Solubilization and reconstitution of chick renal mitochondrial 25-hydroxyvitamin D3 24-hydroxylase

Chick kidney mitochondrial 25-hydroxyvitamin D3 24-hydroxylase has been solubilized with sodium cholate and reconstituted with NADPH, beef adrenal ferredoxin, and beef adrenal ferredoxin reductase, each component being essential for maximal 24-hydroxylase activity. The product 24(R),25-dihydroxyvitamin D3 was identified by cochromatography with synthetic compound on straight-phase and reversed-phase high-performance liquid chromatography and by periodate oxidation. The enzyme has an apparent Km for 25-hydroxyvitamin D3 of 0.67 microM. At 1 microM 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3 production is linear with time for up to 15 min and with protein concentrations of up to 2 mg/mL. The antioxidant diphenyl-p-phenylenediamine (1.3 X 10(-4) M) has no effect on this reaction. Reconstituted 24-hydroxylase activity is enhanced by the addition of NaCl and KCl up to 100 mM, with higher concentrations having an inhibitory effect. 1 alpha-Hydroxylase is not present in this preparation from vitamin D replete chicks. The similarities of this reconstituted system to the 25-hydroxyvitamin D3 1 alpha-hydroxylase and the adrenal systems suggest that the 25-hydroxyvitamin D3 24-hydroxylase is also a cytochrome P-450 type mixed-function oxidase.     

Isolation and characterization of a cytochrome P-450 from rat kidney mitochondria that catalyzes the 24-hydroxylation of 25-hydroxyvitamin D3

A cytochrome P-450 that catalyzes the 24-hydroxylation of 25-hydroxyvitamin D3 (P-450cc24: P-450cholecalciferol24) was purified to electrophoretic homogeneity from the kidney mitochondria of female rats treated with vitamin D3 (Ohyama, Y., Hayashi, S., and Okuda, K. (1989) FEBS Lett. 255, 405-408). The molecular weight was 53,000, and its absorption spectrum showed peaks characteristic of cytochrome P-450. The turnover number was 22 min-1 and the specific content was 2.8 nmol/mg protein. The N-terminal amino acid sequence, Arg-Ala-Pro-Lys-Glu-Val-Pro-Leu-, is different from the N-terminal sequence of any other cytochrome P-450s so far reported. Upon reconstitution with the electron-transferring system of the adrenal mitochondria, the enzyme showed a high activity in hydroxylating 25-hydroxyvitamin D3 as well as 1 alpha,25-dihydroxyvitamin D3 at position 24. However, the purified enzyme hydroxylated neither vitamin D3 nor 1 alpha-hydroxyvitamin D3. The enzyme was also inactive toward xenobiotics. The enzyme hydroxylated 25-hydroxyvitamin D3 at position 24 but not at 1 alpha, indicating that the enzyme is distinct from that catalyzing 1 alpha-hydroxylation. The reaction followed Michaelis-Menten kinetics, and the Km value for 25-hydroxyvitamin D3 was 2.8 microM. Both vitamin D3 and 1 alpha-hydroxyvitamin D3 inhibited the 24-hydroxylation of 25-hydroxyvitamin D3 in a competitive, concentration-dependent manner. 25-Hydroxyvitamin D3 24-hydroxylase activity was significantly inhibited by 7,8-benzoflavone, ketoconazole, and CO, whereas it was only slightly inhibited by aminoglutethimide, metyrapone, and SKF-525A. Mouse antibodies raised against the cytochrome P-450 inhibited the reaction about 70% and reacted with the P-450cc24 in immunoblotting but did not react with other kinds of cytochrome P-450 in rat liver microsomes and mitochondria.     

