Purification and Characterization of Vitamin D 25-Hydroxylase from Rat Liver Mitochondria*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 263, No. 28, Issue of October 5, pp Printed in U.S.A. Purification and Characterization of Vitamin D 25-Hydroxylase from Rat Liver Mitochondria* (Received for publication, April 4, 1988) Osamu Masumoto, Yoshihiko Ohyama, and Kyuichiro Okuda From the Department of Biochemistry, Hiroshima University School of Dentistry, Kasumi, Minami-ku, Hiroshima 734, Japan Vitamin D 25-hydroxylase was purified from female rat liver mitochondria based on the catalytic enzyme activity. The final preparation of cytochrome P-450 was homogeneous judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,500). The specific content of the purified enzyme was 12 nmol/mg of protein. The absorption spectrum of the purified enzyme showed a peak at 417 nm and that of the dithionite-reduced CO complex at 450 nm, indicating the enzyme belongs to the cytochrome P-450 fam- ily. Upon reconstitution with the electron-transferring system of the adrenal system (adrenodoxin and NADPH-adrenodoxin reductase), the enzyme showed a high activity in hydroxylating la-hydroxycholecalciferol at position 25 with a turnover number of 3.8 min- and K,,, of 54 PM. The enzyme activity was completely lost when the electron-transferring system was replaced by that of microsomes (NADPH-cytochrome P-450 reductase purified from rat liver microsomes), confirming that the P-450 enzyme was of the mitochondrial type, not the microsomal type. The omission of cytochrome P-450, adrenodoxin, or NADPH-adre- nodoxin reductase resulted in complete loss of enzyme activity. The liver mitochondrial cytochrome P-450 hydroxylates vitamin D3 and la-hydroxyvitamin D3 at position 25, but did not show any activity toward xenobiotics such as benzphetamine, 7-ethoxycoumarin, and benzo[a]pyrene. The enzyme activity was not inhibited by aminoglutethimide but slightly inhibited by metyr- apone. The enzyme activity was markedly inhibited an atmosphere of CO:Os:Nz, 40:20:40. Vitamin D, although it was identified as an anti-rachitic factor originally, is now considered as a hormone functioning for maintenance of calcium homeostasis in vertebrates. The active form of vitamin D3 was recently established as la,25- dihydroxycholecalciferol by Kodicek s and DeLuca s groups (1). The conversion of vitamin DS into its active form involves two steps, the first step being the hydroxylation at in liver (2) and the second being the hydroxylation at C1 in kidney mitochondria (3). 25-Hydroxylation of vitamin D3 is conducted both in microsomes (2) and mitochondria (3). The microsomal enzyme was purified from male rat liver and characterized as cytochrome P-450 by Andersson et al. (4) and Hayashi et al. (5). * This study was supported in part by Grants-in-Aid for Scientific Research ( to K. 0. and to Y. 0.) from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact However, this cytochrome P-450 was hardly detectable in female rat liver microsomes (6). Recently it was shown that the mitochondrial enzyme activity is severalfold higher in the female rat than in the male rat (7). This fact, taken together with the fact that human liver microsomes are devoid of vitamin D3 25-hydroxylase activity, cast doubt about the physiological importance of the liver microsomal enzyme (8). However, the mitochondrial enzyme has not been purified further than ammonium sulfate fractionation of the solubi- lized extract, although its nature as cytochrome P-450 seemed to be established based on the reconstitution study (4). In view of the fact that there are many homologous species of cytochrome P-450 with similar properties in liver, further purification of the enzyme is essential to allow thorough characterization. We have now obtained the rat liver mitochondrial vitamin D 