De-orphanization of Cytochrome P450 2R1

The conversion of vitamin D into an active ligand for the vitamin D receptor requires 25-hydroxylation in the liver and 1α-hydroxylation in the kidney. Mitochondrial and microsomal vitamin D 25-hydroxylase enzymes catalyze the first reaction. The mitochondrial activity is associated with sterol 27-hydroxylase, a cytochrome P450 (CYP27A1); however, the identity of the microsomal enzyme has remained elusive. A cDNA library prepared from hepatic mRNA of sterol 27-hydroxylase-deficient mice was screened with a ligand activation assay to identify an evolutionarily conserved microsomal cytochrome P450 (CYP2R1) with vitamin D 25-hydroxylase activity. Expression of CYP2R1 in cells led to the transcriptional activation of the vitamin D receptor when either vitamin D2 or D3 was added to the medium. Thin layer chromatography and radioimmunoassays indicated that the secosteroid product of CYP2R1 was 25-hydroxyvitamin D3. Co-expression of CYP2R1 with vitamin D 1α-hydroxylase (CYP27B1) elicited additive activation of vitamin D3, whereas co-expression with vitamin D 24-hydroxylase (CYP24A1) caused inactivation. CYP2R1 mRNA is abundant in the liver and testis, and present at lower levels in other tissues. The data suggest that CYP2R1 is a strong candidate for the microsomal vitamin D 25-hydroxylase. The conversion of vitamin D into an active ligand for the vitamin D receptor requires 25-hydroxylation in the liver and 1α-hydroxylation in the kidney. Mitochondrial and microsomal vitamin D 25-hydroxylase enzymes catalyze the first reaction. The mitochondrial activity is associated with sterol 27-hydroxylase, a cytochrome P450 (CYP27A1); however, the identity of the microsomal enzyme has remained elusive. A cDNA library prepared from hepatic mRNA of sterol 27-hydroxylase-deficient mice was screened with a ligand activation assay to identify an evolutionarily conserved microsomal cytochrome P450 (CYP2R1) with vitamin D 25-hydroxylase activity. Expression of CYP2R1 in cells led to the transcriptional activation of the vitamin D receptor when either vitamin D2 or D3 was added to the medium. Thin layer chromatography and radioimmunoassays indicated that the secosteroid product of CYP2R1 was 25-hydroxyvitamin D3. Co-expression of CYP2R1 with vitamin D 1α-hydroxylase (CYP27B1) elicited additive activation of vitamin D3, whereas co-expression with vitamin D 24-hydroxylase (CYP24A1) caused inactivation. CYP2R1 mRNA is abundant in the liver and testis, and present at lower levels in other tissues. The data suggest that CYP2R1 is a strong candidate for the microsomal vitamin D 25-hydroxylase. Vitamin D regulates calcium and phosphate metabolism by activating the vitamin D receptor, a transcription factor and member of the nuclear receptor family. In the 1920s, McCollum, Mellanby, and Pappenheimer (see Ref. 1Simoni R.D. Hill R.L. Vaughan M. J. Biol. Chem. 2002; 277: 9623Abstract Full Text Full Text PDF Google Scholar) showed that deficiency of vitamin D caused rickets in experimental animals. Subsequently, the structure of vitamin D and its origins from plant and animal steroids were determined, and the roles of the vitamin in the mobilization of minerals from the diet and in bone were defined. The liver was shown to be required for the activation of vitamin D by 25-hydroxylation (2Ponchon G. DeLuca H.F. J. Clin. Invest. 1969; 48: 1273-1279Crossref PubMed Scopus (203) Google Scholar, 3Ponchon G. Kennan A.L. DeLuca H.F. J. Clin. Invest. 1969; 48: 2032-2037Crossref PubMed Scopus (242) Google Scholar). Although 25-hydroxyvitamin D was more active than vitamin D in many bioassays (4Blunt J.W. DeLuca H.F. Schnoes H.K. Biochemistry. 