Purification and NH2-terminal amino acid sequences of human and rat kidney fatty acid ω-hydroxylases

156

BBALIP

53793

Purification and NH2-terminal amino acid sequences of human and rat kidney fatty acid o-hydroxylases Hidenori

Kawashima

I,‘, Emi Kusunose ‘, Ichiro Kubota ‘, Masanobu Masamichi Kusunose L-)

(Received

Key words:

HPLC:

Amino

acid sequence:

Maekawa

’

and

I5 May IOYI)

Fatty acid w-hydroxylase; Cytochrome microsome)

P-450: (Human

kidney microsome):

(Rat kidney

A cytochrome P-450 (P-4501, designated P-450,,,, has been isolated and purified from human kidney microsomes to a specific content of 13 nmoles of P-450/mg of protein. P-450uKw showed an apparent molecular weight of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Absolute spectra of the oxidized form indicated that this P-450 was largely in the low-spin state and partly in the high-spin state. It catalyzed the wand (w - l)-hydroxylation of fatty acids such as laurate, myristate, and palmitate, with no activity toward prostaglandin A,, benzphetamine, 7-ethoxycoumarin, or 7-ethoxyresorufin. The first 35 NH,-terminal amino acid sequence of P-450,,, had about 70% homology with those of rabbit kidney fatty acid w-hydroxylases of the P-450 IVA gene subfamily, P-450 ka_ ,, P-450,, _2, and P-450,,, except for four undetermined residues. Moreover, Western blot and immuno-inhibition studies showed that P-450,,, reacted with an antibody against the rabbit kidney fatty acid w-hydroxylase. The results suggest that P-450,,, is a member of the same P-450 gene family (IVA subfamily) as the rabbit enzymes. In addition, the terminal sequence of P-450,,, also showed 54% homology with that of a fatty acid w-hydroxylase from rat kidney microsomes. To our knowledge, this is the first time that a P-450,_,, P-450 specific for fatty acid w-hydroxylase activity has been isolated to homogeneity from human tissues.

Introduction Microsomes from both kidney cortex and liver catalyze the w-and (o - l)-hydroxylation of fatty acids at similar rates, whereas kidney cortex microsomes are much lower than liver microsomes in their ability to metabolize xenobiotics, suggesting that kidney P-450 system is rather specialized for the w-hydroxylation of fatty acids [l-5]. Earlier, we purified two major forms of P-450 from kidney cortex microsomes of untreated rats, designated P-450,_, and P-45O,p2, both of which are highly active in the w- and (w - 1)-hydroxylation of

Abbreviations: P-450, HPLC, high-performance Correspondence: Toyonaka Osaka

cytochrome P-450: PGA. liquid chromatography.

E. Kusunose. 560. Japan.

Toneyama

Hospital.

prostaglandin

Toneyama

A:

5-l-l.

fatty acids [6]. In addition, P-450,_, was capable of hydroxylating prostaglandin A, (PGA, ) and PGA, at the w-position solely, whereas P-450,-, was inactive toward PGA, and PGA, Furthermore, we also isolated from kidney cortex microsomes of rabbits treated with di(2-ethylhexyllphthalate four distinct forms of P-450, which catalyzed the w- and (w - I l-hydroxylation of fatty acids and were designated P-450,,-, [71, P-450,,-, [7-Y]. P-450,, [lo], and P-450,, [lo]. Like rat P-450,- ,, the former two were also highly active in the w-hydroxylation of PGA, and PGA, [7--g]. In contrast. the latter two had no activity toward PGA, and PGA, [lo]. Furthermore, cDNA clones corresponding to P-450,;, _ ,, P-450,,- ?, and P-450,, have been isolated using a cDNA clone for the lung prostaglandin w-hydroxylase designated P-450,_, (P-450 IVA4) [11,12] as hybridization probe, and sequenced [13,14]. Thus, these three kidney fatty acid w-hydroxylases have been demonstrated to be members of the

