenase is unknown. Further characterization of renal cytochrome P450 and a study of its regulation are needed. The characterization of cytochrome P450 in humans has been limited by the difficulty in obtaining organs in good condition. Hepatic cytochrome P450 of humans has been purified and studied in detail (14-20). It has been compared with rat hepatic cytochrome P450, which has also been studied extensively. The study of extrahepatic cytochrome P450 in humans is only in microsomes, because the specific content of cytochrome P450 in extrahepatic organs of humans is low (28-30) and the purification of cytochrome P450 is difficult. Because of the species differences in cytochrome P450, renal cytochrome P450 in humans must be purified for characterization. In this study, we characterized P450” K-2, K-4, and K5 purified to homogeneity from rat kidney by their NH2terminal sequence, spectral properties, and catalytic activity. Furthermore, we partially purified renal cytochrome P450 of humans and compared it with rat renal cytochrome P45Os. MATERIALS
AND
METHODS
Chemicals. Testosterone, sodium laurate, and w-hydroxylauric acid were obtained from Sigma Chemical Co. (St. Louis, MO). (w-l)Hydroxylauric acid was the kind gift of Dr. Y. Imai of Osaka University. 2n-, 6/j-, 7a-, 16a-, and 16fi-hydroxytestosterone were the kind gift of Dr. Y. Nakamura (Shionogi Research Laboratory, Osaka, Japan). Both 6n- and 15u-hydroxytestosterone were provided by Prof. D. N. Kirk (Queen Mary University of London, UK). Dilauroylphosphatidylcholine was obtained from Sigma. NADPH was obtained from Oriental Yeast Co. (Tokyo, Japan). Emulgen 911 was a gift of the Kao Corp. (Tokyo, Japan). tu-Chymotrypsin was obtained from Miles Diagnostics (Kankakee, IL). 9-Anthryldiazomethane was obtained from Funakoshi Chemical Industry (Tokyo, Japan). Coomassie brilliant blue R-250 was obtained from Bio-Rad Laboratories (Richmond, CA). A Vectastain ABC kit containing the reagent consisting of avidin and biotinylated horseradish peroxidase was obtained from Vector Laboratories (Burlingame, CA). A gradient poiyacrylamide gel (10~20%) for SDS-polyacrylamide gel electrophoresis was obtained from Daiichi Pure Chemicals Co. (Tokyo, Japan). Other reagents and organic solvents were obtained from Wako Pure Chemical Industries (Tokyo, Japan). Purification of renal cytochrome P4.50. Male Sprague-Dawley rats weighing 200-250 g were obtained from
