Ing speed ofPreparation of Inclusion Body and Membrane FractionsAll operations were carried out at 4uC or on ice. Newly harvested cells were treated with osmotic shock to remove the periplasmic fraction [35]. The pellets were resuspended in 5?10 ml/g lysis buffer. Cell lysis was achieved by using either theOPRM from E. coli100 nm/min and a time constant of 0.5 s. For data processing background correction and data accumulation (20 spectra) were used. Data were converted into units of mean residue ellipticity (deg cm2 dmol21) and analyzed for secondary structure with the program K2D [41].was performed between EM-1 injections. Data analysis was carried out by using BIAevaluation software using an 1:1 interaction model.AcknowledgmentsThe authors would like to acknowledge Andreas Kuberl, Dr. Tino Polen ?and Dr. Christian Schultz from Research Center Julich for their assistance ?in identification of OPRM by mass spectrometry, and Qiagen GmbH who provided the synthetic gene for OPRM.Ligand Binding Assays by Surface Plasmon ResonanceThe binding experiments were carried out on 15481974 a Biacore-X instrument (Biacore) at 25uC. OPRM was immobilized in one cell within a Ni-NTA sensor chip to 
obtain around 4000 response units (RU). The second cell was used as a control. Both cells were equilibrated with running Buffer B to establish a stable baseline. EM-1 was dissolved in buffer B and injected (flow rate 5 ml/min) over the captured receptor and the reference cell at concentrations of 10, 30, 50, 60, 80, and 100 nM. Association was monitored for 2 min, and dissociation was monitored for 5 min. No regenerationAuthor ContributionsConceived and designed the experiments: YM JL. Performed the experiments: YM JK. Analyzed the data: YM JK JL. Contributed reagents/materials/analysis tools: YM JK JL. Wrote the paper: YM JL.
Vitamin D plays an important role in the regulation of bone metabolism and immunological reactions [1,2]. In humans, vitamin D, in the form of vitamin D3, is derived from dietary TA-02 sources or made from 7-dehydrocholesterol in the skin by exposure to ultraviolet rays [3,4,5,6,7]. Then, vitamin D3 is metabolized by two-step hydroxylations: first 25-hydroxylation in the liver to form 25-hydroxyvitamin D3 (25OHD3), the major circulating metabolite of vitamin D3, followed by 1,a-hydroxylation in the kidney to form 1a,25-dihydroxyvitamin D3 (1,25OH2D3), the biologically active metabolite of vitamin D3 [6,7]. In the early years of biochemical research, a mitochondrial Gracillin web cytochrome P450 (CYP27A1), an important enzyme in the bile acid synthesis pathway [8,9], was demonstrated to be 25hydroxylase. Afterwards, Cheng et al. identified a microsomal cytochrome P450 (CYP2R1) with vitamin D 25-hydroxylase activity [10,11]. In addition, other cytochrome P450 enzymes, such as CYP2C11, CYP2D25, CYP3A4 and CYP2J2, were all identified as vitamin D 25-hydroxylases [12,13,14], and the two most active 25-hydroxylases were found to be CYP27A1 andCYP2R1 12926553 [10]. It was reported that CYP27A1 was the more abundant 25-hydroxylase in the liver [10,15]. However, mutations in human and mouse genes encoding CYP27A1 protein influenced bile acid synthesis, but had no consequence on vitamin D metabolism [15,16,17,18]. Thus, the question as to which of these proteins is the key 25-hydroxylase in the liver remains controversial. In addition, it was reported that, besides the liver, there are extra-hepatic sites of 25OHD3 synthesis, including the skin [7,19,20,21], prostate [22,23], macrophag.Ing speed ofPreparation of Inclusion Body and Membrane FractionsAll operations were carried out at 4uC or on ice. Newly harvested cells were treated with osmotic shock to remove the periplasmic fraction [35]. The pellets were resuspended in 5?10 ml/g lysis buffer. Cell lysis was achieved by using either theOPRM from E. coli100 nm/min and a time constant of 0.5 s. For data processing background correction and data accumulation (20 spectra) were used. Data were converted into units of mean residue ellipticity (deg cm2 dmol21) and analyzed for secondary structure with the program K2D [41].was performed between EM-1 injections. Data analysis was carried out by using BIAevaluation software using an 1:1 interaction model.AcknowledgmentsThe authors would like to acknowledge Andreas Kuberl, Dr. Tino Polen ?and Dr. Christian Schultz from Research Center Julich for their assistance ?in identification of OPRM by mass spectrometry, and Qiagen GmbH who provided the synthetic gene for OPRM.Ligand Binding Assays by Surface Plasmon ResonanceThe binding experiments were carried out on 15481974 a Biacore-X instrument (Biacore) at 25uC. OPRM was immobilized in one cell within a Ni-NTA sensor chip to obtain around 4000 response units (RU). The second cell was used as a control. Both cells were equilibrated with running Buffer B to establish a stable baseline. EM-1 was dissolved in buffer B and injected (flow rate 5 ml/min) over the captured receptor and the reference cell at concentrations of 10, 30, 50, 60, 80, and 100 nM. Association was monitored for 2 min, and dissociation was monitored for 5 min. No regenerationAuthor ContributionsConceived and designed the experiments: YM JL. Performed the experiments: YM JK. Analyzed the data: YM JK JL. Contributed reagents/materials/analysis tools: YM JK JL. Wrote the paper: YM JL.
Vitamin D plays an important role in the regulation of bone metabolism and immunological reactions [1,2]. In humans, vitamin D, in the form of vitamin D3, is derived from dietary sources or made from 7-dehydrocholesterol in the skin by exposure to ultraviolet rays [3,4,5,6,7]. Then, vitamin D3 is metabolized by two-step hydroxylations: first 25-hydroxylation in the liver to form 25-hydroxyvitamin D3 (25OHD3), the major circulating metabolite of vitamin D3, followed by 1,a-hydroxylation in the kidney to form 1a,25-dihydroxyvitamin D3 (1,25OH2D3), the biologically active metabolite of vitamin D3 [6,7]. In the early years of biochemical research, a mitochondrial cytochrome P450 (CYP27A1), an important enzyme in the bile acid synthesis pathway [8,9], was demonstrated to be 25hydroxylase. Afterwards, Cheng et al. identified a microsomal cytochrome 
P450 (CYP2R1) with vitamin D 25-hydroxylase activity [10,11]. In addition, other cytochrome P450 enzymes, such as CYP2C11, CYP2D25, CYP3A4 and CYP2J2, were all identified as vitamin D 25-hydroxylases [12,13,14], and the two most active 25-hydroxylases were found to be CYP27A1 andCYP2R1 12926553 [10]. It was reported that CYP27A1 was the more abundant 25-hydroxylase in the liver [10,15]. However, mutations in human and mouse genes encoding CYP27A1 protein influenced bile acid synthesis, but had no consequence on vitamin D metabolism [15,16,17,18]. Thus, the question as to which of these proteins is the key 25-hydroxylase in the liver remains controversial. In addition, it was reported that, besides the liver, there are extra-hepatic sites of 25OHD3 synthesis, including the skin [7,19,20,21], prostate [22,23], macrophag.