Characterization of recombinant CYP2C11: a vitamin D 25-hydroxylase and 24-hydroxylase

Studies were performed to further characterize the male-specific hepatic recombinant microsomal vitamin D 25-hydroxlase CYP2C11, expressed in baculovirus-infected insect cells, and determine whether it is also a vitamin D 24-hydroxylase. 25- and 24-hydroxylase activities were compared with those of 10 other recombinant hepatic microsomal cytochrome P-450 enzymes expressed in baculovirus-infected insect cells. Each of them 25-hydroxylated vitamin D2, vitamin D3, 1alpha-hydroxyvitamin D2 (1alphaOHD2), and 1alpha-hydroxyvitamin D3 (1alphaOHD3). CYP2C11 had the greatest activity with these substrates, except vitamin D3, which had the same activity as four of the other enzymes. The descending order of 25-hydroxylation by CYP2C11 was 1alphaOHD3 > 1alphaOHD2 > vitamin D2 > vitamin D3. Each of the recombinant cytochrome P-450 enzymes 24-hydroxylated 1alphaOHD2. CYP2C11 had the greatest activity. 24-Hydroxylation of 1alphaOHD3 was very low, and there was none with vitamin D3. Only CYP2C11 24-hydroxylated vitamin D2. Structures of vitamin D metabolites, including 24-hydroxyvitamin D2, 1,24(S)-dihydroxyvitamin D2, and 1,24-dihydroxyvitamin D3, were confirmed by HPLC and gas chromatography retention times and characteristic mass spectrometric fragmentation patterns. In male rats, hypophysectomy significantly reduced body weight, liver weight, hepatic CYP2C11 mRNA expression, and 24- and 25-hydroxylation of 1alphaOHD2. Expression of CYP2J3 and CYP2R1 mRNA did not change. In male rat hepatocytes, CYP2C11 mRNA expression and 24- and 25-hydroxylation were significantly reduced after culture for 24 h compared with uncultured cells. Expression of CYP2J3 and CYP2R1 either increased or did not change. It is concluded that CYP2C11 is a male-specific hepatic microsomal vitamin D 25-hydroxylase that hydroxylates vitamin D2, vitamin D3, 1alphaOHD2, and 1alphaOHD3. CYP2C11 is also a vitamin D 24-hydroxylase.     

Kinetic properties of 25-hydroxyvitamin D- and 1,25-dihydroxyvitamin D-24-hydroxylase from chick kidney

1,25-Dihydroxyvitamin D3 induces both 25-hydroxyvitamin D3- and 1,25-dihydroxyvitamin D3- 24-hydroxylase activities. However, whether 24-hydroxylation of these substrates is catalyzed by a single enzyme is unknown. We have examined the substrate specificity of the enzyme using the solubilized and reconstituted chick renal mitochondrial 24-hydroxylase enzyme system. The soluble enzyme catalyzes 24-hydroxylation of both substrates. The apparent Km of the 24-hydroxylase for 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 were 1.47 and 0.14 microM, respectively. Kinetic studies demonstrated that 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 act as competitive inhibitors with respect to each other. 1,25-Dihydroxyvitamin D3 inhibited the production of 24,25-dihydroxyvitamin D3 with an apparent Ki of 0.09 microM and 25-hydroxyvitamin D3 inhibited the production of 1,24,25-trihydroxyvitamin D3 with an apparent Ki of 3.9 microM. These results indicate that chick 24-hydroxylase preferentially hydroxylates 1,25-dihydroxyvitamin D3 and support the idea that the 24-hydroxylation of these substrates is catalyzed by a single enzyme.     

Immunopurified 25-hydroxyvitamin D 1 alpha-hydroxylase and 1,25-dihydroxyvitamin D 24-hydroxylase are closely related but distinct enzymes.

