The genetic variants of group-specific component (vitamin D-binding protein) possess different binding characteristics for immobilized Cibacron Blue 3-GA.

The elution profiles of several variants of the Gc protein have been studied after chromatography on immobilized Cibacron Blue 3-GA. The allele products belonging to the Gcl type were retarded and eluted with a Ve/Vo at 1.5, as previously reported for the Gcl-1 phenotype [Chapuis-Cellier, Gianazza & Arnaud (1982) Biochim. Biophys. Acta 709, 353-357]. The allele products belonging to the Gc2 type were further retarded (Ve/Vo at 2.6), and both Gcl and Gc2 allele products were clearly separated in heterozygous individuals. This observation allows the isolation and purification of Gc variants in heterozygous individuals which carry the combination Gcl variant-Gc2, Gcl-Gc2 variant, or Gcl variant-Gc2 variant. In contrast, the corresponding holoproteins did not bind to the gel and were eluted in the void volume. This suggests that the interaction of Gc with immobilized Cibacron Blue 3-GA involves the binding site of the protein for 25-hydroxycholecalciferol and that the dye behaves as a 'pseudoligand' for the protein. In addition, our data suggest that the different elution profiles of the variants could reflect a different affinity of the gene products for the dye.     

Affinity differences for vitamin D metabolites associated with the genetic isoforms of the human serum carrier protein (DBP)

Human vitamin D binding protein (DBP) displays considerable polymorphism with 120 described alleles. Among these, three alleles are frequently observed, Gc 1F (pI 4.94–4.84), Gc 1S (pI 4.95–4.85) and Gc 2 (pI 5.1). Differences between these genetic forms of the protein in affinity for vitamin D metabolites have been detected by electrophoretic methods. The constant affinity (Ka) values determined in this study confirm these differences. The affinities of six rare variants were also examine. Those of the DBP genetic forms to the vitamin D derivatives 25-OH-D₃ and 1,25-(OH)₂-D₃ seem to be related to the isoelectric point of the proteins: a high affinity corresponding to a low isoelectric point. The Gc 1A9 and 1A11 mutants were associated with higher affinity for the vitamin D derivatives and the Gc 1C1 and 1C21 mutants were deficient.     

Determination of the affinity of vitamin D metabolites to serum vitamin D binding protein using assay employing lipid-coated polystyrene beads.

We have developed an assay to measure the affinity of serum vitamin D binding protein for 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and vitamin D3, using uniform diameter (6.4 microns) polystyrene beads coated with phosphatidylcholine and vitamin D metabolites as the vitamin D donor. The lipid metabolite coated beads have a solid core, and thus all of the vitamin D metabolites are on the bead surface from which transfer to protein occurs. After incubating these beads in neutral buffer for 3 h, essentially no 3H-labeled vitamin D metabolites desorb from this surface. Phosphatidylcholine/vitamin D metabolite-coated beads (1 microM vitamin D metabolite) were incubated with varying concentrations of serum vitamin D binding protein under conditions in which the bead surfaces were saturated with protein, but most of the protein was free in solution. After incubation, beads were rapidly centrifuged without disturbing the equilibrium of binding and vitamin D metabolite bound to sDBP in solution was assayed in the supernatant. All three vitamin D metabolites became bound to serum vitamin D binding protein, and after 10 min of incubation the transfer of the metabolites to serum vitamin D binding protein was time independent. The transfer followed a Langmuir isotherm, and the Kd for each metabolite binding to serum vitamin D binding protein was derived by nonlinear least-squares fit analysis. From this analysis the following values for the Kd were obtained: 5.59 x 10(-6) M, 25-hydroxyvitamin D; 9.45 x 10(-6) M, 1,25-dihydroxyvitamin D; and 9.17 x 10(-5) M, vitamin D. In conclusion, we have developed a method which avoids problems encountered in previous assays and allows the precise and convenient determination of binding affinities of vitamin D metabolites and serum vitamin D binding protein.     

Human serum binding protein for vitamin D and its metabolites (DBP): evidence that actin is the DBP binding component in human skeletal muscle

The tissue protein which tightly binds the human serum binding protein for vitamin D and its metabolites (HDBP) was studied in soluble extracts of human skeletal muscle. A tissue protein-HDBP complex was effected in vitro by the addition of human serum Cohn IV to high-speed supernatant from muscle, and the complex was partially purified by ion-exchange chromatography, gel filtration, and affinity chromatography. The faster-sedimenting complex was retained longer than HDBP on DEAE-Sephacel columns, and was estimated to have a size of 100,000 daltons by gel filtration. The complex displayed inhibitory activity to deoxyribonuclease I (DNase I), whereas HDBP alone did not. When the complex was applied to affinity chromatography columns, immunoassayable HDBP was retained by DNase I-agarose and two dominant proteins of ~58,000 and 45,000 M_r were retained by the IgG fraction of anti-HDBP serum covalently bonded to amino-agarose, as revealed by sodium dodecyl sulfate-polyacrylamide electrophoresis. Pure HDBP does not bind to nor inhibit DNase I, but an actin-HDBP complex does. These data suggested that the tissue component with high affinity for HDBP was actin. Incubation of equimolar amounts of polymerized actin and pure HDBP in its apo form resulted in the depolymerization of the actin. This depolymerizing activity was also observed with HDBP saturated with cholecalciferol, 25-hydroxycholecalciferol, 24R, 25-dihydroxycholecalciferol, or 1,25-dihydroxycholecalciferol.     