157

P-450IVA subfamily. Johnson et al. [15] also have isolated and sequenced similar clones encoding three w-hydroxylase P-450s from a rabbit kidney cDNA library. These cDNA clones designated K,A, (IVA61, KoB,, (IVA7), and K,B, (IVAS), correspond to P450 ka_ , , P-450 ka_ *, and P-450 kd, respectively. Although human liver P-450s have been extensively investigated [ 161, only limited information is available on human kidney P-450. Earlier, Jakobsson and Cinti [ 171, and subsequently, Okita et al. [18] reported that human kidney cortex microsomes contained a P-450 which catalyzed the W- and (w - l)-hydroxylation of laurate. Recently, Imaoka et al. [19] partially purified a P-450 active in the w- and (w - l)-hydroxylation of laurate from human kidney microsomes. In the present study, we have first purified to homogeneity a fatty acid w-hydroxylase from human kidney microsomes. The results suggest that this P-450, designated P-450 uKw, is a member of the same P-450 gene family (IVA subfamily) as the rabbit kidney o-hydroxylases. In addition, we have also highly purified P450,_ ,, a rat kidney fatty acid o-hydroxylase. The determination of its NH2-terminal amino acid sequence revealed that this P-450 is equivalent to P-450 IVA2 described by Kimura et al. [20]. Materials

and Methods

Materials

Sepharose 4B and DEAE-Sephadex A-50 were purchased from Pharmacia Fine Chemicals Wppsala); CM HPLC column (ES-5020, from Asahi Kasei (Tokyo); hydroxylapatite HPLC column (KB 12-00121, from Koken (Tokyo); various fatty acids, PGA ,, PGA, and ‘ADAM reagent’ (9-anthryldiazomethane), from Funakoshi Chemicals (Tokyo); 7-ethoxycoumarin, 7ethoxyresorufin, and phospholipids, from Sigma Chemicals (St. Louis). Amino-n-hexyl-Sepharose 4B (AHSepharose 4B) was prepared by the method of Cuatrecasas [21]. Emulgen 911 and 913, and benzphetamine were gifts from Kao Chemicals (Tokyo) and Upjohn (Kalamazoo), respectively. NADPH-cytochrome P-450 reductase and cytochrome b, were prepared from rabbit liver microsomes by the methods of Taniguchi et al. [22], and Spatz and Strittmatter [23], respectively. Rabbit kidney P-450s designated P-450,,_,, P-448,, and P-450,,_, were purified by the methods of Kusunose et al. [7], Ogita et al. [24], and Ogita et al. [8], respectively. Polyclonal antibodies against P-450s were prepared in guinea-pigs as described earlier [2.5]. Purification of somes

P-450,,,from human kidney micro-

Human kidneys were obtained from four patients with renal pelvic tumor or ureteral tumor. Kidney