25-hydroxylase in a highly purified state employing a high performance liquid chromatographic technique, which was successfully applied for purification of cholesterol 7a-hydroxylase (9), an important endogenous cytochrome P-450 catalyzing the initial hydroxylation of cholesterol for catabolism in liver microsomes. In this paper we describe the purification and characterization of a cytochrome P-450 catalyzing vitamin D 25-hydroxylation from female rat liver mitochondria. EXPERIMENTAL PROCEDURES Materials-Adrenodoxin and NADPH-adrenodoxin reductase were purified from bovine adrenocortical mitochondria according to the methods described by Suhara et al. (10) and the modified method of Suhara et al. (11, 12). NADPH-cytochrome P-450 reductase was purified from rat liver microsomes according to the method described by Yasukochi and Masters (13). Metyrapone and aminoglutethimide were a gift from Ciba Geigy Co. (Basel, Switzerland). la-hydroxycholecalciferol and lol,25-dihydroxycholecalciferol were kindly supplied by Teijin Co. (Tokyo) and Chugai Pharmaceutical Co. (Tokyo), respectively. Lubrol PX and cholic acid were purchased from Sigma and hydroxylapatite (Bio-Gel HTP) was from Bio-Rad Co. Sepharose 4B was the product of Pharmacia Fine Chemicals (Uppsala, Sweden). NADPH was obtained from Oriental Co. (Tokyo) and other chemicals used wereof the highest quality commercially available. TSK-gel DEAE-5PW was purchased from Toyo Soda Co. (Tokyo). High performance liquid chromatography (model TRI ROTAR SR2) equipped with UV spectrophotometer (model UVIDEC-100-IV) was the product of JASCO Co., Tokyo. Preparation of Mitochondria-Livers from 12 female rats of Wistar strain weighing about 200 g were excised and thoroughly perfused with 0.9% NaCl solution, and then homogenized with 9 volumes of 20 mm Tris-HC1 buffer (ph 7.4) containing 0.25 M sucrose, 1 mm EDTA, pepstatin (1 pg/ml), and leupeptin (1 pg/ml). The homogenate was centrifuged at 600 X g for 12 min in a refrigerated centrifuge, and the supernatant was again centrifuged at 8000 X g for 15 min. The precipitated pellets were washed twice with the same buffer. The precipitate was then suspended in 60 mm Tris-HC1 buffer (ph 7.4) containing 20% glycerol, 0.5 mm EDTA, 0.5 mm 1,4-dithiothreitol, leupeptin (1 pg/ml), and pepstatin (1 pg/ml). Solubilization and Purification of Vitamin D 25-Hydroxylase-To

2 a mitochondrial suspension 10% cholate was added slowly under mild stirring to give a final cholate/protein ratio of 2/1, W/W. All buffer solutions used in the following purification procedures contained 20% glycerol, 1 pg/ml leupeptin, and 1 pg/ml pepstatin unless otherwise stated. The solubilized mitochondrial suspension was then subjected to polyethylene glycol fractionation according to the method described by Coon et al. (14). A fraction precipitated by 4-12% polyethylene glycol was collected by centrifugation and suspended in 100 mm potassium phosphate buffer (ph 7.4) containing 1 mm EDTA and 0.5 mm dithiothreitol. The suspension was dialyzed against the suspension buffer. The dialysate was then resolubilized with 1% sodium cholate and centrifuged at 100,000 X g for 1 h. The supernatant was applied to an w-aminohexyl-sepharose 4B column (2.1 X 16 cm) equilibrated with 100 mm potassium phosphate buffer (ph 7.4) containing 0.5% sodium cholate, 1 mm EDTA, and 0.5 mm dithiothreitol. The column was washed with the equilibration buffer and then eluted with 100 mm potassium phosphate buffer containing 0.4% sodium cholate, 0.1% Lubrol PX, 1 mm EDTA, and 0.5 mm dithiothreitol. The fraction with la-hydroxycholecalciferol 25-hydroxylase activity was collected and dialyzed against 20 mm potassium phosphate buffer (ph 7.4) containing 0.5 mm EDTA, 0.4 mm dithiothreitol, 0.1% sodium cholate, and 0.08% Lubrol PX. The fraction was then applied to a hydroxylapatite column (1.2 X 