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Keywords:

  • 25-hydroxyvitamin D3 24-hydroxylase;
  • 24R,25-dihydroxyvitamin D3;
  • 1α,24,25-trihydroxyvitamin D3;
  • adrenodoxin;
  • NADPH-adrenodoxin reductase

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

An accurate assay method of 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) was established. Kidney mitochondria prepared from vitamin D-replete rats were treated with polyoxyethylenesorbitan monolaurate. The solubilized suspension was ultracentrifuged at 100,000g for 60 minutes and an aliquot of the supernatant was incubated under the saturating concentrations of substrate NADPH and the mitochondrial-type electron transferring proteins, adrenodoxin and NADPH-adrenodoxin reductase. Products were analyzed by high-performance liquid chromatography (HPLC) monitoring effluents at a wavelength of 265 nm. The maximal velocity of the enzyme in vitamin D-replete rats was 400 pmol/minute per mg of protein, which was considerably higher than those reported by previous authors who used intact kidney mitochondria as the enzyme source. In applying the new assay method, an interesting property was found; Michaelis constant of 24-hydroxylase for 25-hydroxyvitamin D3 [25(OH)D3] was 0.6 μM, which was 35-fold lower than that for 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] which was 20.9 μM. This fact indicates that affinity of the enzyme to 25(OH)D3 is 35-fold higher than that to 1α,25(OH)2D3. These data suggest that 25(OH)D3 is the preferred substrate to 1α,25(OH)2D3.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

IT IS well known that 25-hydroxyvitamin D3 [25(OH)D3] formed in the liver is hydroxylated into 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] by 25-hydroxyvitamin D3 1α-hydroxylase (1α-hydroxylase) or into 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3] by 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) in the kidney, depending on the calcium and/or vitamin D status of animals.(1,2)

Although it generally is accepted that 1α,25(OH)2D3 is the active form of vitamin D,(1) there is some controversy as to the physiological function of 24,25(OH)2D3. Some authors contend that it plays important roles in bone forming,(3) repair of bone fracture,(4) cartilage development,(5) and egg hatchability.(6) Other authors contend that the real substrate of 24-hydroxylase is 1α,25(OH)2D3 instead of 25(OH)D3, and the major role of 24-hydroxylase is inactivation of excess 1α,25(OH)2D3. The enzyme hydroxylates 1α,25(OH)2D3 into 1α,24R,25-trihydroxyvitamin D3 [1α,24,25(OH)3D3], which is subjected to further degradation(7–9); this assumption is based on a kinetic study of the enzyme, in which the Michaelis constant of 24-hydroxylase for 1α,25(OH)2D3 was reported to be 5- to 30-fold lower than that for 25(OH)D3.(8,9) Because affinity of the enzyme to a substrate is inversely related to the Michaelis constant, the enzyme must have much higher affinity to 1α,25(OH)2D3 than 25(OH)D3.

The kinetic property of 24-hydroxylase has been assessed by using an assay method. However, there are two problems in assaying this enzyme in mammals: (1) 24-hydroxylase in mitochondria does not seem to be saturated with the electron transferring proteins as observed with 1α-hydroxylase,(10) and (2) there is an inhibitory factor in mammalian tissues that binds to 25(OH)D3, reduces the availability of the substrate, and inhibits the enzyme reactions employing 25(OH)D3 as substrates 1α-hydroxylase and 24-hydroxylase.(10–12) These facts suggest that it is not adequate to use intact mitochondria for assay of 24- and 1α-hydroxylases. In addressing these problems, we established an assay method and reexamined kinetic properties of 24-hydroxylase using the new method. Consequently, we found that some properties of the enzyme so far reported should be amended.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Materials

Adrenodoxin was prepared from bovine adrenals according to the method described by Suhara et al.,(13) and NADPH-adrenodoxin reductase was prepared by a modified method of Suhara et al.(14) 25(OH)D3, 24,25(OH)2D3 and 1α,24,25(OH)3D3 were purchased from Duphar (Weesp, The Netherlands). 1α,25(OH)2D3 was procured from Wako (Osaka, Japan). When 1α,25(OH)2D3 was used as substrate, the commercial product was purified by high-performance liquid chromatography (HPLC). Polyoxyethylenesorbitan monolaurate (Tween 20) was obtained from Bio-Rad (Richmond, CA, USA), and NADPH was obtained from Oriental Co. (Tokyo, Japan). EDTA, pepstatin, and leupeptin were produced by Wako. Other reagents were of the highest grade commercially available.