microsomes were prepared and suspended in 0.1 M potassium phosphate buffer (pH 7.51 containing 20% glycerol, 1 mM dithiothreitol (DIT), and 1 mM EDTA (designated 0.1 M ‘KP Buffer A’) to give a concentration of 4 mg protein per ml. A 10% sodium cholate solution was added dropwise with stirring to this suspension to give a final concentration of 0.8% (w/v). After stirring at 4” C for 60 min, the mixture was centrifuged at 140,000 X g for 60 min. The supernatant was diluted with 0.1 M KP Buffer A to give a concentration of 0.6% sodium cholate, and applied to an AH-Sepharose 4B column (3 X 9 cm), which had been equilibrated with the buffer containing 0.6% sodium cholate. After the column had been washed with 650 ml of 0.13 M KP Buffer A containing 0.3% sodium cholate, P-450 was eluted with 0.1 M KP Buffer A containing 0.4% sodium cholate and 0.1% Emulgen 913. The eluate was concentrated, and dialyzed against 10 mM potassium phosphate buffer (pH 7.5) containing 20% glycerol, 0.1 mM DTT, 0.1 mM EDTA, 0.1% sodium cholate, and 0.2% Emulgen 913 (designated 10 mM ‘KP Buffer B’). The dialyzed materials were applied to a DEAE-Sephadex A-50 column (1.5 x 17 cm) which had been equilibrated with the same buffer. A part of P-450 passed through on the column wash (‘Fraction I’). Most of P-450 were eluted sequentially with 30 mM KP Buffer B (‘Fraction II’), 60 mM KP Buffer B (‘Fraction III’), and 80 mM KP Buffer B (‘Fraction IV’). Fraction III was concentrated, dialyzed, and applied to a second DEAE-Sephadex A-50 column (1.3 x 20 cm). After the column had been washed with 100 ml of 15 mM KP Buffer B, P-450 was eluted with 50 mM KP Buffer B. The eluate was concentrated, and dialyzed against 10 mM KP Buffer B, then applied to a third DEAE-Sephadex A-50 column (1.3 X 20 cm>. After the column had been washed with 140 ml of 15 mM KP Buffer B, P-450 was eluted with 50 mM KP Buffer B. The eluate was concentrated, dialyzed against 20 mM sodium phosphate buffer (pH 6.5) containing 20% glycerol and 0.4% Emulgen 911 (‘CM-A Buffer’), and applied to a CM HPLC column (ES-502C, 0.76 X 10 cm). P-450was eluted at the flow rate of 0.6 ml/min with a linear gradient of CM-A Buffer, and CM-A Buffer containing 1 M sodium acetate (pH 7.0) (designated ‘CM-B Buffer’) (l%/min), using a solvent programmer at room temperature, with monitoring at 417 nm. The eluates at around 30% CM-B Buffer in CM-A Buffer were pooled, concentrated, then dialyzed against 10 mM sodium phosphate buffer (pH 6.5) containing 20% glycerol, 0.2% sodium cholate, and 0.4% Emulgen 911 (‘KB-A Buffer’), and applied to a. hydroxylapatite HPLC column (KB column, 0.6 x 10 cm> equilibrated with the same buffer. After the column had been washed, P-450 was eluted at the flow rate of 0.5 ml/min with a linear gradient, between KB-A Buffer

158 and 350 mM sodium phosphate buffer (pH 7.5) containing 20% glycerol, 0.2% sodium cholate, 0.4% Emulgen 911 (designated ‘KB-B Buffer’) (35%/70 min). Fractions showing high purity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) were pooled and concentrated on a small hydroxylapatite column. The detergent was removed by washing the column with 20 mM potassium phosphate buffer (pH 7.25) containing 20% glycerol until there was no absorption at 210 nm due to the detergent. P-450 was eluted with 0.35 M potassium phosphate buffer (pH 7.5) containing 20% glycerol. The final preparation had a specific content of 13 nmol of P450/mg of protein and was designated ‘P-450,,,,,,‘.

Purification

of P-450, ~ 2 from

rat kidney microsomes

Kidney microsomes from 100 rats were used for the purification of P-45O,_2 [6]. The microsomes were solubilized with 800 ml of 0.8% sodium cholate in 0.1 M KP Buffer A, then applied to an AH-Sepharose 4B column (3.6 X 10 cm), equilibrated with the buffer containing 0.6% cholate. After the column had been washed with 780 ml of 0.13 M KF’ Buffer A containing 0.3% cholate. P-450 was eluted with 0.1 M KP Buffer A containing 0.4% cholate and 0.1% Emulgen 913. After the eluate had been concentrated and dialyzed against 10 mM KP Buffer B, the dialyzed materials were applied to a DEAE-Sephadex A-50 column (1.4 x 23 cm) equlibrated with the same buffer, and washed with the buffer. P-450 was eluted with 20 mM KP Buffer B, concentrated, then dialyzed, and applied to a second DEAE-Sephadex A-50 column ( 1.0 x 15 cm). After the column had been washed with the same buffer, P-450 was eluted with 30 mM KP Buffer B. The eluate was concentrated, dialyzed against CM-A Buffer. and applied to a CM HPLC column. P-450 was eluted with a linear gradient of CM-A Buffer and CM-B Buffer. The eluates at around 46% CM-B Buffer in CM-A Buffer were pooled, concentrated, then dialyzed against KB-A Buffer, and applied to a KB column. After the column had been washed, P-450 was eluted at a flow rate of 0.5 ml/min with a linear gradient, between KB-A Buffer and KB-B Buffer. Fractions showing high purity on SDS-PAGE were pooled and concentrated. The detergent was removed. The final P-45O,_2 preparation had a specific content of 15 nmol of P-450/mg of protein. Analytical