10.6 cm) equilibrated with the same buffer as used for dialysis. The column was washed with 40 mm potassium phosphate buffer (ph 7.4) containing 0.5 mm EDTA, 0.4 mm dithiothreitol, 0.1% sodium cholate, and 0.08% Lubrol PX, and then eluted with stepwise increase of the potassium phosphate concentration to 60 mm at first and then to 90 mm. The fraction with the enzyme activity was collected and dialyzed against 30 mm Tris- HC1 buffer (ph 7.4) containing 0.4 mm EDTA, 0.4 mm dithiothreitol, 0.4% Lubrol PX, and 0.1% sodium cholate. From this step on, protease inhibitors were omitted. Aliquots were injected into a TSKgel DEAE-5PW column (7.5 X 75 mm) equilibrated with 30 mm Tris- HC1 (ph 7.4) containing 0.4 mm EDTA, 0.4 mm dithiothreitol, 0.4% Lubrol PX, and 0.1% sodium cholate. The column was eluted with linear gradients of sodium chloride (0-70 min, M; min, M). The flow rate was 0.4 ml/min, and the effluent was monitored at 417 nm. Chromatographic conditions were essentially the same as described by Ogishima et al. (9). Enzyme Assay-lor-Hydroxycholecalciferol 25-hydroxylase activity was assayed by two methods. Mitochondrial enzyme activity was assayed as follows. 0.5 ml of the standard reaction mixture contained 20 pmol of potassium phosphate (ph 7.4), 10 pmol of MgCh, 0.1 pmol of EDTA, 5 pmol of isocitrate, 20 nmol of la-hydroxycholecalciferol dissolved in 4 p1 of ethanol, and an appropriate amount of mitochondria. The mixture was preincubated for 2 min and then the reaction was started by adding isocitrate. The incubation was conducted for 20 min at 37 "C. Then the reaction was terminated by adding 5 ml of benzene and the products were extracted with the same solvent. A 4- ml aliquot was pipetted out and evaporated to dryness and the residue was dissolved in chloroform. An aliquot of chloroform solution was subjected to HPLC' analysis using Finepak Si1 column (4.6 X 250 mm, JASCO Co. Ltd., Tokyo) with a solvent mixture of isopropanokmethanokhexane, 8884 (v/v) at a flow rate of 1.3 ml/min (5). (For identification of the products reversed phase HPLC was also performed using Finepak Si1 CIS with a solvent mixture of methanokwater, 8020, v/v.) The effluents were monitored by absorbance at 265 nm. After the enzyme was solubilized the enzyme activity was assayed by the reconstitution method A typical mixture contained pmol of cytochrome P-450,4 nmol of adrenodoxin and 0.1 unit of NADPH-adrenodoxin reductase, 40 pmol of potassium phosphate (ph 7.4), 10 pmol of NADPH, and 100 nmol of vitamin D3 or 20 nmol of la-hydroxycholecalciferol in a final volume of 1 ml. The reaction was initiated by adding NADPH after preincubating the mixture for 2 min at 37 "C. The incubation was conducted for 15 min and then terminated by adding benzene. The products were analyzed as described above. Cholecalciferol 25-hydroxylase activity was assayed in a similar manner except that the solvent system used was 6.5% isopropyl alcohol in n-hexane for normal phase HPLC and 7% water in methanol for reversed phase HPLC. 7-Ethoxycoumarin O-deethylase activity was assayed according to the method of Greenlee and Poland (15) and aryl hydrocarbon hydroxylase was assayed by the method of Nebert and Gelboin (16). Benzphetamine demethylase was assayed according to the method of Miwa et al. (17). Other Methods-Sodium dodecyl sulfate-polyacrylamide gel elec- The abbreviation used is: HPLC, high performance liquid chromatography. Mitochondrial Vitamin D 25-Hydroxylase trophoresis was performed according to the method of Laemmli (18) and the protein bands were visualized by silver staining (19). cytochrome P-450 concentration was determined by a reduced CO difference spectrum using an extinction coefficient of 91 mm" cm" (20). Protein concentration was determined by the method of Lowry et al. (21) using bovine serum albumin as the standard. Data