1968; 7: 3317-3322Crossref PubMed Scopus (405) Google Scholar), the most potent hormone was 1α,25-dihydroxyvitamin D (5Haussler M.R. Myrtle J.F. Norman A.W. J. Biol. Chem. 1968; 243: 4055-4064Abstract Full Text PDF PubMed Google Scholar, 6Holick M.F. Schnoes H.K. DeLuca H.F. Suda T. Cousins R.J. Biochemistry. 1971; 10: 2799-2804Crossref PubMed Scopus (372) Google Scholar), which was synthesized from 25-hydroxyvitamin D in the kidney (7Fraser D.R. Kodicek E. Nature. 1970; 228: 764-766Crossref PubMed Scopus (924) Google Scholar). The molecular mechanism of vitamin D action was manifest with the identification (8Haussler M.R. Norman A.W. Proc. Natl. Acad. Sci. U. S. A. 1969; 62: 155-162Crossref PubMed Scopus (175) Google Scholar) and cDNA cloning (9McDonnell D.P. Mangelsdorf D.J. Pike J.W. Haussler M.R. O'Malley B.W. Science. 1987; 235: 1214-1217Crossref PubMed Scopus (420) Google Scholar) of the vitamin D receptor. Hydroxylation reactions catalyzed by cytochrome P450s (CYP) 1The abbreviations used are: CYP, cytochrome P450; Adx, adrenodoxin; h, human; m, mouse; GAL4, galactose 4; VDR, vitamin D receptor; VDRE, vitamin D response element; TK, thymidine kinase; HEK, human embryonic kidney; C T, threshold value; CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus.1The abbreviations used are: CYP, cytochrome P450; Adx, adrenodoxin; h, human; m, mouse; GAL4, galactose 4; VDR, vitamin D receptor; VDRE, vitamin D response element; TK, thymidine kinase; HEK, human embryonic kidney; C T, threshold value; CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus. activate and inactivate vitamin D as a ligand for the receptor. 25-Hydroxylation is performed in the liver by two different enzymes, one located in the mitochondria (10Bjorkhem I. Holmberg I. J. Biol. Chem. 1978; 253: 842-849Abstract Full Text PDF PubMed Google Scholar), identified as the CYP27A1 sterol 27-hydroxylase (11Guo Y.-D. Strugnell S.A. Back D.W. Jones G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8668-8672Crossref PubMed Scopus (141) Google Scholar, 12Dahlback H. Wikvall K. Biochem. J. 1988; 252: 207-213Crossref PubMed Scopus (53) Google Scholar, 13Masumoto O. Ohyama Y. Okuda K. J. Biol. Chem. 1988; 263: 256-260Abstract Full Text PDF Google Scholar, 14Su P. Rennert H. Shayiq R.M. Yamamoto R. Zheng Y. Addya S. Strauss III, J.F. Avadhani N.G. DNA Cell Biol. 1990; 9: 657-665Crossref PubMed Scopus (107) Google Scholar, 15Usui E. Noshiro M. Okuda K. FEBS Lett. 1990; 262: 135-138Crossref PubMed Scopus (110) Google Scholar), and a second in microsomes (16Bhattacharyya M.H. DeLuca H.F. Arch. Biochem. Biophys. 1974; 160: 58-62Crossref PubMed Scopus (127) Google Scholar, 17Madhok T.C. DeLuca H.F. Biochem. J. 1979; 184: 491-499Crossref PubMed Scopus (85) Google Scholar), which has not been identified in most species. A second mitochondrial P450 (CYP27B1), for which an encoding cDNA was isolated by expression cloning (18Takeyama K. S. T. M. J. S. Science. 277: PubMed Scopus Google Scholar), 1α-hydroxylation of 25-hydroxyvitamin D in the and a mitochondrial vitamin D 24-hydroxylase the vitamin in Y. Noshiro M. Okuda K. FEBS Lett. PubMed Scopus Google Scholar, DeLuca H.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar, S. T. O. E. K. Y. T. Biophys. PubMed Scopus Google Scholar). enzymes the and levels of vitamin D in by the vitamin D receptor G. Strugnell S.A. DeLuca H.F. PubMed Scopus Google Scholar). The and of the two hepatic vitamin D 25-hydroxylase enzymes in U. D.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar, I. E. A.L. The and of Scholar) and mice H. A. A. S. G. I. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. D.W. J. Biol. Chem. Google Scholar) by in the mitochondrial CYP27A1 vitamin D 25-hydroxylase of enzyme has as CYP27A1 an in In vitamin D metabolism is in and mice with that the microsomal vitamin D 25-hydroxylase for of the mitochondrial activity. In the a cDNA library from hepatic mRNA of mice in the encoding the mitochondrial CYP27A1 enzyme was screened a vitamin D ligand activation assay (18Takeyama K. S. T. M. J. S. Science. 