Effect of ligand binding on the conformation of human plasma vitamin D binding protein (group-specific component).

Several techniques have been used to demonstrate that the binding of specific ligands to human plasma vitamin D binding protein induces a change in protein conformation. Apoprotein and holoprotein show circular dichroism spectra of similar form in the peptide region with double minima at 207 and 218 nm. The minimum mean residue ellipticity of apoprotein (20.6 X 10(3) degrees.cm2.dmol-1) is decreased by about 8% after vitamin D3 binding, suggesting a small change in the backbone conformation. Spectrofluorimetric studies showed that 25-hydroxycholecalciferol causes a saturable enhancement of intrinsic fluorescence of human vitamin D binding protein and alters the pH profile of protein fluorescence, suggesting that there are alterations in the local environment of tryptophan residue(s) after ligand binding. Furthermore, in the presence of 25-hydroxycholecalciferol, the rate of chemical modification of the amino groups in human vitamin D binding protein is decreased and the susceptibility of intact vitamin D binding protein to proteolytic degradation is reduced, suggesting that some surface sites in the vitamin D binding protein molecule are less accessible to external agents. In addition, although the absorbance of vitamin made if difficult to interpret the ultraviolet spectra of holoprotein and apoprotein, the presence of vitamin D binding protein appears to stabilize the vitamin in an aqueous environment, a phenomenon that may be of physiological importance.     

The uptake and metabolism of 25-hydroxyvitamin D3 and vitamin D binding protein by cultured porcine kidney cells (LLC-PK1).

1. Uptake of 3H-25OHD3, 3H-25OHD3-DBP, 125I-holo-DBP and 125I-apo-DBP by LLC-PK1 cells was linearly related to the concentration of each in the culture media. The presence of DBP in the medium significantly reduced the amount of 3H-25OHD3 taken up by cells. 2. Free 25OHD3 and 25OHD3 bound to DBP were both metabolized by the cells to 24,25(OH)2D3 and an unidentified product of apparent lower polarity than 25OHD3. 3. A significant amount of DBP taken up by the LLC-PK1 cells was metabolized to a TCA-soluble form. 4. Uptake of DBP was similar to horseradish peroxidase, but higher than inulin, indicative of a non-specific endocytic mechanism with an adsorptive component. 5. It is suggested that both free circulating 25OHD3 and that derived from lysosomal degradation of 25OHD3-DBP are available for hydroxylation by the kidney.     

Species differences in the binding kinetics of 25-hydroxyvitamin D3 to vitamin D binding protein

The specific binding of 25-hydroxyvitamin D3 to its binding protein was studied in serum of the human, rhesus monkey, cow, horse, and rat. The free fraction of 25-hydroxyvitamin D3 in the rat was 0.34 +/- 0.15 pmol free/nmol total (+/- SD) and this was lower than in any of the other species (p less than 0.01). In the human, the free fraction was 1.5 +/- 0.32 pmol free/nmol total, which was higher than in any of the other species (p less than 0.001). The differences in the free fraction were mainly due to differences in dissociation constant. The relative levels of free 25-hydroxyvitamin D should be taken into account when extrapolating findings about vitamin D metabolism in animals to the human. A technical outcome of this study is that of the species tested, vitamin D binding protein from rat serum is the most suitable as a reagent component for methods used to measure total 25-hydroxyvitamin D by competitive protein binding assay.     

Identification of the sterol- and actin-binding domains of plasma vitamin D binding protein (Gc-globulin).

The mammalian plasma vitamin D binding protein (DBP), or Gc-globulin, is recognized to have at least two functional properties: sterol binding and G-actin sequestration. Affinity labeling of the sterol binding site with the radioactive electrophilic ligand, 3 beta-(bromoacetoxy)-25-hydroxycholecalciferol, followed by limited proteolysis, permitted the isolation and identification of three overlapping peptides in the amino terminus of the molecule. When G-actin affinity chromatography was applied to other proteolytic fragments, two fragments from the carboxy terminus of the molecule were isolated and identified. Another, large, tryptic fragment displayed both sterol- and actin-binding properties. The amino-terminal assignment of the sterol-binding domain was confirmed by demonstrating sterol-specific binding by an in vitro transcribed and translated product of a mutated rat DBP cDNA encoding a protein truncated in its carboxy terminus. The sterol-binding domain was localized to the region between the first-amino-terminal disulfide bond, and the actin-binding domain was found between residues 350 and 403. A high degree of sequence conservation in these regions was found among human, rat, and mouse DBP's. These functional domain assignments confirm the apparent independence of these two binding activities and help to explain the observed triprotein complex of DBP-actin-DNase I and the competition between DBP and profilin for G-actin binding. Our findings should facilitate more precise delineation of the binding domains by site-directed mutagenesis experiments.     