Processing-Maximum velocities and Michaelis constants were computed by Wilkinson's hyperbolic method (22). RESULTS Purification of Vitamin D 25-Hydroxylase-Table I summarizes the results of the purification procedures. Vitamin D 25-hydroxylase was solubilized by cholate from liver mitochondria of female rats and fractionated with polyethylene glycol. The fraction was further purified by chromatography on w-aminohexyl-sepharose 4B, and then hydroxylapatite, and finally on high performance anion-exchange columns. Fig. 1 shows the separation of vitamin D 25-hydroxylase by HPLC, in which the enzyme activity was detected only in one peak. A sodium dodecyl sulfate-polyacrylamide gel electrophoretogram of the fraction with the activity from HPLC is shown in Fig. 2. The active fraction revealed a single major band. Molecular weight of the enzyme was calculated from this electrophoretogram as 52,500. Absorption Spectra of Vitamin D 25-Hydroxylase-Absorpption spectra of vitamin D 25-hydroxylase exhibited absorption maxima at 417,535, and 570 nm in the oxidized form, and in the reduced state at 417 and 550 nm. The absorption maximum for the reduced CO complex was at 450 nm. Reconstitution of Enzyme Activity-Vitamin D 25-hydroxylase activity was reconstituted with the purified cytochrome P-450, adrenodoxin, NADPH-adrenodoxin reductase, and NADPH. The omission of either cytochrome P-450 or the electron-carrying system (adrenodoxin and NADPH-adrenodoxin reductase) resulted in complete loss of activity (Table 11). The addition of rat liver NADPH-cytochrome P-450 reductase to the latter incubation mixture in the absence of adrenodoxin and NADPH-adrenodoxin reductase did not restore any activity. Kinetic Properties of Vitamin D 25-Hydroxylase-The enzyme reaction proceeded in a time-linear fashion up to 20 min under the assay condition described, a in manner proportional to the concentration of the enzyme up to 20 nm. The ph activity curve showed a broad peak at ph 7.8. When the substrate concentration was varied, the reaction followed Michaelis type kinetics, as shown in Fig. 3. The Michaelis constant calculated from Lineweaver-Burk plot was 54 PM. The turnover number calculated from the V, value was 3.8 min". Identification of the Reaction Product-The reaction product of vitamin D was identified by two different HPLC methods, one being a straight phase chromatography and the other being a reversed phase HPLC. The reaction product of la-hydroxycholecalciferol gave the same retention time as la,25-dihydroxycholecalciferol in both systems and the admixture of the reaction mixture and the authentic sample did not produce any new peak or shoulder in both HPLC systems (data not shown). The reaction product of cholecalciferol was identified as 25-hydroxycholecalciferol in a similar manner. Substrate Specificity-Substrate specificity is shown in Table 111. As shown in the table, the mitochondrial cytochrome P-450 did not show any activity toward benzo[a]pyrene, 7- ethoxycoumarin, or benzphetamine. Inhibition of Enzyme Activity-Table IV summarizes the effects of inhibitors on vitamin D 25-hydroxylase. Aminoglutethimide, which inhibits the activity of beef adrenal mito-

3 14258 Mitochondrial Vitamin D 25-Hydroxylase TABLE I Purification of vitamin D 25-hydroxylase Assays were carried out as described under Experimental Procedures using la-hydroxycholecalciferol as substrate. Steps activity Total Total Specific Total activity protein content P-450 Turnover number nmol mg nmollmg nmollmin pmollminlmg nmol/min/nmol protein P-450 Mitochondria 3, Polyethylene glycol w-aminohexyl-sepharose Hydroxylapatite DEAE-5PW , Time (mill) FIG. 1. Separation of vitamin D 25-hydroxylase by high performance anion-exchange chromatography. An aliquot of the eluate from the hydroxylapatite column with the catalytic activity was injected into a TSK-gel