277: PubMed Scopus Google Scholar). A cDNA a microsomal P450 enzyme CYP2R1 with was The and of CYP2R1 with enzyme the microsomal vitamin D 25-hydroxylase. Expression cDNA of was by the from hepatic the and The DNA was with the enzymes and and into The expression the enzyme activity in as by the of the to the vitamin D 25-hydroxylase activity of sterol A vitamin D 24-hydroxylase cDNA of was by from kidney The for cDNA was isolated from a with of 1α,25-dihydroxyvitamin of for to for the and The DNA was with the enzymes and and into the cytochrome P450 of and cytochrome P450 were by from liver cDNA for the cDNA and The DNA was with the enzymes and and into the The expression enzyme activity in human embryonic kidney as by layer for the cDNA and The DNA was with the enzymes and and into the A DNA the of the of was from and The DNA product was with the enzymes and and into the K. R.M. Mangelsdorf D.J. 9: PubMed Scopus Google Scholar). A 25-hydroxyvitamin D3 1α-hydroxylase cDNA was a of of The sterol 27-hydroxylase cDNA used in was J. D.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Expression cDNA library was from of hepatic isolated from mice in sterol 27-hydroxylase H. A. A. S. G. I. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) the for cDNA cDNA was by the cDNA were to with the enzyme by and into and in the were with and in of cells were with of the The was into of with of the was added to of a and with for DNA was isolated from the the cells were with of the The cells were in at in an of in Dulbecco's modified Eagle's with and of an expression cloning were in in a of of with cells were with a and of DNA of M. K. J. Mangelsdorf D.J. Full Text Full Text PDF PubMed Scopus Google Scholar), of K. R.M. Mangelsdorf D.J. 9: PubMed Scopus Google Scholar), of (see of R.J. T. Biochem. 1987; PubMed Scopus Google Scholar), of and of cDNA were to the for h, and or D3 was added to a of an of cells were by the of CHAPS, and The were for at and of from were to a assay of and was added to and activity was in a the the of of in enzyme activity was in a the an of was to of a by the of of and and an of at to the at A were by the by the in the as of from Vitamin D and was of the K. R.M. Mangelsdorf D.J. 9: PubMed Scopus Google Scholar), and which a of of the galactose transcription factor by of and of the human vitamin D receptor by of The D response used of the M. Yamamoto T. M. Mangelsdorf D.J. DeLuca H.F. S. Chem. Biol. 10: Full Text Full Text PDF PubMed Scopus Google Scholar) and a the vitamin D receptor of M. H. R.M. Haussler M.R. Mangelsdorf D.J. Science. 2002; PubMed Scopus Google Scholar). The was of the DNA the into the K. R.M. Mangelsdorf D.J. 9: PubMed Scopus Google and the DNA with the were as the and the DNA of DNA of of or of of and of expression or used in the vitamin D3, D3, 25-hydroxyvitamin D3, and 1α,25-dihydroxyvitamin were from and added to the in of of in from to and were at Thin cells were at a of in of with and cells were with and a of of and either of of of and of or of and of h, the was from the and with of as and D3 cells with the the was to a of vitamin D3 by the of and were and vitamin D were with of and a of The were in of and by layer chromatography of and were vitamin D were by a of of D3, 25-hydroxyvitamin D3, D3, and 1α,25-dihydroxyvitamin in were in the and by of Thin Scholar). D was used for the of 25-hydroxyvitamin D in from cells in the were the with of DNA of of of of and either of or or of and of h, or vitamin D3 at of or was added to the medium. were performed of from by the The of for the was was and were performed