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.

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.

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.