results strengthen the concept of the complicated role of TGF W signaling in normal bone biology. That Vitamin D3, 2 hydroxypropyl W cyclodextrin, NADPH, dioleoyl phosphatidylcholine, bovine heart cardiolipin and cholesterol were from Sigma Aldrich Pty. The e3 ubiquitin pGro7 plasmid was from Takara Bio Inc. The silica gel plates were from Alugram Sil H, Macherey Nagel, Inc.. The cholesterol and emulsifier secure scintillant were from PerkinElmer Life Science. 26 Hydroxycholesterol cholest 5 ene 3B,26 diol was purchased from Research Plus Inc.. 2Human adrenodoxin and adrenodoxin reductase were expressed in Escherichia coli together with the coexpression of molecular chaperones, GroEL/ES, and purified as previously described. The cDNA sequence of individual CYP27A1 useful for expression was as reported by Cali et al., together with the addition of the C final 6 His tag and the 5 modifications as reported by Pikuleva et al. Escherichia coli JM109 containing the plasmid was transformed using the CYP27A1 pTrc99A construct. The growth and induction of bacteria, together with the purification of the indicated CYP27A1 were performed in a similar way to that described for the expression of mouse CYP27B1, except the soap cholate was used in place of CHAPS. The term level measured Lymph node after nickel affinity chromatography was 126 nmol/L tradition. After octyl Sepharose chromatography, the ultimate preparation of stated CYP27A1 had a 414/280 absorbance ratio of 0 and was largely free from P420. 80. 2Phospholipid vesicles were prepared from bovine heart cardiolipin and dioleoyl phosphatidylcholine at a molar ratio of 15. Vitamin D3, cholesterol or D3 were added to the phospholipids as required and the ethanol solvent removed under nitrogen. For incubations involving cholesterol, both cholesterol natural product library and unlabelled cholesterol were present. Barrier containing 20 mM HEPES, 100 mM NaCl, 0. 1 mM dithiothreitol and 0. 1 mM EDTA was put into the dry lipid mixture and sonicated for 10 min in a bath type sonicator. Reactions were carried in a concentration of 510 uM phospholipid in the above mentioned buffer to which 15 uM individual adrenodoxin, 0. 5 uM human adrenodoxin reductase, 2 mM glucose 6 phosphate, 2 U/mL glucose 6 phosphate dehydrogenase and 50 uM NADPH were added, similar to reactions explained for CYP27B1 and CYP11A1. The filtered CYP27A1 was preincubated with the vesicles for 6 min at 37 C. Adrenodoxin was added last to initiate the response. For kinetic studies, the incubations were an average of 0. 5 mL and were carried out on the original linear amount of the effect D3. Ice-cold dichloromethane was included with stop the reactions and samples were then taken as before for HPLC analysis. The kinetic parameters were determined by fitting hyperbolic curves described by the Michaelis Menten equation using Kaleidagraph 3. 6, similar to the thing that was described previously.