Rapid analysis of 25-hydroxyvitamin D(2) and D(3) by liquid chromatography-tandem mass spectrometry and association of vitamin D and parathyroid hormone concentrations in healthy adults.

Measurement of 25-hydroxyvitamin D (25OH-vitD) is used to assess vitamin D status. We developed a high-sensitivity measurement method for 25OH-vitD and assessed the relationship between 25OH-vitD and parathyroid hormone (PTH) in healthy adults. Aliquots (100 microL) of serum were spiked with internal standard, proteins were precipitated, and samples were analyzed by liquid chromatography-tandem mass spectrometry using 2-dimensional chromatographic separation. Total imprecision was less than 10%, and the limit of quantitation was 1.0 ng/mL. We determined the distribution of concentrations of 25OH-vitD(2) and 25OH-vitD(3) in healthy adults using samples collected during winter and summer and evaluated the association between 25OH-vitD and PTH. The difference between median concentrations of 25OH-vitD in samples collected during winter and summer was 11 ng/mL (27 nmol/L). Statistically significant differences in concentrations of PTH were observed between groups of samples with 25OH-vitD less than 11 (27 nmol/L) and 11 to 15 ng/mL (27-37 nmol/L) and between groups with 25 to 30 (62-75 nmol/L) and more than 40 ng/mL (100 nmol/L). Among the advantages of this method are its high sensitivity and specificity.

The TATA-binding protein core domain in solution variably bends TATA sequences via a three-step binding mechanism.

Studies of the binding and bending of the AdMLP TATA sequence (TATAAAAG) by the core domain of yeast TBP allow quantitation of the roles of the N-terminal domains of yeast and human TBP. All three proteins bind DNA via a three-step mechanism with no evidence for an initially bound but unbent DNA. The large enthalpy and entropy of activation for the first step in yTBP binding can now be assigned to movement of the NTD from the DNA binding pocket and not to energetics of DNA bending. The energetic patterns for hTBP and cTBP suggest that the 158-amino acid NTD in hTBP does not initially occupy the DNA binding pocket. Despite the appearance of similar energetics for hTBP and cTBP, order of magnitude differences in rate constants lead to differing populations of intermediates during DNA binding. We find that the NTDs destabilize the three bound forms of DNA for both yTBP and hTBP. For all three proteins, the DNA bend angle (theta) depends on the TATA sequence, with theta for cTBP and hTBP being greater than that for yTBP. For all three proteins, theta for the G6 variant (TATAAGAG) varies with temperature and increases in the presence of osmolyte to be similar to that of AdMLP. Crystallographic studies of cTBP binding to a number of variants had shown no dependence of DNA bending on sequence. The results reported here reveal a clear structural difference for the bound DNA in solution versus the crystal; we attribute the difference to the presence of osmolytes in the crystals.

TATA-binding protein recognition and bending of a consensus promoter are protein species dependent.

The structure and behavior of full-length human TBP binding the adenovirus major late promoter (AdMLP) have been characterized using biophysical methods. The human protein induces a 97 degrees bend in DNA AdMLP. The high-resolution functional data provide a quantitative energetic and kinetic description of the partial reaction sequence as native human TBP binds rapidly to a consensus promoter with high affinity. The reaction proceeds with successive formation of three bound species, all having strongly bent DNA, with the concurrence of binding and bending demonstrated by both fluorescence and anisotropy stopped flow. These results establish the protein species dependence of the TBP-DNA AdMLP structure and recognition mechanism. Additionally, the strong correlation between the DNA bend angle and transcription efficiency demonstrated previously for yeast TBP is shown to extend to human TBP. The heterologous NH 2-terminal domains are the apparent source of the species-specific differences. Together with previous studies the present work establishes that TBP wt-DNA TATA function and structure depend both on the TATA box sequence and on the TBP species.