methods

The concentration of P-450 was determined by the method of Omura and Sato [26]. SDS-PAGE was performed as described by Laemmli [27] with slight modification. The NH,-terminal amino acid sequence of P-450 was analyzed by Edman degradation using an automatic gas phase sequencer (Applied Biosystems.

Model 477AJ after P-450 had been dialyzed throughly against water. Fatty acid w- and (w - 1)-hydroxylase activities wcrc measured in a reconstituted system containing Tris-HCI buffer (pH 7.5, 30 pmol), P-450 (10 pmol), NADPHcytochrome P-450 reductase (0.15 unit). cytochromc hi (10 pmol), NADPH (70 nmol). dilauroylphosphatdylcholine (3 pg). @cyclodextrin (5 pug), and fatty acids (50 nmol), in a total volume of 0.3 ml. The reaction mixture was incubated at 37 ’ C for 10 min. The w- and (w - I)-hydroxy fatty acids were determined as described [2X]. The w- and (w - I)-hydroxylase activities of PGA, were determined by a modification [3Y] of Theoharidcs and Kupfer [30]. The activities for henzphetaminc Ndemethylase. 7-ethoxycoumarine O-dcethylase. and 7cthoxyresorufin O-deethylasc were determined by the methods of Nash [31]. Greenlee and Poland [32] and Prough et al. [33], respectively. Western blotting was carried out according to the method of Towbin et al. [34]. Results Purification

of P-450,,,C,, jiorn

latma,l

kidney micro-

SOlllCS

Table I shows that kidney microsomes from different human subjects display significant activity toward the w- and (w - l)-hydroxylation of laurate except one case (Case 8). The mean values of the specific activities and the ratio of w- to (w - l)-hydroxylation activities were 143 + 32 pmol/min/mg of protein and 13.0, respectively. The specific contents of P-450 in these human kidney microsomes were found to be only 2-5 pmoles of P-450/mg of protein, or in many instances

TABLE

I

The specific activity was expressed protein. ( ). Ratio of the specific hydroxylation.

as pmol of products/min/mg of activity of iaurate CO- to (0) -- I )-

-be

Sex

Cause for nephrectomy

Activity

I

67

7 3 4 5 h 7 8 Y 10

SO 6s 67 15 10 55 h‘l 3’) Sh

M M F F F M M M M M

Renal pelvic tumor Staghorn calculi Ureteral tumor Ureteral tumor Renal tumor Renal pelvic tumor Ureteral tumor Ureteral tumor Renal artery aneurysm Renal tumor

71.9(11.4) 81.3 (1I.Y) 178.1 ( 10.X) 2YY.3 (13.4) I 13.‘) (13.4) 373.’ ( 14.1) lhS.1 (15.3l Il.0 (6) h5.9 (12.1) 170.3 ( 16.9)

” The values

are single determinations.