Animals

Two-month-old male rats, Sprague-Dawley strain, were given a single intraperitoneal injection of 1α,25(OH)2D3 (1000 ng/100 g body weight), dissolved in 1 ml of olive oil, 1 day before the experiment.

The experimental manipulations were approved by the Committee on Animal Experimentation of Miyazaki Medical College.

Preparation of mitochondria

Kidneys were homogenized with 9 vol of 15 mM Tris-HCl (pH 7.4) containing 0.19 M sucrose, 0.5 mM EDTA, 1 mM 1,4-dithiothreitol (DTT), 20% glycerol, and 1 μg/ml each of leupeptin and pepstatin. The homogenate was centrifuged at 200g for 1 minute. The precipitate was homogenized again with the same buffer and mixed with the above supernatant. The mixture was centrifuged again at 200g for 10 minutes and the nuclear pellet was washed twice. Washings were added to the supernatant and the mixture was centrifuged at 12,000g for 20 minutes. The mitochondrial pellet was washed once with the same buffer and suspended in 100 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol, 0.5 mM EDTA, 1 mM DTT, and 1 μg/ml each of leupeptin and pepstatin to give a protein concentration of 15 mg/ml.

Solubilization of 25(OH)D3 24-hydroxylase

Ten percent Tween 20 dissolved in the suspension buffer was added to the mitochondrial suspension to give a final Tween 20/protein ratio of 1:1 (wt/wt) under mild stirring on ice in the course of 1 minute, and stirring was continued for 30 minutes longer while cooling. The solubilized suspension was centrifuged at 100,000g for 60 minutes, and the supernatant was used as the enzyme source.

Enzyme assay

A typical incubation mixture contained an appropriate amount of enzyme (about 150 μg of protein), 0.8 nmol of adrenodoxin, 0.02 U of NADPH-adrenodoxin reductase, 0.2 μmol of EDTA, 20 μmol of HEPES (pH 8.0), and 4 nmol of 25(OH)D3 dissolved in 2 μl of ethanol, in a total volume of 0.2 ml. After the mixture was preincubated for 2 minutes at 37°C, incubation was initiated by adding 6 μmol NADPH and then continued for 3 minutes at the same temperature. Reaction was terminated by adding 0.4 ml of 0.25 M NaOH and 0.3 ml of methanol. An incubation mixture was extracted twice with 4 ml of benzene, and the extract was washed with water. The extract was transferred to a new tube and the solvent was evaporated under a stream of nitrogen. The residue was dissolved in 20 μl of chloroform/ethyl acetate (4:1, vol/vol) and an aliquot was subjected to HPLC analysis.

Analysis of the product

When 25(OH)D3 was used as substrate, the products were analyzed by straight-phase HPLC on a silica gel column (Wakosil 5SIL, 4.6 mm × 250 mm) using a solvent system, hexane/isopropanol/methanol, 88/6/6 (vol/vol/vol), at a flow rate of 1.4 ml/minute. Effluents were monitored at 265 nm. When 1α,25(OH)2D3 was used as substrate, another solvent system, dichloromethane/isopropanol, 92/8 (vol/vol) was used. The HPLC was performed using a product of Shimadzu (Kyoto, Japan; model LC-10A) equipped with a UV detector (model SPD-10A; Shimadzu) and an integration system (CLASS-LC10; Shimadzu). For further confirmation of the products, reversed-phase HPLC was performed using an octadecane-coated column (Finepak Sil C18, 4.6 mm × 150 mm; JASCO, Tokyo, Japan), and a solvent system of methanol/water, 80/20 (vol/vol), at a flow rate of 1.0 ml/minute. Product amounts were determined by comparing the peak area with those of known amounts of the authentic samples.