Pancreatic vitamin D-dependent calcium binding protein: biochemical properties and response to vitamin D

The biochemical properties of a chick pancreatic calcium binding protein (CaBP) and its response to vitamin D status and dietary calcium and phosphorus levels were studied and compared with the known vitamin D-dependent CaBPs present in the chick intestine and kidney. Pancreatic CaBP is homologous to the intestinal CaBP on the basis of immunological cross-reactivity, molecular size (28,200 Da), and charge properties (chromatographic mobility on DEAE-Sephadex in the presence of either EDTA or Ca2+). Pancreatic levels of CaBP respond to changes in vitamin D status and dietary Ca and P level in a fashion similar to the intestinal CaBP. Thus, in the absence of dietary vitamin D, both pancreatic and intestinal CaBPs were essentially undetectable, while in the presence of dietary vitamin D, a low dietary P (0.05%) elevated the pancreatic and intestinal CaBP 1.5X and 1.6X, respectively, compared to the CaBP levels present with normal dietary Ca and P (1.0%, 1.0%). The tissue levels of pancreatic CaBP (6-10 ng/mg protein) are about 0.2% of the intestine (5000 ng/mg protein) and 1% of the kidney CaBP (700 ng/mg protein). However, when corrections are made for the CaBP distribution in the tissues and expressed as CaBP concentration per CaBP-containing cells, the pancreatic CaBP level was 30% of the intestine and 10% of the kidney. Collectively, these results suggest that the chick pancreatic vitamin D-dependent CaBP is a homologous protein to the intestinal CaBP, both with regards to its relative cellular concentration as well as in its response to changing dietary levels of Ca and P.     

The low affinity binding sites of the vitamin D-dependent calcium-binding protein and their functional role in calcium transport

Calcium- and other divalent cation-binding properties of the vitamin D-dependent calcium-binding protein isolated from the duodenal mucosa of chicks were studied using the flow dialysis technique and ⁴⁵Ca. It was found that the calcium-binding protein along with the high affinity binding sites has approximately 40 low affinity binding sites with K_a of about 1000 M^?1. The low affinity sites possess of certain specificity towards binding of Ca. The affinity of the calcium-bindin protein for other divalent cations depends on the ionic radius. It is suggested that the low affinity binding sites of the calcium-binding protein take part in calcium transport organization across the intestinal epithelium.     

Influence of cholecalciferol (vitamin D(3)) on the initial kinetics of the uptake of calcium ions by rat small-intestinal mucosa (Short Communication)

The uptake of Ca²⁺ by isolated small-intestinal mucosa from vitamin D-depleted and -repleted rats was analysed for the effects of vitamin D on initial kinetics. The rapid association of Ca²⁺ with the tissue, which is complete within 1min, was unaffected by the vitamin, whereas the subsequent, linear, uptake was significantly increased. Neither the tissue space accessible to inulin nor the permeability to thiourea was influenced by vitamin D treatment.     

Evidence that sodium deprivation influences vitamin D dependent rat renal calcium binding protein

In order to provide some insight concerning the role of renal calcium binding protein (CaBP) in the functioning of the mammalian kidney, the response of renal CaBP to dietary alterations was examined. Three week old rats were fed diets deficient in calcium, phosphorous or sodium supplemented with vitamin D for a four week period. The specific activity of renal CaBP (as measured by the chelex resin assay; Ca²⁺ bound protein/Ca²⁺ bound resin per mg protein) in the 28,000 M_r region was found to increase four fold in rats fed the low phosphorus diet and two fold rats fed the low calcium diet when compared to rats fed the control diet. Renal CaBP/mg protein from rats fed the low sodium diet decreased 50% from the control values. Changes in renal CaBP were confirmed by polyacrylamide gel analysis of the 28,000 M_r fraction by densitometric tracing using a purified CaBP marker. The greater response to dietary phosphorus restriction suggests that renal CaBP may be regulated by a mechanism different from that of intestinal CaBP. The decrease in renal CaBP in rats fed the low sodium diet suggests for the first time that sodium is required for vitamin D dependent distal tubular calcium transport processes.     

Effect of dietary calcium and phosphorus depletion on vitamin D metabolism and calcium binding protein in the growing pig

Twenty-four young pigs were divided into three groups and each fed a replete, low calcium (Ca) or low phosphorus (P) diet. It was found that the deficient diets induced rises in renal 25 hydroxy-vitamin D 1,hydroxylase (1-hydroxylase) activity, circulating 1,25 dihydroxy-vitamin D3 (1,25 (OH)2-D3) and Ca binding protein (CaBP) and intestinal 1,25(OH)2D3 and CaBP. All these rises were statistically significant in the low Ca group but only the rises in the 1-hydroxylase activity and intestinal 1,25(OH)2D3 were significant in the low P group. A high degree of correlation existed between the parameters. There was no enhancement of intestinal 1,25(OH)2D3 or CaBP concentration relative to the 1-hydroxylase activity in the low P pigs as occurs in the chick. The low-P-induced rise in 1-hydroxylase activity was independent of parathyroid hormone.