The chick kidney mitochondrial cytochrome P-450 1,25-dihydroxyvitamin D3 24-hydroxylase was partially purified by sequential polyethylene glycol precipitation, aminohexyl-Sepharose 4B, and hydroxylapatite chromatography. The specific activity of the final preparation, when reconstituted with NADPH, adrenodoxin, and adrenodoxin reductase, was 245 pmol/min/mg of protein or 0.56 pmol/min/pmol of P-450. The specific cytochrome P-450 content was 0.45-0.73 nmol/mg of protein. BALB/c mice immunized with this preparation developed serum polyclonal antibodies to the 24-hydroxylase, as demonstrated by immunoprecipitation. Splenic lymphocytes from an immunized mouse were fused with myeloma NSI/1-Ag-4-1 cells, and hybridomas secreting monoclonal antibodies to the 24-hydroxylase were detected by immunoprecipitation. The hybridoma lines were cloned by limiting dilution and further characterized as IgG1, IgG3, and IgM subclasses. In one-dimensional immunoblots of soluble 24-hydroxylase preparations, the monoclonal antibodies revealed a single band with an apparent molecular weight of 59,000. The monoclonal antibodies did not cross-react with cytochrome P-450s from other species but immunoprecipitated and immunoblotted a soluble chick renal mitochondrial 25-hydroxyvitamin D3 1 alpha-hydroxylase preparation, demonstrating the close similarity of these two hydroxylases. These antibodies were coupled to Sepharose CL-4B and used to isolate to homogeneity the two enzymes from chick kidney mitochondria. Amino-terminal sequences and amino acid composition data demonstrate that these enzymes are different but homologous.     

Characteristics of the 25-hydroxyvitamin D3- and 1,25-dihydroxyvitamin D3-24-hydroxylase(s) from HL-60 cells.

1,25-Dihydroxyvitamin D3 induces the human promyelocyte leukemia cell line, HL-60, to differentiate into macrophages/monocytes via a steroid-receptor mechanism. This system is a relevant one for an investigation of the molecular mechanism of 1,25-dihydroxyvitamin D3. We have now examined the effect of 1,25-dihydroxyvitamin D3 on the induction of 1,25-dihydroxyvitamin D3- and 25-hydroxyvitamin D3-24-hydroxylase activities in HL-60 cells. The hydroxylase activities were measured by a periodate-based assay, which was validated by comparison with well-established HPLC analysis. HPLC analysis also suggested that 1,25-dihydroxyvitamin D3 induces a 23-hydroxylase in addition to the 24-hydroxylase. 1,25-Dihydroxyvitamin D3- and 25-hydroxyvitamin D3-24-hydroxylase activities were stimulated as early as 4 h after the addition of 10(-7) M 1,25-dihydroxyvitamin D3 and became maximal by 24 h. 1,25-Dihydroxyvitamin D3 stimulated both activities in a dose-dependent manner up to 10(-6) M. The Km of 24-hydroxylase for 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were 2 x 10(-8) M and 5.2 x 10(-7) M, respectively. Cycloheximide (5 microM) inhibited 1,25-dihydroxyvitamin D3-mediated stimulation of 24-hydroxylase activity. Other differentiation inducers, such as retinoic acid and phorbol ester, did not induce either activity. 1,25-Dihydroxyvitamin D3-24-hydroxylase in HL-60 mitochondria was solubilized with 0.6% cholate and reconstituted with NADPH, beef adrenal ferredoxin, and beef adrenal ferredoxin reductase, each component being essential for 24-hydroxylase activity. These results strongly suggest that the 24-hydroxylase in HL-60 cells is a three-component cytochrome P450-dependent mixed-function oxidase.     

Separation of the cytochromes P-450 in pig kidney mitochondria catalyzing 1 alpha-, 24- and 26-hydroxylations of 25-hydroxyvitamin D3

The cytochromes P-450 in pig kidney mitochondria catalyzing 1 alpha-, 24- and 26-hydroxylations of 25-hydroxyvitamin D3 have been separated. The cytochrome P-450 fractions required NADPH, mitochondrial ferredoxin and ferredoxin reductase for catalytic activity. The present report demonstrates that different forms of cytochrome P-450 are involved in 1 alpha-, 24- and 26-hydroxylations of 25-hydroxyvitamin D3 and provides a basis for further purification and characterization of these enzymes.     

Purification of a cytochrome P-450 from pig kidney microsomes catalysing the 25-hydroxylation of vitamin D3.

Cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from pig kidney microsomes. The enzyme fraction contained 7 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 50,500 upon SDS/polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 1,000 times more efficiently, and 25-hydroxylation of 1 alpha-hydroxyvitamin D3 up to 4000 times more efficiently, than the microsomes. The cytochrome P-450 required microsomal NADPH-cytochrome P-450 reductase for catalytic activity. Mitochondrial ferredoxin and ferredoxin reductase could not replace microsomal NADPH-cytochrome P-450 reductase. The enzyme preparation showed no detectable 25-hydroxylase activity towards vitamin D2 or 1 alpha-hydroxylase activity towards 25-hydroxyvitamin D3. CO inhibited the 25-hydroxylation by more than 85%. Mannitol, hydroquinone, catalase and superoxide dismutase did not affect the 25-hydroxylation. The possible role of the kidney microsomal cytochrome P-450 in the metabolism of vitamin D3 is discussed.     

Is steroid-16α-hydroxylase supported by more than one monooxygenase?

Steroid-16α-hydroxylase activities have been measured in normal and induced rat livers using four different substrates. The male/female activity ratio as well as the induction factor vary with the substrate indicating that steroid-16α-hydroxylase activity is a heterogenous enzyme. Experiments using specific inhibitors led to the conclusion that steroid-16α-hydroxylase is supported by at least two cytochrome P-450 forms, different from the cytochrome P-448.     

25-Hydroxyvitamin D3-24-hydroxylase in rat kidney mitochondria

Assay conditions for the measurement of 25-hydroxyvitamin D3-24-hydroxylase activity in rat kidney mitochondria have been worked out. The product, 24,25-dihydroxyvitamin D3 was quantitated either by high pressure liquid chromatography or by isotope dilution-mass spectrometry. By these procedures, the enzyme activity could be measured with saturating concentration (greater than 2.5 X 10(-6) M) of substrate. Pretreatment of the animals by aminophylline (Kulkowski, J. A., Chow, T., Martinez, J., and Ghazarian, J. G. (1979) Biochem. Biophys. Res. Commun. 90, 50-57) stimulated the 24-hydroxylase activity in vitro at least 2 to 3-fold. The identity of the product was verified by gas chromatography-mass spectrometry. The rates of the reaction varied between 1.5 and 5 pmol/mg of mitochondrial protein.min (at 25 degrees C), and the K'm was determined to be 4.2 X 10(-7) M. Malate, succinate, and isocitrate were all able to support the reaction. Low O2 tension, CO, KCN, and the uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited the reaction, while the respiratory inhibitor rotenone had no effect. Metyrapone inhibited the reaction with 50% inhibition at a concentration of 2.5 mumol/ml. The enzyme was found to be localized inside the inner mitochondrial membrane. The results indicate that in the rat the renal mitochondrial 25-hydroxyvitamin D3-24-hydroxylase is a cytochrome P-450 and that the reducing equivalents are primarily supplied by NADPH via the energy-dependent transhydrogenase.     

Effects of two oral antimycotics, ketoconazole and fluconazole, upon steroidogenesis in rat adrenal cells in vitro

Rat adrenal cells were incubated with various concentrations of two orally active azole antimycotics in order to evaluate the effects on steroidogenesis. The first compound was ketoconazole, a well-known inhibitor not only of fungal cytochrome P-450 but at higher concentrations also of mammalian cytochrome P-450 dependent enzymes. The second was fluconazole, a newly developed oral antimycotic with a triazole structure, which likewise inhibits fungal cytochrome P-450. The influence of both drugs on mammalian cytochrome P-450 dependent enzymes was investigated in this study. Ketoconazole inhibited ACTH-stimulated corticosterone (IC50 = 0.9 microM) and aldosterone secretion (IC50 = 1.4 microM) and enhanced 11-deoxycorticosterone output at low concentrations but reduced it at higher concentrations. Radiotracer experiments with [3H]pregnenolone or [3H]11-deoxycorticosterone as exogenous substrates revealed a 50% inhibition of the oxidative substrate metabolism at about 1 microM ketoconazole. These effects could also be observed with fluconazole but occurred at concentrations approximately two orders of magnitude higher as compared to ketoconazole. We conclude that fluconazole has a much higher selectivity for fungal cytochrome P-450 than ketoconazole. The order of sensitivity of the cytochrome P-450 dependent enzymes of rat adrenal steroidogenesis to ketoconazole was the 11 beta/18-hydroxylase, the cholesterol side chain cleavage enzyme and the 21-hydroxylase with decreasing sensitivities.     