Data processing

A Lineweaver-Burk plot of initial velocities was obtained by the weighted method of Wilkinson.(15)

Other method

Proteins were determined by the method of Lowry et al.(16) using bovine serum albumin (BSA) as a standard. Periodate oxidation of the products was carried out as described by Burgos-Trinidad et al.(17)

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Assay of 24-hydroxylase

When incubation was carried out with the mitochondrial suspension or the Tween 20-treated mitochondria without ultracentrifugation, the specific enzyme activity of the peak at the retention time corresponding to authentic 24,25(OH)2D3 was only 0.95 pmol/minute per mg or 1.6 pmol/minute per mg of protein, respectively ( Figs. 1A and 1B). After ultracentrifugation, a marked increase in enzyme activities was observed; the specific activity was 50-fold higher than that without ultracentrifugation although Tween 20-treated mitochondria were used (Fig. 1C). Similarly, a 62-fold increase of the specific activity was observed when 1α,25(OH)2D3 was used as substrate. To confirm the structure of the reaction product of 25(OH)D3, the following experiments were carried out: (1) the absorption spectrum of the product showed a peak at 265 nm, indicating that the triene structure characteristic of the vitamin D group was intact (data not shown); (2) when the product peak corresponding to authentic 24,25(OH)2D3 in the straight-phase HPLC was isolated and subjected to reversed-phase HPLC, the product gave the same elution time as the 24,25(OH)2D3; and (3) when the product obtained by reversed-phase HPLC was treated with periodate, it could no longer be detected, suggesting that the newly introduced hydroxyl group was vicinal to that in the substrate. When 1α,25(OH)2D3 was used as substrate, the structure of the product was similarly confirmed as 1α,24,25(OH)3D3.

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Figure FIG. 1. HPLC chromatograms of products obtained by incubation of 25(OH)D3 with the kidney mitochondrial preparations of vitamin D-replete rats that were given a single intraperitoneal injection of 1α,25(OH)2D3 (40 ng/100 g body weight) 1 day before the experiment: (A) the mitochondrial suspension, (B) Tween 20-treated mitochondria without ultracentrifugation, and (C) Tween 20-treated mitochondria with ultracentrifugation. The same amounts of proteins were used in each preparation. Other incubation conditions were the same as the standard method. Open arrow indicates the substrate; solid arrow indicates the products. D shows an HPLC of the authentic 24,25(OH)2D3, 10 pmol, with arrow.

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Optimization of assay conditions for 24-hydroxylase

The pH of the incubation mixture:

As shown in Fig. 2 the enzyme activity depended on the kind of buffer, the best being HEPES at varied pH. The highest activity was at pH 8.0. Accordingly, HEPES, pH 8.0, was used as the standard assay.

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Figure FIG. 2. Effect of pH on 24-hydroxylase activity. The enzyme activity was determined as described in Materials and Methods except that pH of the incubation mixture was varied. Potassium phosphate (○), Tris-HCl (▵), and HEPES (•) buffers were used. Values were means of triplicate experiments.

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Incubation temperature:

The incubation was conducted for 3 minutes by changing the temperature from 30°C to 50°C (Fig. 3). The highest enzyme activity was observed at 37°C.

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Figure FIG. 3. Effect of incubation temperature on 24-hydroxylase activity. Incubation was conducted as described in Materials and Methods except that incubation temperature was varied. Values were means of triplicate experiments.

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Incubation time:

As shown in Fig. 4, the amount of the product 24,25(OH)2D3 was proportionate to the incubation period of up to 3 minutes at 37°C. Accordingly, incubation was conducted routinely for 3 minutes at 37°C.

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Figure FIG. 4. Effect of incubation time on the production of 24,25(OH)2D3. Incubation was conducted as described in Materials and Methods except that incubation time was varied at different temperatures: 30°C (□), 37°C (•), and 40°C (▵). Values were means of triplicate experiments.

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Concentrations of the electron transferring proteins:

Figure 5A shows the effect of concentration of adrenodoxin on the reaction velocity at different concentrations of NADPH-adrenodoxin reductase. The rate of product formation increased with higher concentrations of adrenodoxin, and reached maximum velocity with 2 nmol/ml of adrenodoxin at varying concentrations of NADPH-adrenodoxin reductase (0.04–0.2 U/ml). Figure 5B shows the effect of concentration of NADPH-adrenodoxin reductase on the velocity at different concentrations of adrenodoxin. Reaction velocity leveled off at 0.04 U/ml of the reductase. Four nanomoles of adrenodoxin and 0.1 U of NADPH-adrenodoxin reductase per milliliter therefore were adopted for routine assay.

