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1.
The interaction of fatty acids with bovine vitamin D-binding protein (DBP) was studied using a partition equilibrium method. This protein has one high affinity site for binding of fatty acids with an association constant Ka = 7 x 10(5) M-1 for palmitic acid and Ka = 6 x 10(5) M-1 for arachidonic acid. Competition experiments showed that palmitic acid hardly competes with 25-hydroxycholecalciferol for binding to DBP. However, arachidonic acid showed comparatively a stronger competition for binding to this protein. The great difference in competition of palmitic and arachidonic acids with 25-hydroxycholecalciferol may be related to changes in DBP conformation promoted by the binding of different ligands. 相似文献
2.
The transporter of vitamin D and its metabolites in blood has received increasing attention in recent years, and is recognized to be a member of a gene family that includes albumin and -fetoprotein. Identical to the group specific component (Gc-globulin) of serum, the protein is a single-chain polypeptide constitutively synthesized in liver that circulates in amounts in far excess of normal vitamin D metabolite concentrations in blood. It plays the major role in the egress of endogenously synthesized vitamin D 3 from skin and appears to restrain D-sterols from too rapid/excessive cell entry. Along with plasma gelsolin, it comprises the plasma actin-scavenger system that facilitates removal of actin, liberated from lysed cells, by depolymerization and prevention of polymerization. Recently, the protein has been shown to behave as a co-chemotaxin specific for the complement peptide C5a, and its sialic acid-free form has been reported to play a role in macrophage activation. The latter functions strongly implicate its participation in inflammation responses. A unifying hypothesis might also suggest the protein to provide focal D-sterol delivery to cells that are important to the resolution of tissue injuries. 相似文献
3.
In the absence of vitamin D-binding protein (DBP), 1,25-(OH)2D3 at 10(-12) M significantly inhibited the [3H]thymidine incorporation in human lymphocytes during mixed lymphocyte cultures (MLC) or after phyto-hemaglutinin (PHA) stimulation. In the presence of a physiological concentration of DBP (5 x 10(-6) M), the concentration of 1,25-(OH)2D3 required for inhibition was 10(-10) M (for PHA-cultures) and 10(-9) M (for MLC). Several vitamin D analogs were compared for their inhibitory action on PHA stimulation. In the absence of DBP, the concentration necessary for 50% inhibition of [3H]thymidine incorporation ranged from 10(-12) M [1,25-(OH)2D3 and 24,24-F2-1,25-(OH)2D3], over 10(-10) M [1,24R, 25-(OH)3D3; 1,25S, 26-(OH)3D3 and 26,27-F6-1,25-(OH)2D3] and 10(-8) M [25 OHD3 and 24,25-(OH)2D3] to 10(-6) M [calcitriol-lactone]. This rank order correlates with the binding affinity of the various analogs to the cytoplasmic 1,25-(OH)2D3-receptor. DBP counteracted the inhibitory effect of all analogs and the degree of counteraction was directly proportional to the binding affinity between DBP and the vitamin D analog. DBP thus decreased the in vitro inhibitory action of 1,25-(OH)2D3 and its analogs on lymphocyte proliferation. Of all analogs tested, only 1,25-(OH)2D3 had a significant effect at a physiological concentration. 相似文献
6.
The vitamin D-binding protein in human serum (the group-specific component) is an alpha 2-globulin which is genetically polymorphic in all populations studied. Previous work (J. Svasti and B. H. Bowman (1978) J. Biol. Chem. 253, 5188-5194, and J. Svasti, A. Kurosky, A. Bennett, and B. H. Bowman (1979) Biochemistry 18, 1611-1617) has shown that the electrophoretic variations of the proteins controlled by two allelic genes, Gc1 and Gc2, are due to at least three amino acid substitutions between Gc1 and Gc2 (Svasti et al. (1979] and to heterogeneity in the Gc1 phenotype arising from carbohydrate dissimilarities. Gc1 migrates electrophoretically as two protein bands, while Gc2 migrates cathodally as a single band. This study demonstrates a post-translational glycosylation difference occurring in a single area of the Gc1 sequence which accounts for the heterogeneity observed previously. The glycosylation site, a threonine residue, appears to be in a sequence which differs between Gc1 and Gc2. The O-glycosidic bond, which is typical of mucins, is rare in plasma proteins. The cyanogen bromide fragment containing the galactosamine-containing carbohydrate in Gc1 was partially sequenced through 20 residues from the amino terminus. No detectable galactosamine could be found in the homologous cyanogen bromide fragment in Gc2. A new purification procedure for the vitamin D-binding protein in human plasma has been developed. Three chromatographic steps provide purified protein. 相似文献
7.