The role of calcium binding protein in the mechanism of action of cholecalciferol (vitamin D3).

A role has been sought for the calcium binding protein (CaBP) which is synthesised de novo after giving cholecalciferol (CC, vitamin D3) to rachitic chicks. After homogenation of mucosal cells in sucrose media, the CaBP was found in the 78,000 X g supernatant. Therefore, the CaBP is either present in the cytoplasm or in some labile membrane structure, e.g. the microvilli, that is disrupted by homogenation. This intracellular CaBP may facilitate diffusion of Ca into intestinal cells. No secretion of CaBP into the lumen could be detected nor did excess CaBP placed in the lumen increase Ca absorption of rachitic chicks. The mitochondria of duodenal mucosal cells contained most of the Ca being translocated by the small intestine. CaBP caused release of Ca already present in mitochondria and diminished Ca uptake by mitochondria and it appreared to do this by increasing the rate of Ca flux across the mitochondrial membrane. This would explain the greater "turnover" of Ca in mucosal cells of cholecalciferol-treated chicks. These and previous findings have been used to propose a scheme for the effect of cholecalciferol on Ca transport from the small intestine.     

Nuclear and cytoplasmic binding components for vitamin D metabolites.

Specific binding of 1α,25-dihydroxyvitamin D₃ to macromolecular components of small intestinal nuclei and cytosol is demonstrated. The nuclear 1α,25-dihydroxyvitamin D₃ complex can be extracted from chromatin by 0.3 M KCl and sediments at 3.7S in sucrose density gradients. The cytoplasmic 1α,25-dihydroxyvitamin D₃-binding components also sediment at 3.7S, identically to the nuclear complex under the ultracentrifugation procedures employed.Macromolecular binding components with a high affinity for 25-hydroxyvitamin D₃ (K_d = 4.5 × 10⁻⁹ M) were also identified in intestinal cytosol which differ from the 1α,25-hydroxyvitamin D₃ receptor in that: 1) they sediment at 5–6S in sucrose gradients, 2) they are observed in organs other than the intestine, and 3) while they do bind 1α,25-dihydroxyvitamin D₃ at higher concentrations than 25-hydroxyvitamin D₃, they are not observed to transfer either 25-hydroxyvitamin D₃ or 1α,25-dihydroxyvitamin D₃ to the nucleus, $\text{in vitro}$.     

Polymorphism of 14C vitamin D3 binding protein in cattle and water buffalo serum.

Cattle and water buffalo sera labelled with vitamin D3[14C] (300 and 480 individual samples respectively) were subjected to starch gel electrophoresis followed by autoradiography in an attempt to identify a possible polymorphism of the proteins capable of binding this vitamin. Three phenotypes controlled by two codominant autosomal alleles were identified in cattle while in water buffalo six phenotypes controlled by three codominant autosomal alleles were observed.     

Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3

3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3.     

Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum.

Horizontal polyacrylamide gel electrophoreses, on 10% separation gel, of horse serum revealed polymorphism of the vitamin D binding protein (Gc protein) and another post-albumin protein (Pa). Family data supported the hypothesis that Gc and Pa types were controlled by autosomal codominant alleles. For both Gc and Pa proteins, the homozygous types showed a single fraction while the heterozygous type had two fractions. Pa types were found to be identical to the post-albumin types reported earlier by starch gel electrophoresis. Two Gc alleles, GcF and GcS, and three Pa alleles, Pa D, Pa F and Pa S, were observed in samples from Swedish (four breeds), Lipizzaner and Arab horses. The frequency of the more common allele at the two loci, i.e. GcF and PaF, ranged from 0.72-0.93 and from 0.58-0.99, respectively, in the different breeds studied. Plasma samples showed an extra protein fraction near the GcS fraction and thus were found unsuitable for Gc typing.     

