Imawari M, Akanuma Y, Muto Y, Itakura H, Kosaka K
J Biochem. 1980 Aug;88(2):349-60. doi: 10.1093/oxfordjournals.jbchem.a132980.
Two immunologically similar, probably identical, binding proteins for vitamin D and its metabolites (DBP1 and DBP2) were isolated separately from rat serum after approximately 180-fold purification by novel procedures using Blue Sepharose CL-6B chromatography. The freshly purified DBP1 and DBP2 each showed a single band of protein on polyacrylamide gel electrophoresis, and had alpha-mobility, although DBP1 moved slightly faster than DBP2. DBP1 and DBP2 had the same molecular weight, which was estimated as approximately 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric points of DBP1 and DBP2 were estimated as 4.9 and 5.0, respectively, from the results of isoelectric focusing experiments. DBP1 and DBP2 both appeared to have one binding site for 25-hydroxyvitamin D3 per molecule of protein, with apparently similar association constants at 4 degrees C of 5--7 x 10(9) M-1. The amino acid compositions of DBP1 and DBP2 were also determined and compared. A monospecific antiserum against rat DBP2 was prepared in a rabbit and was used for immunological studies of rat DBP. On double immunodiffusion, anti-DBP2 antiserum produced precipitin lines of complete reaction-of-identity against the purified DBP1, the purified DBP2, and rat whole serum. There was no immunological cross-reactivity between rat DBP and sera from man, dog, and rabbit, but mouse serum showed a pattern of partial identity with rat DBP. When rat serum samples were analyzed by immunoelectrophoresis using anti-DBP antiserum, three patterns of precipitin line were observed: a pattern showing the existence of only DBP1, designated as DBP 1-1; a pattern showing the existence of only DBP2, designated as DBP 2-2; and a pattern showing the existence of both DBP1 and DBP2, designated as DBP 2-1. Using single radial immunodiffusion assay for rat serum DBP, the mean (+/- S.D.) serum DBP concentrations were found to be 461 +/- 59 microgram/ml in adult male rats and 328 +/- 16 microgram/ml in adult female rats, and the difference was significant (p < 0.001). In molar terms, DBPs are present in normal rat serum in large excess relative to vitamin D and its metabolites, and most of the serum DBP, therefore, circulates as apo-DBP, not containing a bound molecule of vitamin D or of its metabolites. The immunoprecipitation studies of DBP in rat serum showed that DBPs were common main transport proteins for naturally occurring vitamin D and its metabolites, and that DBP played some, but not a principal, role in the transport of synthetic 1 alpha-hydroxyvitamin D3.
通过使用蓝色琼脂糖凝胶CL - 6B色谱的新方法，经过约180倍的纯化后，从大鼠血清中分别分离出两种免疫学上相似、可能相同的维生素D及其代谢物结合蛋白（DBP1和DBP2）。新鲜纯化的DBP1和DBP2在聚丙烯酰胺凝胶电泳上均显示出一条蛋白带，且具有α迁移率，尽管DBP1的迁移速度比DBP2略快。DBP1和DBP2具有相同的分子量，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳估计约为54,000。根据等电聚焦实验结果，DBP1和DBP2的等电点分别估计为4.9和5.0。DBP1和DBP2每分子蛋白质似乎都有一个25 - 羟基维生素D3结合位点，在4℃时的缔合常数明显相似，为5 - 7×10⁹ M⁻¹。还测定并比较了DBP1和DBP2的氨基酸组成。用兔制备了抗大鼠DBP2的单特异性抗血清，并用于大鼠DBP的免疫学研究。在双向免疫扩散中，抗DBP2抗血清针对纯化的DBP1、纯化的DBP2和大鼠全血清产生了完全同一性反应的沉淀线。大鼠DBP与人、狗和兔的血清之间没有免疫交叉反应，但小鼠血清与大鼠DBP呈现部分同一性模式。当用抗DBP抗血清对大鼠血清样品进行免疫电泳分析时，观察到三种沉淀线模式：仅显示DBP1存在的模式，称为DBP 1 - 1；仅显示DBP2存在的模式，称为DBP 2 - 2；以及显示DBP1和DBP2都存在的模式，称为DBP 2 - 1。使用单向放射免疫扩散法测定大鼠血清DBP，发现成年雄性大鼠血清DBP平均浓度（±标准差）为461±59微克/毫升，成年雌性大鼠为328±16微克/毫升，差异显著（p < 0.001）。以摩尔计算，相对于维生素D及其代谢物，DBP在正常大鼠血清中大量过剩，因此，大多数血清DBP以脱辅基DBP形式循环，不含有结合的维生素D或其代谢物分子。大鼠血清中DBP的免疫沉淀研究表明，DBP是天然存在的维生素D及其代谢物常见的主要转运蛋白，并且DBP在合成的1α - 羟基维生素D3的转运中起了一定但非主要的作用。