I’

159 TABLE

II

Laurate w- and (w - I)-hydroxylation, and PGA, o-hydroxylation activities of four fractions separated by DEAE-Sephadex A-50 column chromatography The assay conditions are given in Materials and Methods. Activities are expressed as nmol products/min/nmol P-450. Activity less than 0.01 nmol/min/nmol P-450 is expressed as -. Substrate

Laurate PGA,

Fraction

w w-l w

94-

-

a

I

II

III

IV

0.10 _ 0.12

0.38 _ 0.22

1.96 0.10 0.09

0.09 _ _

67-

’ The values are single determinations.

could not be quantitated spectrophotometrically, because of the presence of interferring hemoglobin. In the present study, the kidneys obtained from Cases 3, 4, 6, and 7 (Table I) were used for the purification of P-450. AH-Sepharose 4B chromatography of the cholate extracts from the combined kidney microsomes of four patients yielded 40 nmoles of P-450. Subsequent chromatography on a DEAE-Sephadex A-50 column separated P-450 into four different fractions (Fraction I, II, III, and IV). Fraction III had the highest activity toward laurate w-hydroxylation, whereas PGA, w-hydroxylation was highest with Fraction II (Table II), suggesting the presence of two forms of w-hydroxylating P-450 in the human kidney microsomes. Additionally, significant 7-ethoxyresorufin Odeethylase activity was detected in Fraction III. In order to remove a bulk of contaminated proteins, Fraction III was rechromatographed twice on DEAE-Sephadex A-50 columns, followed by CM column HPLC. The final preparation of P-450 (P-450,,,) had a specific content of 13 nmols of P-450/mg of protein. Table III summarizes the results of the purification of P-450.,,.

TABLE

III

Purification of P-450,,,

from human kidneys

Step

Microsomes AH-Sepharose 4B First DEAE-Sephadex A50 (Fraction III) Second DEAE-Sephadex A50 Third DEAE-Sephadex A50 CM HPLC Hydroxylapatite HPLC

Total protein (mg)

Total P-450 (nmol)

3,200 130

- a 40.0

0.31

25.2 17.3 11.5 6.3 1.3

1.08 1.40 2.10 6.63 13.0

23.3 12.4 5.48 0.95 0.10

Specific content (nmol/mg)

a Microsomal contents of P-450 can not be measured because of the presence of interferring hemoglobin.

Yield (%o)

100 63 43 29 16 3.3 accurately,

30-

-

20-e

v_

-__I

1

2

3

Fig. 1. SDS-PAGE of P-450,,,. 1. molecular weight markers; phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and soybean trypsin inhibitor (20 kDa); 2. P-450,,_, (5 pmol); 3. P-450,,, (2 pmol). Electrophoresis was performed on 10% acrylamide gel, and proteins were stained with 0.1% Coomassie brilliant Blue.

When Fraction III was applied to a CM HPLC column, a P-450 with 7-ethoxyresorufin 0-deethylase activity, passed through on the column wash. Western immunoblot analysis showed that the antibody raised to a kidney P-450 designated as P-448, [241 cross-reacted with a protein with an apparent molecular weight of 54,000 on SDS-PAGE in this fraction (Data not shown). P-448, was inducible with 3-methylcholanthrene, and it was very similar, if not identical, to rabbit liver P-450 form 6 (P-450 IA11 [35]. Further characterization of this P-450 remains to be undertaken. Properties

of P-450,,,

The purified P-450.., was found to be essentially homogenous, as judged by SDS-PAGE. When its migration was compared with those of several standard proteins, a value of 52,000, less than that of P-450,,_, (53,000), a rabbit kidney fatty acid w-hydroxylase was

TABLE

IV

.4 compurison of NH,-terminal amino acid sequences of fkzty ucid w -hydro).~ylases from humun, rut. und rabbit kidney microsonwv

Ala

LKU

400

500

Wavelength

600

700

( nm )

Fig. 2. Absolute spectra of the oxidized form of P-450,,,,. (1.2 FM) was used. The baseline was automatically Shimazu UV 3100 spectrometer.