Expression of a rat liver microsomal cytochrome P-450 catalyzing testosterone 16 alpha-hydroxylation in Saccharomyces cerevisiae: vitamin D3 25-hydroxylase and testosterone 16 alpha-hydroxylase are distinct forms of cytochrome P-450

Rat cytochrome P-450(M-1) cDNA was expressed in Saccharomyces cerevisiae TD1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450(M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator. The yeast cells synthesized up to 2 X 10(5) molecules of P-450(M-1) per cell. The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein. The expressed cytochrome P-450 catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone in accordance with the catalytic activity of P-450(M-1), but did not hydroxylate vitamin D3 or 1 alpha-hydroxycholecalciferol at the 25 position. The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3. These results indicated that P-450(M-1) cDNA coded the 2 alpha- and 16 alpha-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450. Vitamin D3 25-hydroxylase and testosterone 16 alpha- and 2 alpha-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.     

Reciprocal post-translational regulation of renal 1 alpha- and 24-hydroxylases of 25-hydroxyvitamin D3 by phosphorylation of ferredoxin. mRNA-directed cell-free synthesis and immunoisolation of ferredoxin.

We have used a cell-free rabbit reticulocyte translational system programmed with polyadenylated [poly(A)+] RNA prepared from chick kidney tissue to study the synthesis of nascent ferredoxin, a class of iron-sulphur-containing proteins functional in the renal mitochondrial 1 alpha- and 24-hydroxylases of 25-hydroxyvitamin D3. The synthesis of ferredoxin was monitored by determining [35S]methionine incorporation into ferredoxin and quantified by SDS/PAGE and autoradiography after immunoprecipitation from the total translation products. Compared with normal controls, vitamin D deprivation caused a significant increase in the net synthesis of nascent ferredoxin with an Mr of 12,000-13,000. [3H]Orotate incorporation as uridine into kidney poly(A)+ RNA was stimulated by aminophylline, a potent inducer of 25-hydroxyvitamin D3 24-hydroxylase; however, the amount of nascent ferredoxin synthesis was the same as in normal controls. Also, partially purified chick kidney mitochondrial cyclic AMP-stimulated protein kinase catalysed the phosphorylation of ferredoxin in vitro. The catalytic activity of the ferredoxin in 1 alpha- and 24-hydroxylations of 25-hydroxyvitamin D3 in reconstituted systems consisting of cytochrome P-450 and ferredoxin reductase was altered with ferredoxin phosphorylation. The phosphorylation caused inhibition of the 1 alpha-hydroxylase activity while at the same time it stimulated the 24-hydroxylase. Authentic 1 alpha,25- and 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were used as standards to monitor the separation of the enzymic products by h.p.l.c. using methanol/water (4:1, v/v) as solvent. These results indicate that, in the absence of vitamin D or its metabolites in the deficient state, the synthesis of ferredoxin necessary for the 1 alpha-hydroxylase is accentuated, whereas the stimulation of the 24-hydroxylase requires the phosphorylation of existing ferredoxin without a net gain in its synthesis. This would suggest a post-translational regulation of the 1 alpha- and 24-hydroxylases. A model delineating the various aspects of this study is presented.     

Evidence for the formation of 26-hydroxycholesterol by cytochrome P-450 in pig kidney mitochondria

Pig kidney mitochondria were found to catalyze the formation of 26-hydroxycholesterol, an inhibitor of cholesterol biosynthesis. The cholesterol 26-hydroxylase was purified 600-fold. It was present in a mitochondrial enzyme fraction enriched in cytochrome P-450. The cytochrome P-450 fraction required NADPH, mitochondrial ferredoxin and ferredoxin reductase for 26-hydroxylase activity. The mitochondria and the purified 26-hydroxylase preparation also catalyzed 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, and intermediate in cholic acid biosynthesis, and of 25-hydroxyvitamin D3. The role of extra-hepatic formation of 26-hydroxycholesterol is discussed.     

Assay and properties of 25-hydroxyvitamin D3 23-hydroxylase. Evidence that 23,25-dihydroxyvitamin D3 is a major metabolite in 1,25-dihydroxyvitamin D3-treated or fasted guinea pigs.