The affinity of purified human vitamin D-binding protein from serum (DBP) for 25-hydroxyvitamin D 3 (25-OHD 3) and 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2D 3] was measured in the presence of free fatty acids (FFA), cholesterol, prostaglandins and several drugs. Mono- and polyunsaturated fatty acids markedly decreased the affinity of both 25-OHD 3 and 1,25-(OH) 2D 3 for DBP, whereas saturated fatty acids (stearic and arachidic acid), cholesterol, cholesterol esters, retinol, retinoic acid and prostaglandins (A 1 and E 1) did not affect the apparent affinity. Several chemicals known to decrease the binding of thyroxine to its plasma-binding protein did not affect the affinity of DBP. The apparent affinity of DBP for both 25-OHD3 and 1,25-(OH)2D3 decreased 2.4- to 4.6-fold in the presence of 36 μM of linoleic or arachidonic acid, respectively. Only a molar ratio of FFA:DBP higher than 10,000 was able to decrease the binding of 25-OHD3 to DBP by 20%. Much smaller ratio's of FFA:DBP (25 for arachidonic and 45 for oleic acid), however, decreased the binding of 1,25-(OH)2D3 to DBP. These latter ratio's are well within the physiological range. The addition of human albumin in a physiological albumin:DBP molar ratio did not impair the inhibitory effect of linoleic acid on the binding of [3H]25-OHD3 to DBP. The binding and bioavailability of vitamin D metabolites thus might be altered by mono- and polyunsaturated but not by saturated fatty acids. 相似文献
8.
An interaction of vitamin D-binding protein to immobilized Cibacron Blue F3-GA was studied under urea containing buffers. In these buffers, this protein was adsorbed to the immobilized dye and was eluted with salt gradients as in the same buffer without urea. The protein was also adsorbed to immobilized diethylaminoethyl but not to immobilized carboxymethyl. It is implicated that a combination of pseudo-ligand affinity and/or hydrogen bonding interaction plays a large role whereas ionic, hydrophobic and lipophilic interactions act little between the protein and the immobilized blue dye. 相似文献
9.
Summary Vitamin D-binding protein (DBP) concentrations were determined in the sera of 90 cystic fibrosis homozygotes, 57 obligate heterozygotes, and 46 normal controls. Very significantly lower mean concentrations were found in the sera of CF homozygotes compared with both heterozygotes and controls ( P<0.01, Wilcoxon Rank Sums Test). Subdivision of the samples by Gc phenotype showed that this relationship held true both in the Gc1 and Gc2-1 phenotypes. The small sample size of the Gc2 genotype makes the significance levels of limited usefulness, but the pattern of variation of DBP levels among CF homozygotes, heterozygotes, and controls was consistent with that observed for the Gc1 and Gc2-1 classes. Haptoglobin levels showed high coefficients of variation when compared among CF homozygotes, obligate heterozygotes, and controls, presumably because of nonspecific elevation in the acute-phase response. Alpha 2-macroglobulin levels were, if anything, slightly elevated in CF homozygotes compared with controls, while albumin levels showed no significant mean differences between these groups. Since the DBP concentration does not vary with age nor with levels of vitamin D and its metabolites, we interpret our results to mean that DBP levels are specifically decreased in cystic fibrosis, perhaps as the result of impaired glycosylation of the protein.A preliminary report of this work appeared in the Proceedings of the 8th International Congress on Cystic Fibrosis. 相似文献
10.
Polymorphism at the GC locus in goats was detected using isoelectric focusing (pH 4.5-5.4) and immunoblotting with antiserum to human GC. Three variants, designated A, B and C in order of decreasing mobility to the anode, were detected and were shown to be controlled by three codominant alleles, GCA, GCB and GCC. GCA and GCB occurred in all four breeds (Australian and Texan Angora, Cashmere and Dairy) with GCA being the most common and having gene frequencies ranging from 0.851 to 0.993. GCC was found only in Australian Angora and Cashmere animals. The products of the three GC alleles had isoelectric points in the range pH 4.63-4.95 and M(r) of approximately 54,375. The major isoforms of the three alleles were shown to contain sialic acid. Linkage between the GC and albumin loci was unable to be demonstrated due to the low frequency of ALBA (0.02) in the Cashmere breed. 相似文献
11.
Vitamin D-binding protein (DBP), a multi-functional serum glycoprotein, has a triple-domain modular structure. Mutation of Trp145 (in Domain I) to Ser decreased 25-OH-D(3)-binding by 80%. Furthermore, recombinant Domain I (1-203) and Domain I + II (1-330) showed specific and strong binding for 25-OH-D(3), but Domain III (375-427) did not, suggesting that only Domains I and II might be required for vitamin D sterol-binding. Past studies have suggested that Domain III is independently capable of binding G-actin. We exploited this apparently independent ligand-binding property of DBP to purify DBP-actin complex from human serum and rabbit muscle actin by 25-OH-D(3) affinity chromatography. Competitive (3)H-25-OH-D(3) binding curves for native DBP and DBP-actin complex were almost identical, further suggesting that vitamin D sterol- and actin-binding activities by DBP might be largely independent of each other. Trypsin treatment of DBP produced a prominent 25 kDa band (Domain I, minus 5 amino acids in N-terminus), while actin was completely fragmented by such treatment. In contrast, tryptic digestion of purified DBP-actin complex showed two prominent bands, 52 (DBP, minus 5 amino acids in the N-terminus) and 34 kDa (actin, starting with amino acid position 69) indicating that DBP, particularly its Domains II and III were protected from trypsin cleavage upon actin-binding. Similarly, actin, except its N-terminus, was also protected from tryptic digestion when complexed with DBP. These results provided the basis for our studies to crystallize DBP-actin complex, which produced a 2.5 A crystal, primitive orthorhombic with unit cell dimensions a=80.2A, b=87.3A, and c=159.6A, P2(1)2(1)2(1) space group, V(m)=2.9. Soaking of crystals of actin-DBP in crystallization buffer containing various concentrations of 25-OH-D(3) resulted in cracking of the crystal, which was probably a reflection of a ligand-induced conformational change in the complex, disrupting crystal contacts. In conclusion, we have provided data to suggest that although binding of 25-OH-D(3) to DBP might result in discrete conformational changes in the holo-protein to influence actin-binding, these binding processes are largely independent of each other in solution. 相似文献
12.