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Figure FIG. 5. Effect of concentration of (A) adrenodoxin and (B) NADPH-adrenodoxin reductase on 24-hydroxylase activity. Incubation was carried out as described in Materials and Methods except that concentrations of adrenodoxin and NADPH-adrenodoxin reductase were varied. Amounts of the reductase used in A were 0 U (○), 0.008 U (•), 0.02 U (▴), and 0.04 U (▵) in a total volume of 0.2 ml. Amounts of adrenodoxin used in B were 0 nmol (○), 0.04 nmol (•), 0.12 nmol (▵), 0.4 nmol (▴), 0.8 nmol (□), and 1.2 nmol (▪).

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Enzyme concentration:

As shown in Fig. 6, the rate of product formation was proportionate to the amount of the enzyme from 0.3 to 1.6 mg of protein per milliliter. Above the amount of 1.6 mg protein per milliliter there was no further linearity in the calibration curve. Therefore, for routine assay, 150 μg of protein/0.2 ml was used.

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Figure FIG. 6. Effect of enzyme concentration on the in vitro production of 24,25(OH)2D3. Incubation was conducted as described in Materials and Methods except that amount of enzyme was varied.

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Substrate concentration:

When the substrate concentration was varied, the reaction followed Michaelis-Menten kinetics. The Michaelis-Menten constant, Km, was calculated from the Lineweaver-Burk plot. The Km values for 25(OH)D3 and 1α,25(OH)2D3 were 0.6 μM and 20.9 μM, respectively (Fig. 7).

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Figure FIG. 7. Effect of substrate concentration on 24-hydroxylase activity toward (A) 25(OH)D3 and (B) 1α,25(OH)2D3. Incubation was conducted as described in Materials and Methods except that the concentration of substrate was varied. The inset shows the Lineweaver-Burk plot. The points are experimental and the line is a weighted least-square fit of the data to hyperbola.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

The fact that 24-hydroxylase activity toward 25(OH)D3 was barely observed in the Tween 20-treated mitochondria that were not ultracentrifuged (even if they were fortified with the electron transferring proteins) indicates there was an inhibitory factor for this enzyme in mitochondria. This could be expected from the report of Botham et al. who postulated that the inhibitory factor for 1α-hydroxylase preferentially binds the substrate 25(OH)D3 and reduces its availability.(11) To our knowledge, however, there are few reports describing the inhibitory factor for 24-hydroxylase.(12) This may be caused by the fact that a residual activity could be observed even in the presence of the inhibitory factor, because 24-hydroxylase activity is very strong.

We established the assay method for 24-hydroxylase by removing the inhibitory factor and optimizing assay conditions as listed here. (1) Solubilization of the enzyme in renal mitochondria was carried out most effectively with Tween 20. Other detergents, cholic acid, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), polyethylene glycol monododecyl ether (Lubrol PX), and n-octyl β-D-glucopyranoside (n-octyl glucoside), also were examined. Tween 20 solubilized the enzyme effectively and did not inhibit enzyme activity, while various degrees of inhibition were observed with other detergents. (2) Solubilized preparation was ultracentrifuged. (3) Incubation was conducted at the optimum pH and incubation temperature. (4) Reaction times were selected to obtain initial velocity. (5) Adrenodoxin and NADPH-adrenodoxin reductase were used instead of kidney ferredoxin and NADPH-ferredoxin reductase, respectively. Thus, removal of the inhibitory factor, optimization of other reaction conditions, and saturation of the enzyme with electron transferring proteins markedly increased the specific activity of 24-hydroxylase about 250-fold higher than the activity reported by Knutson and DeLuca,(18) who used kidney mitochondria as an enzyme source.

This study disclosed interesting properties of 24-hydroxylase as follows. It has been believed up to now that the Michaelis constant of the enzyme is 5- to 30-fold higher for 25(OH)D3 than that for 1α,25(OH)2D3, suggesting that the true substrate is 1α,25(OH)2D3 instead of 25(OH)D3.(8,9) However, the present result shows that the enzyme exhibits much lower Km value (35-fold lower) to 25(OH)D3 than to 1α,25(OH)2D3, indicating that the affinity of the enzyme to 25(OH)D3 is much higher than that to 1α,25(OH)2D3. This finding suggests that 25(OH)D3 is the preferred substrate.