Incubation of 25-hydroxyvitamin D3 with kidney cortex mitochondria from 1,25-dihydroxyvitamin D3-treated guinea pigs resulted in the formation of 23,25-dihydroxyvitamin D3 as the major product. The identity of the product was verified by g.c.-m.s. and quantification was performed by h.p.l.c. The rates of the reaction were in the range 1.0-1.8 pmol/min per mg of mitochondrial protein (at 37 degrees C), which were 5-10 times the rates of formation of 24,25-dihydroxyvitamin D3. In mitochondrial preparations from untreated guinea pigs, the rate of 23-hydroxylation was below detection limit (0.02 pmol/min per mg of mitochondrial protein). Fasting the animals for 24 h induced the 23-hydroxylase almost as efficiently as treatment with 1,25-dihydroxyvitamin D3, with a concomitant depression of the 1 alpha-hydroxylase. The 23-hydroxylase reaction required oxidizable substrate, was decreased by low O2 partial pressures and inhibited by CO or the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It was stimulated by the respiratory-chain inhibitors rotenone, antimycin A and KCN. These results indicate that the guinea-pig renal mitochondrial 23-hydroxylase is a cytochrome P-450 and that the reducing equivalents are primarily supplied by NADPH via the energy-dependent transhydrogenase.     

Oxidative stress limits vitamin D metabolism by bovine proximal tubule cells in vitro

When bovine proximal tubule cells are placed in primary culture, they are subject to elevated oxidative stress which acts to limit the expression of mitochondrial vitamin D3 1 alpha- and 24-hydroxylase activities. This increased oxidative stress was demonstrated by increased production of cell and mitochondrial membrane lipid hyperperoxides (LOOH). This increased production was prevented by the addition of the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Cell and mitochondrial membrane LOOH increased from 1 to 2 pmol/mg protein on the day of plating to 70-90 pmol/mg protein after 6 days in culture. Pretreatment of cultures with BHA and BHT resulted in membrane LOOH of 15-20 pmol/mg protein after 6 days. Mitochondrial LOOH production was greater than total cell LOOH after 6 days. The increase in cellular oxidative stress was paralleled by decreases in both 1 alpha- and 24-hydroxylase activities toward 25-OH D3. Mitochondrial hydroxylase activities were inversely proportional to the increase in mitochondrial membrane LOOH production. Mitochondrial cytochrome P-450 content, determined spectrophotometrically, was decreased over time in culture. Mitochondrial cytochrome P-450 content determined by a specific polyclonal antibody in an enzyme-linked immunosorbant assay also decreased over time in culture. Specificity of polyclonal antibodies, raised against rat liver microsomal cytochrome P-450 RLM5, was demonstrated by the immunosequestration of both 1 alpha- and 24-hydroxylase activities from a partially purified preparation of renal mitochondrial cytochrome P-450. BHA showed the loss of 1 alpha- and 24-hydroxylase activities and mitochondrial P-450 content measured by all criteria. These experiments indicate that oxidative stress-mediated changes in hydroxylase activities are mediated directly by changes in hydroxylase content and not at distal sites. A partially purified preparation of bovine proximal tubule mitochondrial cytochrome P-450, with purified renal ferredoxin, ferredoxin reductase, and NADPH, expressed both 1 alpha- and 24-hydroxylase activities toward 25-OH D3. LOOH, derived from mitochondrial membranes of 5-day-old cultures, when added to this mixture, caused a dose-dependent decrease in both activities. These experiments suggested that an increase in mitochondrial LOOH production resulted in a loss of 1 alpha- and 24-hydroxylase activities. 1 alpha-Hydroxylase was more sensitive to the effects of LOOH treatment than 24-hydroxylase. At a ratio of LOOH:P-450 of 5:1 (molar), all 1 alpha-hydroxylase activity was lost but 50% of the 24-hydroxylase activity remained.(ABSTRACT TRUNCATED AT 400 WORDS)     

25-Hydroxyvitamin D-24-hydroxylase (CYP24A1): its important role in the degradation of vitamin D