to that were was prepared from of were with cDNA was from of and transcription which was in cDNA synthesized from of was with were an used in CYP2R1 and and CYP27A1 and and and expression cloning assay was in cells and to encoding enzymes of activating vitamin D3 by The vitamin of a encoding a of the DNA of the transcription factor to the ligand of the human vitamin D receptor, and a in which expression was by a of DNA response to expression from the is to as The of into cells by the of levels of 1α,25-dihydroxyvitamin D3 to the led to a of enzyme activity that in the of ligand not the of that be at one in the expression were performed with the CYP27A1 expression the and the D3. A enzyme activity that was was when the sterol 27-hydroxylase was with a a cDNA indicated that a cDNA of be screened in the expression cloning assay when D3 was used as the that the sterol 27-hydroxylase enzyme is of vitamin D3 and D3 (11Guo Y.-D. Strugnell S.A. Back D.W. Jones G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8668-8672Crossref PubMed Scopus (141) Google Scholar, K. Biophys. PubMed Scopus Google Scholar, I. T. S. I. J. Clin. Invest. PubMed Scopus (53) Google Scholar). encoding the sterol 27-hydroxylase, the cDNA library used in was mRNA isolated from of mice sterol 27-hydroxylase H. A. A. S. G. I. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The library was to with an cDNA of of the library were with the into The data from one is shown in cDNA a enzyme activity that was that in of cDNA and were performed the to identify a cDNA that the vitamin activity. A of in were cDNA were different for the receptor factor and the a by the receptor a of vitamin D receptor in the by the factor is most the of the receptor and transcription The mechanism by which the expression the was The cDNA in the that vitamin D receptor activity cytochrome P450 (CYP2R1) The of the CYP2R1 was from the isolated cDNA and the of the human enzyme was from a cDNA isolated from liver mRNA The two enzymes identity at the and the of the two identity at the The human CYP2R1 is located and The a The CYP2R1 and in with the more abundant than the not The of and human CYP2R1 and the of enzyme to activate D3, that be a vitamin D 25-hydroxylase. was in a of with different cytochrome P450 vitamin D receptor and Vitamin D3 than D3 was as a 25-hydroxyvitamin D3 the vitamin D receptor, with with the (18Takeyama K. S. T. M. J. S. Science. 277: PubMed Scopus Google Scholar). of the or human CYP2R1 cDNA expression in the A was when a encoding the sterol 27-hydroxylase was into the the expression a the human cytochrome P450 cDNA the expression of enzyme activity were when the was to one of the vitamin D receptor and a by of a vitamin D response from the to a to as the expression of the human or CYP2R1 led to of enzyme activity and as expression of the cDNA The was of the vitamin D receptor and a DNA the of the to the showed that the a vitamin S. 2002; PubMed Scopus Google Scholar). The expression in in the of the or was of the human or CYP2R1 a of activity. of by vitamin D3 is to that in other with the S. 2002; PubMed Scopus Google Scholar). the of the CYP2R1 enzymes to in with the vitamin D 1α-hydroxylase to activate vitamin D for D3 by the enzyme has activity the vitamin D receptor (18Takeyama K. S. T. M. J. S. Science. 277: PubMed Scopus Google Scholar). 