P-450,,,w

corrected

with a

Srr Pro Ser Ark2 Letl LttU Gly ASP VLII Ser GlY

obtained (Fig. 1). The carbon monoxide difference spectrum of the reduced form of P-450,,,, showed a peak at 450 nm. The oxidized form gave a Soret band at 417 nm, and (Y and p bands at 572 and 534 nm. respectively, characteristic of the low-spin state (Fig. 2). In addition, the oxidized form also had a shoulder between 390 and 400 nm, and a weak band at 650 nm, indicative of the high-spin state. Thus, the P-450 appears to be largely in the low-spin state and partly in the high-spin state. the w- and (w - l)-hydroxylP-450 HKw catalyzed ation of laurate, myristate, and palmitate with turnover rates of 4.3, 1.4, and 0.44 nmol/min/nmol of P-450, respectively. The ratios of w- and (w - l)-hydroxylation activities toward these fatty acids were 17.0, 4.5, and 3.9, respectively. Earlier, we observed that pcyclodextrin markedly enhanced the activity of rabbit prostaglandin w-hydroxylase (unpublished data). Lau-

2 Fig. 3. Western P-450,,mL. Lane

immunoblot 1, P-450..,;

3

Ile LCU Gln Ala X X Leu Leu Ile Leu Leu Leu Leu Lru Ile X Ala Val Gln Leu X Leu

5

Val Phe Se1 Pro Thr Arg

Ala Leu Asn Pro Thr Arg LCU Pro Gly Ser Leu Ser Gly Leu Leu Gln Val ,413

Srr LetI Asp Gl!, Val Ser CJlY

Phe Phe x Cl) Ala Phc Leu Lell X Letl Phe X X Leu Phe X Ala

G]Y

LtW Leu Gly Leu Leu Leu Leu I,ell LCU LVS Ala Ala Gin Leu Ty1 Leu

Ala

LCU Srr Ser Thr Aw Lcu Pi71 Gly Ser Phe Ser GIV Phe Lru Gln Ala Ala Ala Leu LetI Gly LW Leu Lsu Let1 Leu Lcu LyS Ala Ala Gin LCU Tyr Leu

Ala LSU

Scr Pi-0 Thr Arg LCU

Pro (;I!. Srr Leu Ser Gil\Lcu Lru Gln Val Al:1 Ala LCU LCU Gl) Lru Leu

LcLl I.CU Leu LW I .yh i\lS Al2 Gin

LCU TylL.eu

6

of highly purified human, rabbit and rat kidney fatty acid w-hydroxylases with a guinea-pig antibody against lane 2, rabbit P-450,,_, [7]; lane 3, rabbit P-450,,_, [7]; lane 4, rabbit P-450,, [lo]; lane 5, rabbit P-450,, [IO]: lane 6, rat P-450,_, [6]; lane 7. rat P-450, ‘. 2 pmol of each P-450 was used.

161 rate w- and (w - 1)-hydroxylase activity of P-450,,, was also stimulated 50-100% by /3-cyclodextrin in a reconstituted system. The enzyme had negligible or no activity toward PGA r, benzphetamine, 7ethoxycoumarin, 7-ethoxyresorufin, or benzo(a)pyrene, since the values for activities were less than 0.01 nmol/min/nmol P-450. NH,-terminal amino acid sequence immunochemical studies

of P-450,,,

and

The NH ,-terminal amino acid sequence of P-450 uKw was determined and compared with those of three forms of rabbit kidney fatty acid w-hydroxylases (Table IV). Excluding four undetermined residues, P-450,,, was identical in 22/31, 21/31, and 22/31 positions with P-450,, [10,14], P-450,,_, 1131, and P-450,,-, [13], respectively. In addition, P-450,,, was identical in 17/31 positions with P-450,-,, a rat kidney fatty acid w-hydroxylase [6]. Western blot analysis showed that a guinea-pig antibody to P-450,,_ 2 reacted strongly with P-450,,, as well as with rabbit and rat kidney fatty acid w-hydroxylases [6-101 (Fig. 3). This antibody also inhibited the w- and (w - 1)-hydroxylation of laurate by human kidney microsomes, indicating that the majority of the microsomal fatty acid w-hydroxylation activity is responsible for P-450 uKw or closely related isoenzyme (Fig. 4). In contrast, no inhibition was observed with an antibody to P-450,,_, [8]. P-450 kb_, was an inducible