DEAEdPW column (7.5 X 75 mm). Equilibration and elution of the column was performed as described under Experimental Procedures. la-hydroxycholecalciferol was used as substrate for assaying enzyme activity. -, absorbance at 417 nm; - --, la-hydroxycholecalciferol 25-hydroxylase activity. TABLE I1 Recorwtitution of vitamin D 25-hydroxylase activity with the mitochondrial P-450, adrenodoxin, and NADPH-adrenodoxin reductase Reconstitution was performed as described under Experimental Procedures. Liver NADPH- NADPH- Product Exp. mitochondrial Adreno- adreno- cytochrome no. cytochrome doxin doxin P-450 dihydroxychole- P-450 reductase reductase calciferol) pmol nml unit unit nmol/20 min , not detectable. 30 K- - A B FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoretogram of the HPLC fraction showing vitamin D 25- hydroxylase activity. Migration was from top to bottom. The gel was stained with silver. Molecular weight standards used (lane A) were phosphorylase b (M, 94,000), bovine serum albumin (M, 67,0001, ovalbumin (M, 43,000), and carbonic anhydrase (Mr 30,000). The amount of cytochrome P-450 applied (lune B) was 0.3 pg of protein. chondrial cytochrome P-450,,, (23), showed no inhibitory effect on the activity at a concentration of 40 pm, whereas metyrapone, which is known to inhibit beef adrenal mito- FIG. 3. Effect of substrate concentration on vitamin D 25- hydroxylase. Enzyme activity was assayed as described under Experimental Procedures except that the concentration of the substrate, la-hydroxycholecalciferol, was varied. chondrial cytochrome P-45Oll8 (24), inhibited the enzyme activity slightly. The enzyme activity was strongly inhibited by sulfhydryl reagents such as p-chloromercuribenzoate and iodoacetamide. However, it was not attempted at present to determine which of the three proteins involved in the reaction (cytochrome P-450, adrenodoxin, NADPH-adrenodoxin reductase) was responsible for the sensitivity, since dithiothreitol present in the enzyme preparation prevented simple analysis. The enzyme activity was also highly inhibited by carbon monoxide, the sensitivity being comparable to that of cholesterol 7a-hydroxylase (9).

4 Mitochondrial Vitamin D 25-Hydroxylase TABLE 111 Substrate and functional specificities of the rat liuer mitochondrial vitamin D 25-hydroxyhe Reaction mixture contained an indicated amount of the mitochondrial cytochrome P-450, 2 nmol of adrenodoxin and 0.05 unit of NADPH-adrenodoxin reductase and 10 *mol of NADPH, in a final volume of 0.5 ml. Incubations were conducted for 15 min at 37 'C. Position Substrate Concentration hydroxylated Product PM pmollmin la-hydroxycholecalciferol" Cholecalciferol" Benzphetamineb 1000 ' 7-Ethoxycoumarin* 100 Benzo[ajpyreneb 80 Testosterone" 40 2a 601 6P 7a 1601 Amount of cytochrome P-450 used was nmol. Amount of cytochrome P-450 used was 0.05 nmol. Not detectable. TABLE IV Effect of inhibitors on the rat liver mitochondrial vitamin D 25- hydroxylase Incubations were conducted as described in the legend to Table 11. Inhibitors Concentration Inhibition PM % Aminoglutethimide 40 0 Metyrapone SKF 525A p-chloromercuribenzoate Iodoacetamide 2.5" " 40 Tetraethylthiuram disulfide Carbon monoxideb 81 Expressed in millimolar. 'Experiment was performed under a gas phase of NJO&O, 40:2040. DISCUSSION Throughout these experiments, the enzyme activity was assayed using la-hydroxyvitamin D3 (lcu-hydroxycholecalciferol) as substrate except in some cases where hydroxylation activity toward vitamin Da was of interest. The reason was that 25-hydroxylation activity toward la-hydroxyvitamin D3 was severalfold higher than that toward vitamin D3 (cholecalciferol). When 25-hydroxylation activity was surveyed with vitamin D3, the activity was found only in the fraction where the activity toward la-hydroxyvitamin D3 was observed. Generally, the particulate