25-hydroxyvitamin D3 by the CYP2R1 or CYP27A1 enzymes has activity is a of CYP2R1 enzyme activity in the which is active D3 in with the data were in with the P450 expression shown in when the was vitamin D3 as a of the 1α-hydroxylase cDNA led to a of enzyme activity. the and activation of the receptor by the of vitamin D3 by and the 25-hydroxylation of product by the with the and the human CYP2R1 cDNA the of to and were when the and CYP27A1 were The were to when cells with the were with 1α,25-dihydroxyvitamin D3 were when the and were used to vitamin D activation and The of the vitamin D 1α-hydroxylase and 25-hydroxylase were than with the vitamin D 1α-hydroxylase cDNA the of the of the to activate or inactivate for other nuclear was Expression of human CYP2R1 cDNA was to the of the to human hormone receptor liver receptor human human receptor and receptor not The in and used of expression and of vitamin D3 to activation of the the of the of were in two different In the which the the of P450 or expression was and the of D3 was The data from showed an in enzyme expression with of human or CYP2R1 expression added to the of the response was when of P450 expression was added to the A was when the CYP27A1 expression was used the of enzyme activity at was which be a of an lower activity of the CYP27A1 enzyme D3 (11Guo Y.-D. Strugnell S.A. Back D.W. Jones G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8668-8672Crossref PubMed Scopus (141) Google Scholar). in enzyme activity was when or human were in cells In the second levels of vitamin D3 were and the of the P450 expression were In CYP2R1 was more than CYP27A1 in an active receptor at levels of vitamin D3, was at of the Expression of the 1α-hydroxylase led to an of enzyme activity for the The most potent of was of the human CYP2R1 and which a at the of vitamin D3 Mitochondrial vitamin D 25-hydroxylase a for vitamin D3 vitamin D2 as a (11Guo Y.-D. Strugnell S.A. Back D.W. Jones G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8668-8672Crossref PubMed Scopus (141) Google Scholar, I. T. S. I. J. Clin. Invest. PubMed Scopus (53) Google Scholar). and the of to the roles of the the of vitamin D G. Strugnell S.A. DeLuca H.F. PubMed Scopus Google Scholar). the of the CYP2R1 activation of vitamin D was the that the human CYP2R1 vitamin D3 and D2 and as CYP27A1 showed a for vitamin D3 in was when the were used in with the activation of either vitamin D was when the cells were with a The of vitamin D is by the vitamin D 24-hydroxylase a microsomal P450 (CYP24A1) that is for G. Strugnell S.A. DeLuca H.F. PubMed Scopus Google Scholar). the activation of vitamin D by the CYP2R1 enzyme was caused by of a 24-hydroxylase cDNA with the CYP2R1 cDNA to of the and a in enzyme activity. shown in of cells with the and the 24-hydroxylase cDNA activity in response to ligand from vitamin D3 by the human CYP2R1 enzyme 25-hydroxyvitamin and by a of the CYP2R1 and vitamin D 1α-hydroxylase enzyme 1α,25-dihydroxyvitamin The with the vitamin D 24-hydroxylase expression that the active vitamin D ligand by the CYP2R1 enzyme was 25-hydroxyvitamin was in and of a CYP2R1 cDNA into cells by with vitamin D3 led to the of a product that a layer chromatography at the as 25-hydroxyvitamin D3. expression of the sterol 27-hydroxylase, a vitamin D3 an product with the or with a cDNA encoding not to of were when different were used to the layer chromatography not A was used to the of 25-hydroxyvitamin D3 Expression of or human CYP2R1 in the of vitamin D3 led to the of 25-hydroxyvitamin D3. A of was expression of the sterol 27-hydroxylase cDNA the of vitamin D3 to led to a in 25-hydroxyvitamin D3 with In the sterol 27-hydroxylase enzyme was more active at than was were when the of 1α,25-dihydroxyvitamin D3 from D3 was in the of added to the was cells CYP2R1 1α,25-dihydroxyvitamin D3 for CYP27A1 the data shown in indicated that CYP2R1 is a vitamin D 25-hydroxylase. The of the CYP2R1 mRNA was in the by and with of the CYP27A1 sterol 27-hydroxylase, vitamin D and vitamin D 24-hydroxylase that CYP2R1 mRNA was most abundant in the liver and C for CYP2R1 mRNA in the liver and were The in the levels of mRNA the two shown in as a of the of which the of mRNA