Relative activity (%)

Fig. 4. Inhibition of the w- and (o - ll-hydroxylation of laurate by an antibody against P-450,,_,. Human kidney microsomes (300 kg of protein) were preincubated with the antibodies at 37 ’ C for 10 min. 0, anti-P-450,,_, I&; A, anti-P-450,,_, IgG; 0, non-immune o-hydroxylation; - - -, (w - ll-hydroxylation. The IgG; -, 100% (uninhibited) values for both the microsomal laurate o- and (w - ll-hydroxylase activities were 0.27 and 0.03 nmol/min/mg protein, respectively.

form with phenobarbital, and it was very similar, if not identical, to liver P-450 form 2 (P-450 IIB4) [361. Discussion

Several forms of human liver P-450 have been isolated and characterized in many laboratories 1161. In contrast, only limited data are available on human kidney P-450 [17-191. In the present work, we have first purified to homogeneity a P-450 from human kidney microsomes. This P-45O.k, catalyzed the wand (w-1)-hydroxylation of fatty acids with no activity for prostaglandins or xenobiotics such as benzphetamine and 7-ethoxycoumaline. The first 35 NH,terminal amino acid sequence of this P-450 showed about 70% homology with those of rabbit kidney fatty acid w-hydroxylases (P-450 k. _ , , P-450 ka_ 2, and P450,,) excluding four undetermined residues. In addition, the antibody raised against P-450,, _Z cross-reacted with P-45Ouk,. These results suggest that Pis a member of the same P-450 gene family wIK, (IVA subfamily) as the rabbit o-hydroxylases. The cDNA cloning for P-450,,, is now in progress in our laboratory, using the cDNA for P-450,,_, as a hybridization probe. The characterization of the purified P-450 Hkw provides useful information for the identification of cDNAs isolated from a human kidney cDNA library. As far as we know, no P-450 of the IVA subfamily has yet been isolated from other human tissues. In the present study, a rat kidney fatty acid w-hydroxylase, P-450,_ 2 [6], has also been sequenced for the first 31 NH,-terminal amino acid sequence, which showed about 54% homology with that of P-450..,. Based on the sequence data and other properties, this P-450 appears to correspond to P-450 K-5 purified by Imaoka et al. [19,37], and to the P-450 IVA2 gene isolated by Kimura et al. [20]. The physiological significance of kidney fatty acid w-hydroxylase is not known. The findings that fatty acid w-hydroxylases predominate over other forms of P-450 in kidney cortex microsomes of several species such as human [17-191, pig [1,38], rat [2,4-6,191 and rabbit [7-101 suggest that the w-hydroxylation of fatty acids plays a role in the renal function. In the kidney, arachidonic acid is actively metabolized by P-450 system to give 20- and 19-hydroxyeicosatetraenoic acids 139-411. Recently, Schwartzman and colleagues [42-441 have reported that the former metabolite is a vasoconstrictor, and the latter metabolite is a potent renal Na+,K+-ATPase stimulator, suggesting that both the metabolites promote the development of hypertension by causing vasoconstriction and sodium retention. The observation that the w- and (w - 1)-hydroxylation of arachidonic acid in human kidney microsomes is inhibited by an antibody against lauric acid o-hydroxylase

162 [45] suggests that P-450HK, or closely related enzyme is involved in these arachidonic acid hydroxylations.

IGmura.

S., Hanioka,

( 19X’)) DNA

X. 51)3-S

C’uatrecasas,

Acknowledgements

P. (lY70)

1I.,

Taniguchi,

N..

Matsunapa.

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