Characterization of the human plasma binding protein for vitamin D and its metabolites synthesized by the human hepatoma-derived cell line, Hep 3B

Screening of three human hepatoma-derived cell lines revealed the presence of an immunologically similar plasma binding protein for vitamin D and its metabolites in media from Hep 3B cells. Approximately 3% of protein synthesized and secreted by these cells was immunoprecipitated by specific antiserum to the D-binding protein. Medium content of the protein increased over 11 days following cell seeding, and negligible amounts of 125I-labeled binding protein added to cultures were degraded over 48 h. The hepatoma-derived binding protein was indistinguishable from plasma binding protein or reference pure protein in gel filtration, sucrose gradient ultracentrifugation, and polyacrylamide gel electrophoresis systems. The Hep 3B cell product was found to bind mole/mol with monomeric actin, and bind vitamin D sterols with an affinity and specificity characteristic of the human plasma binding protein. The findings argue strongly for the identity of the Hep 3B cell product and the human plasma protein. The continuous availability of the Hep 3B cell line provides a reasonable model for investigations of biosynthesis and release of this important plasma protein.     

Group-specific component (vitamin D binding protein) prevents the interaction between G-actin and profilin

Profilin purified from human platelets formed a 1:1 molar ratio complex with rabbit skeletal muscle G-actin but was displaced by purified serum Gc (vitamin D binding protein) in a dose-dependent fashion as assessed by chromatography and ultrafiltration. This suggested that Gc and profilin competed for the same binding area on G-actin, with Gc-G-actin complexes being more stable than profilin-G-actin complexes in vitro. The binding domain for Gc on G-actin was localized to a 16,000-Da C-terminal fragment of G-actin generated by Staphylococcus aureus V8 protease, as judged by comigration on two-dimensional electrophoresis and also by overlaying electrophoresis gels with 125I-Gc. Previous studies have reported that residues 374 and 375 of G-actin are essential for binding of profilin. In this study, experiments involving tryptic removal of Cys-374 labeled with the fluorescent probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)-ethylenediamine showed that these C-terminal amino acids were not necessary for interaction with Gc.     

Linkage between the loci for serum albumin and vitamin D binding protein (GC) in sheep

Evidence for close genetic linkage between the loci for serum albumin (ALB) and vitamin D binding protein (GC) in sheep is presented. No recombinants were found in 28 informative offspring of a single ram family. The recombination frequency between the two loci was estimated to be in the range of 0 to 10%. No sign of linkage was observed between the ALB-GC complex and transferrin.     

A new chromatographic system for vitamin D3 and its metabolites: resoluation of a new vitamin D3 metabolite

A simple yet powerful new chromatographic procedure for vitamin D(3) and its metabolites is described. Liquid-gel partition chromatography on Sephadex LH-20 using a solvent of various percentages of CHCl(3) in Skellysolve B (petroleum ether, bp 67-69 degrees C) permits excellent resolution of vitamin D(3), 25-hydroxyvitamin D(3), and their more polar metabolites. Of special importance is the resolution of the metabolites of vitamin D(3) more polar than 25-hydroxycholecalciferol. Because of this resolution, a new metabolite of vitamin D(3) has been demonstrated in the plasma of rats and in the intestines of chicks given 100 IU of vitamin D(3)-1,2-(3)H.     

Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum

Horizontal polyacrylamide gel electrophoresis, on 10% separation gel, of horse serum revealed polymorphism of the vitamin D binding protein (Gc protein) and another post-albumin protein (Pa). Family data supported the hypothesis that Gc and Pa types were controlled by autosomal codominant alleles. For both Gc and Pa proteins, the homozygous types showed a single fraction while the heterozygous type had two fractions. Pa types were found to be identical to the post-albumin types reported earlier by starch gel electrophoresis. Two Gc alleles, Gc F and Gc S , and three Pa alleles, Pa D, Pa F and Pa S , were observed in samples from Swedish (four breeds), Lipizzaner and Arab horses. The frequency of the more common allele at the two loci, i.e. Gc F and Pa F , ranged from 0.72–0.93 and from 0.58–0.99, respectively, in the different breeds studied. Plasma samples showed an extra protein fraction near the Gc S fraction and thus were found unsuitable for Gc typing.     

Linkage between the loci for serum albumin and vitamin D binding protein (GC) in the Japanese quail

Vitamin D binding protein ( GC ) and serum protease inhibitor ( PI ) have been added to genetic markers in the Japanese quail. Both loci were controlled by autosomal codominant alleles named GC^A, GC^B and PI^A, PI^B, PI^C, respectively. Close linkage between the loci for serum albumin ( ALB ) and GC protein is reported. Two recombinants were observed in 145 informative offspring of 14 families. The recombination frequency between the loci was estimated as 0.014±0.006. Thus, GC was assigned to linkage group II in the Japanese quail. No signs of linkage were observed among the loci for the ALB-GC complex, PI. serum prealbumin 2 ( PA2 ), phosphoglucose isomerase ( PG1 ), 6-phosphogluconate dehydrogenase ( PGD ) and esterase-D ( ESD ).