as a and present in lower levels in other and The CYP27A1 mRNA was present at levels in liver and and in other the and were most abundant in the kidney and present in lower in other tissues. as threshold for different with different be is to that the C for the and in the liver and kidney in the whereas that for the hepatic CYP27A1 mRNA is In for which the data not the C for the CYP2R1 mRNA in and liver were and the of in the expression of the and of a cDNA encoding a vitamin D 25-hydroxylase. Expression cloning a vitamin D receptor ligand activation assay identified one cDNA from in a liver library that enzyme activity. DNA a conserved cytochrome P450 which is to in the microsomal The product of the CYP2R1 enzyme with 25-hydroxyvitamin D3 layer and was in a of vitamin D3. Expression of the CYP2R1 enzyme in cells the transcriptional activity of the vitamin D receptor in different with the vitamin D 1α-hydroxylase to a more potent and was by co-expression of the vitamin D the CYP27A1 sterol 27-hydroxylase, which showed for vitamin D3, the CYP2R1 enzyme vitamin D2 and vitamin D3. CYP2R1 mRNA is present in the is to an in the activation of vitamin the identified vitamin D 25-hydroxylase in microsomal (16Bhattacharyya M.H. DeLuca H.F. Arch. Biochem. Biophys. 1974; 160: 58-62Crossref PubMed Scopus (127) Google Scholar, 17Madhok T.C. DeLuca H.F. Biochem. J. 1979; 184: 491-499Crossref PubMed Scopus (85) Google Scholar) and mitochondrial (10Bjorkhem I. Holmberg I. J. Biol. Chem. 1978; 253: 842-849Abstract Full Text PDF PubMed Google Scholar) that were associated with cytochrome P450 of the mitochondrial H. Wikvall K. Biochem. J. 1988; 252: 207-213Crossref PubMed Scopus (53) Google Scholar, 13Masumoto O. Ohyama Y. Okuda K. J. Biol. Chem. 1988; 263: 256-260Abstract Full Text PDF Google Scholar), and cDNA cloning P. Rennert H. Shayiq R.M. Yamamoto R. Zheng Y. Addya S. Strauss III, J.F. Avadhani N.G. DNA Cell Biol. 1990; 9: 657-665Crossref PubMed Scopus (107) Google Scholar, 15Usui E. Noshiro M. Okuda K. FEBS Lett. 1990; 262: 135-138Crossref PubMed Scopus (110) Google Scholar, S. H. H. D.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the CYP27A1 sterol 27-hydroxylase, in to vitamin catalyzed of sterol in the D.W. Biochem. PubMed Scopus Google Scholar). of a of sterol 27-hydroxylase deficiency in and I. E. A.L. The and of Scholar) and H. A. A. S. G. I. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) not of vitamin D that the microsomal for the of vitamin D of with the that the CYP2R1 enzyme identified is the microsomal vitamin D 25-hydroxylase. CYP2R1 has associated with a P450 of the a and in the with the cytochrome P450 Full Text Full Text PDF PubMed Scopus Google Scholar). CYP2R1 not a associated with mitochondrial P450 which is located the and the ligand of the 2002; PubMed Scopus Google Scholar). The CYP2R1 is in to the human enzyme and is conserved at the D.R. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). conserved P450 enzymes with as and and CYP2R1 In vitamin D receptor for different of T. and and that CYP2R1 a activation in A second of is that the of CYP2R1 that of the microsomal vitamin D 25-hydroxylase. in cells with different that CYP2R1 vitamin vitamin D3, and D3 The to and is a of the microsomal vitamin D 25-hydroxylase not the mitochondrial CYP27A1 sterol 27-hydroxylase (11Guo Y.