Human serum binding protein for vitamin D and its metabolites. II. Specific, high affinity association with a protein in nucleated tissue.

The physicochemical and immunological characteristics of the human tissue binding protein for 25-hydroxycholecalciferol (25-OH-D3) were studied. The serum 25-OH-D3 binding protein sedimentation in 5 to 20% linear sucrose gradients was 3 to 4 S whether detected by 25-OH-[3H]D3 binding, the radioiodination of the purified protein, or by specific radioimmunoassay. All 25-OH-[3H]D3 binding by serum was attributable to immunoassayable material in the 3 to 4 S region. In contrast, all 25-OH-[3H]D3 binding and immunoassayable material in high speed supernatants of tissue extracts migrated in the 5 to 6 S region. The serum and tissue binding proteins were revealed to be indistinguishable by immunoassay and double immunodiffusion studies. In contrast to serum binding protein (58,000), the tissue binding protein was estimated to have a molecular weight of 95,000 by calibrated gel filtration analyses and was not altered by treatment with reducing agents, 0.5 M KC1 and Triton X-100. lz51labeled serum binding protein, when incubated with extracts of cultured human skin fibroblasts at physiological pH, formed a ‘251-labeled complex which was indistinguishable from the tissue binding protein labeled with 25-OH-r3H]D3. The ‘251-labeled complex was dissociated by 6 M guanidine HCl or sodium dodecyl sulfate (SDS) treatment, resulting in “‘1 sucrose gradient sedimentation and SDS-polyacrylamide gel electrophoresis migration characteristic of the serum binding protein. Collectively, these data provide clear evidence that the tissue binding protein represents an interionic association between an unidentified tissue factor and the serum binding protein.