CYP24A1 is the cytochrome P450 component of the 25-hydroxyvitamin D(3)-24-hydroxylase enzyme that catalyzes the conversion of 25-hydroxyvitamin D(3) (25-OH-D(3)) and 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) into 24-hydroxylated products, which constitute the degradation of the vitamin D molecule. This review focuses on recent data in the CYP24A1 field, including biochemical, physiological and clinical developments. Notable among these are: the first crystal structure for rat CYP24A1; mutagenesis studies which change the regioselectivity of the enzyme; and the finding that natural inactivating mutations of CYP24A1 cause the genetic disease idiopathic infantile hypercalcemia (IIH). The review also discusses the emerging correlation between rising serum phosphate/FGF-23 levels and increased CYP24A1 expression in chronic kidney disease, which in turn underlies accelerated degradation of both serum 25-OH-D(3) and 1,25-(OH)(2)D(3) in this condition. This review concludes by evaluating the potential clinical utility of blocking this enzyme with CYP24A1 inhibitors in various disease states.     

Development of vitamin D3 25-hydroxylase activity in rat liver microsomes

The ontogeny of vitamin D3 25-hydroxylase activity has been determined in liver microsomes of rat fetuses and neonates. Production of 25-hydroxyvitamin D3 was low (0.11 pmol/g liver/h) 3 days prior to birth. Production rates were 1.2, 2.2, 1.8, and 2.8 pmol/g liver/h on Day 0, Day 2, Day 7, and Day 15, respectively. 25-Hydroxyvitamin D3 production in neonates increased sixfold from Day 15 to Day 22 to a value twice that of the mothers (17.6 pmol/g liver/h compared with 7.3 pmol/g liver/h). Activity in the maternal microsomes was constant (0.22 to 0.30 pmol/mg protein/h) except for the day of parturition (0.54 pmol/mg protein/h) and Day 22 postpartum (0.44 pmol/mg protein/h). A cytosolic factor, present as early as 3 days prior to birth, was required for vitamin D3 25-hydroxylase activity in the fetuses and stimulated the 25-hydroxylase reaction (up to 2.5-fold) in neonates and mothers. The ability of cytosol to prevent degradation of vitamin D3 was also present in the fetal stage. These data suggest that microsomal vitamin D3 25-hydroxylase activity in rat liver microsomes develops slowly and reaches full activity near the weaning stage. Since the cytosolic factor(s) is/are present in the fetal stage, the limiting component in the maturation of vitamin D3 25-hydroxylase activity in liver microsomes is the development of the cytochrome P-450 vitamin D3 25-hydroxylase.     

Androgen enhances the antiproliferative activity of vitamin D3 by suppressing 24-hydroxylase expression in LNCaP cells

25-Hydroxyvitamin D3-24-hydroxylase (24-hydroxylase, CYP24) is an important inactivating enzyme controlling the concentrations of both active metabolites 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3. In this paper, we demonstrate that 25-hydroxyvitamin D3 at 500 nM significantly increases the expression of 24-hydroxylase mRNA and the increase is significantly decreased by 5alpha-dihydrotestosterone (DHT) at concentrations of 1-100 nM in androgen-sensitive prostate cancer cells LNCaP. 25-Hydroxyvitamin D3 at 500 nM and 1alpha,25-dihydroxyvitamin D3 at 10 nM inhibit LNCaP cell growth, and the growth inhibition is enhanced by 1 nM DHT. Neither 25-hydroxyvitamin D3 nor 1alpha,25-dihydroxyvitamin D3 at physiological concentrations has growth effect. However, in the presence of 1 nM DHT, both 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 at physiological concentrations are clearly antiproliferative. These data demonstrate that DHT enhances the antiproliferative activity of Vitamin D3 hormones by inhibiting their inactivating enzyme. Most previous studies on Vitamin D3 action in cell cultures have used pharmacological concentrations of 1alpha,25-dihydroxyvitamin D3, the present results demonstrate, for the first time, that both 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 at physiological concentrations are active in the presence of physiological concentration of androgen. The combined use of androgen and Vitamin D3 metabolites could be a promising treatment for prostate cancer.