-D. Strugnell S.A. Back D.W. Jones G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8668-8672Crossref PubMed Scopus (141) Google Scholar). Although the of vitamin D is to be 25-hydroxylation in the liver by 1α-hydroxylation in the kidney or other G. Strugnell S.A. DeLuca H.F. PubMed Scopus Google Scholar), data that the microsomal 25-hydroxylase is of the of the vitamin as a K. Biophys. PubMed Scopus Google Scholar). In and the mitochondrial CYP27A1 enzyme was more active at of vitamin D3 than was a of in of CYP2R1 the microsomal vitamin D 25-hydroxylase in data M. Y. Y. Y. M. S. Suda T. FEBS Lett. PubMed Scopus Google Scholar) and in data (10Bjorkhem I. Holmberg I. J. Biol. Chem. 1978; 253: 842-849Abstract Full Text PDF PubMed Google Scholar, 17Madhok T.C. DeLuca H.F. Biochem. J. 1979; 184: 491-499Crossref PubMed Scopus (85) Google Scholar, I. Holmberg I. H. J. Biol. Chem. Scholar) suggest that the mitochondrial is a whereas the microsomal is a Although the for different were not with the CYP27A1 and CYP2R1 enzymes to of the vitamin into the or in the of the two enzymes, or other The of the encoding mRNA a of that CYP2R1 is the microsomal 25-hydroxylase In the and the the liver is the of vitamin D as by the of to of vitamin D (2Ponchon G. DeLuca H.F. J. Clin. Invest. 1969; 48: 1273-1279Crossref PubMed Scopus (203) Google Scholar, 3Ponchon G. Kennan A.L. DeLuca H.F. J. Clin. Invest. 1969; 48: 2032-2037Crossref PubMed Scopus (242) Google Scholar). In with CYP2R1 mRNA is most abundant in the liver The mRNA is present in the at levels C for CYP2R1 mRNA in the liver and were The in the levels of mRNA the two shown in as a of the of which the of mRNA as a and in lower in other and present in the PubMed Scopus Google Scholar), and not been in the CYP2R1 to the enzyme is present in the experimental used to hepatic 25-hydroxyvitamin D in the as a of the of Scholar). is not CYP2R1 enzyme activity is in the testis, or the of expression microsomal P450 enzymes with vitamin D 25-hydroxylase activity been identified in other D.W. D.W. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is a enzyme with activity is to and not vitamin D2 S. Holmberg I. Wikvall K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. H. J. Biol. Chem. Scholar). vitamin D2 and D3 is for P450 in Wikvall K. J. Biol. Chem. Scholar). The human the identity with the the enzyme not vitamin D 25-hydroxylase activity Wikvall K. J. Biol. Chem. Scholar). In to the CYP2R1 enzyme is not is conserved from to and of vitamin identity the human CYP2R1 and other and human vitamin D 25-hydroxylase enzymes and as be from in the D.R. T. R. D.J. M.R. O. D.W. PubMed Scopus Google Scholar). In the human CYP2R1 identity with a P450 enzyme isolated from the which has vitamin D 25-hydroxylase activity H. J. T. K. T. S. Biophys. PubMed Scopus Google Scholar). the identity enzymes be used to of the to be in The of two enzymes in and mice of vitamin D the the of CYP2R1 and CYP27A1 to In to is to the of 25-hydroxylation in the microsomal and mitochondrial for were by and Holmberg (10Bjorkhem I. Holmberg I. J. Biol. Chem. 1978; 253: 842-849Abstract Full Text PDF PubMed Google Scholar), used a assay to a activity from to of 25-hydroxyvitamin D3 for the microsomal and from to for the mitochondrial a and the of 25-hydroxyvitamin D in that the microsomal enzyme is for of activity and the mitochondrial enzyme A of CYP2R1 and CYP27A1 mRNA levels into in the C for CYP2R1 mRNA in liver was whereas that for the CYP27A1 mRNA was that CYP27A1 is the more abundant enzyme in the of which suggest that CYP27A1 a of 25-hydroxyvitamin to the in of the vitamin be with The the of in the used in the to an and of the two enzymes The mRNA the of and of the used in the and that to with Although a to the of the of CYP2R1 and CYP27A1 to vitamin D is from of I. E. A.L. The and of Scholar, H. A. A. S. G. I. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and mice H. A. A. S. G. I. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) in sterol 27-hydroxylase that the microsomal which the data suggest is is to in the of mice with in the CYP2R1 to into the of P450 in vitamin D and for for nuclear receptor and for vitamin for P450 enzyme and for of the