In conclusion, by solubilizing the enzyme of kidney mitochondria, removing the inhibitory factor by ultracentrifugation, and fortifying the solubilized enzyme with the electron transferring proteins, we established a simple, sensitive, and accurate assay method for 25-hydroxyvitamin D3 24-hydroxylase. In employing the new assay method, it was shown that the Michaelis constant of the enzyme for 25(OH)D3 was much lower than that for 1α,25(OH)2D3, indicating the affinity of the enzyme for 25(OH)D3 is much higher than that for 1α,25(OH)2D3.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
  • 1
    McCary LC, DeLuca HF 1999 Functional metabolism and molecular biology of vitamin D action. In: HolickMF (ed.) Vitamin D. Humana Press, Totowa, NJ, USA, pp. 3956.
  • 2
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  • 3
    Ono T, Tanaka H, Yamate T, Nagai Y, Nakamura T, Seino Y 1996 24R,25-dihydroxyvitamin D3 promotes bone formation without causing excessive resorption in hypophosphatemic mice. Endocrinology 137:26332637.
  • 4
    Seo EG, Einhorn TA, Norman AW 1997 24R,25-dihydroxyvitamin D3: An essential vitamin D3 metabolite for both normal bone integrity and healing of tibial fracture in chicks. Endocrinology 138:38643872.
  • 5
    Schwartz Z, Dean DD, Walton JK, Brooks BP, Boyan BD 1995 Treatment of resting zone chondrocytes with 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] induces differentiation into a 1,25-(OH)2D3-responsive phenotype characteristic of growth zone chondrocytes. Endocrinology 136:402411.
  • 6
    Henry HL, Norman AW 1978 Vitamin D: Two dihydroxylated metabolites are required for normal chicken egg hatchability. Science 201:835837.
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    Makin G, Lohnes D, Byford V, Ray R, Jones G 1989 Target cell metabolism of 1,25-dihydroxyvitamin D3 to calcitroic acid. Evidence for a pathway in kidney and bone involving 24-oxidation. Biochem J 262:173180.
  • 8
    Shinki T, Jin CH, Nishimura A, Nagai Y, Ohyama Y, Noshiro M, Okuda K, Suda T 1992 Parathyroid hormone inhibits 25-hydroxyvitamin D3-24-hydroxylase mRNA expression stimulated by 1α,25-dihydroxyvitamin D3 in rat kidney but not in intestine. J Biol Chem 267:1375713762.
  • 9
    Burgos-Trinidad M, DeLuca HF 1991 Kinetic properties of 25-hydroxyvitamin D- and 1,25-dihydroxyvitamin D-24-hydroxylase from chick kidney. Biochim Biophys Acta 1078:226230.
  • 10
    Eto TA, Nakamura Y, Taniguchi T, Miyamoto K, Nagatomo J, Maeda Y, Higashi S, Okuda K, Setoguchi T 1998 Assay of 25-hydroxyvitamin D3 1α-hydroxylase in rat kidney mitochondria. Anal Biochem 258:5358.
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    Tanaka Y, DeLuca HF 1981 Measurement of mammalian 25-hydroxyvitamin D3 24R- and 1α-hydroxylase. Proc Natl Acad Sci USA 78:196199.
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    Suhara K, Takemori S, Katagiri M 1972 Improved purification of bovine adrenal iron-sulfur protein. Biochim Biophys Acta 263:272278.
  • 14
    Suhara K, Ikeda Y, Takemori S, Katagiri M 1972 The purification and properties of NADPH-adrenodoxin reductase from bovine adrenocortical mitochondria. FEBS Lett 28:4547.
  • 15
    Wilkinson GN 1961 Statistical estimation in enzyme kinetics. Biochem J 80:324332.
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    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265275.
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    Burgos-Trinidad M, Brown AJ, DeLuca HF 1986 Solubilization and reconstitution of chick renal mitochondrial 25-hydroxyvitamin D3 24-hydroxylase. Biochemistry 25:26922696.
  • 18
    Knutson JC, DeLuca HF 1974 25-Hydroxyvitamin D3-24-hydroxylase. Subcellular location and properties. Biochemistry 13:15431548.