fractions of liver homogenate prepared by differential centrifugation are more or less contaminated by the other fractions. Accordingly, when an enzyme activity is observed in a particulate fraction, it is often uncertain whether the observed activity is due to an enzyme occurring normally in that very fraction or to an enzyme contaminated. We are convinced, however, that the present preparation of rat liver mitochondrial vitamin D 25-hydroxylase was purified from mitochondria and not from microsomes possibly contaminating our mitochondrial preparation, for the following reasons. First, in assaying the mitochondrial enzyme activity, we employed isocitrate as an electron donor, which functions in mitochondria but not in microsomes, since isocitrate dehydrogenase does not exist in microsomes (25). Second, we chose female rat liver instead of male as an enzyme source since the former lacks P-45OCee6, a major cytochrome P-450 catalyzing vitamin D 25-hydroxylation in male rat liver microsomes (5). Third, in the reconstitution experiment, the purified preparation did not reveal any activity when the electron transfer system (adrenodoxin and NADPH-adrenodoxin reductase) was replaced with NADPH-cytochrome P- 450 reductase prepared from rat liver microsomes (Table 11). It is interesting that the mitochondrial vitamin D 25- hydroxylase does not oxidize xenobiotics such as benzphetamine, 7-ethoxycoumarin, and benzo[ajpyrene Iike P-450,, and P-45OIl8 of adrenal cortex mitochondria (26). In this connection it maybe interesting to refer to our previous observation that cholesterol 7a-hydroxylase does not show any activity of xenobiotics oxidation even if it is a microsomal enzyme (9). It was recently shown in this laboratory that female rat liver microsomes contain a species of cytochrome P-450 catalyzing vitamin D 25-hydroxylase which is immunochemically different from that of male (27). However, the total activity of vitamin D 25-hydroxylase of female rat liver microsomes is 4-5 fold lower than that of male when measured with la-hydroxyvitamin D3 as substrate. It may therefore be unable to play the major role in the conversion of vitamin D3 into 25-hydroxyvitamin D3. In contrast, according to our calculation from Tables I and 111, the mitochondrial vitamin D 25-hydroxylase shows activity 1.5-fold higher than that of male rat liver microsomes (5). It may therefore be considered that the mitochondrial vitamin D 25-hydroxylase is the major enzyme responsible for 25-hydroxylation of vitamin D in female rat liver. Recently, Saarem and Pedersen (28) have demonstrated that the mitochondrial vitamin D3 25-hydroxylase activity is sex hormone-dependent. Injection of testosterone into female rats decreases the enzyme activity. In contrast, injection of estradiol into male rats resulted in an increase of activity. Such findings suggest that sex hormones exert a regulatory control on the mitochondrial vitamin D3 25-hydroxylase. These results, taken together with the fact that, in human liver, vitamin D 25-hydroxylase exists only in mitochondria, suggest an important role of the mitochondrial enzyme in maintaining calcium homeostasis in vertebrate. Acknowledgment-We wish to thank Dr. Minor J. Coon of The University of Michigan Medical School for his reading our manuscript and correcting the style of English. REFERENCES 1. DeLuca, H. F., and Schnoes, H. K. (1976) Annu. Reu. Biochem. 45, Madhok, T. C., and DeLuca, H. F. (1979) Biochem. J. 184, Bjorkhem, I., Holmberg, I., Oftebro, H., and Pedersen, J. 1. (1980) J. Biol. Chem. 256, Andersson, S., Holmberg, I., and Wikvall, K. (1983) J. Bwl. Chem. 258, Hayashi, S., Noshiro, M., and Okuda, K. (1986) J. Biochem. (Tokyo) 99, Andersson, S., and Jornvall, H. (1986) J. Bwl. Chen. 261, Saarem, K., and Pedersen, J. I. (1987) Biochem. 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