Vitamin D-binding protein (DBP), a member of a multigene family including alpha-fetoprotein (AFP) and albumin, is a serum glycoprotein that reversibly binds and transports vitamin D and its metabolites to target cells. In this work, we demonstrate that normal and malignant human B-lymphocytes specifically bind and internalize DBP. Radioiodinated DBP (125I-DBP) was used to follow the uptake of the protein by Raji cells, a human pre-B-lymphoma cell line. Time course studies of DBP uptake by these cells exhibited a saturable profile at both 4 and 37 degrees C. The binding saturation curve obtained by incubating Raji cells at 4 degrees C with different concentrations (1.5 nM to 1.5 microM) of 125I-DBP showed two saturation plateaus; Scatchard analysis showed the presence of two groups of receptor sites with a Kd1 of 2.04 x 10(-7) M (n1 = 42,161 +/- 4,336 sites/cell) and a Kd2 of 1.01 x 10(-6) M (n2 = 198,000 +/- 48,000 sites/cell). After incubation of Raji cells at 37 degrees C with both fluorescein isothiocyanate (FITC) and horseradish peroxidase conjugates, DBP was internalized and could be localized in the cytoplasm. DBP-horseradish peroxidase conjugates were used to follow the uptake and to determine the endocytic pathway of the protein in Raji cells. The initial steps, contrary to those observed for AFP, did not apparently involve coated pits and vesicles. Small vesicles (approximately 50-60 nm) with electron-dense DBP-horseradish peroxidase reaction products were observed that could fuse with large endosomes. These endosomes appeared dispersed in the cytoplasm with some preferential localization in the Golgi centrosphere region. Pulse-chase experiments showed that only 10% of the uptaken protein was released in a nondegraded form. Accordingly, most DBP molecules accumulated in endosomes should be degraded in lysosomes, instead of being recycled back to the surface, as in the case of AFP. Contrary to malignant B-cells (Raji), the uptake ability for DBP of normal quiescent B-lymphocytes was very low. Specific binding and internalization of DBP-FITC by these cells were observed following mitogen-induced activation. Significant values of uptake were obtained at 37 degrees C after 72 h of incubation in the presence of pokeweed mitogen. The binding of DBP-FITC was partially inhibited in the presence of an excess of unlabeled protein. Taken together, the actual results suggest that DBP receptors are constitutively expressed by malignant B-cells and in a transitory form by normal B-lymphocytes undergoing mitogen-induced activation.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
13.
Vitamin D-binding protein (DBP) and albumin (ALB) are abundant serum proteins and both possess high-affinity binding for saturated and unsaturated fatty acids. However, certain differences exist. We surmised that in cases where serum albumin level is low, DBP presumably can act as a transporter of fatty acids. To explore this possibility we synthesized several alkylating derivatives of 14C-palmitic acid to probe the fatty acid-binding pockets of DBP and ALB. We observed that N-ethyl-5-phenylisooxazolium-3′-sulfonate-ester (WRK-ester) of 14C-palmitic acid specifically labeled DBP; but p-nitrophenyl- and N-hydroxysuccinimidyl-esters failed to do so. However, p-nitrophenyl ester of 14C-palmitic acid specifically labeled bovine ALB, indicating that the micro-environment of the fatty acid-binding domains of DBP and ALB may be different; and DBP may not replace ALB as a transporter of fatty acids. 相似文献
15.
Complexing of serum Gc (Vitamin D-binding protein) with cellular actin can occur in the extracellular space as a result of cell turnover, and particularly cell necrosis. The clearance of such complexes is significantly more rapid than that of Gc alone, and several tissues are involved in their uptake, but the mechanisms involved are unknown. We present evidence here that interaction with actin results in alteration of certain physicochemical properties of Gc. Fluorescence of the hydrophobic probe 2-p-toluidinylnaphthylene-6-sulfonate was abolished by complex formation with actin. In addition, isoelectric focusing of complexes between Gc, and actin from different tissues, revealed that complexes were generally more acidic than either protein individually. These findings indicate that complexing of Gc with actin results in altered conformation. 相似文献
17.
Selective removal of 6S plasma-generated vitamin D-binding protein from mouse mammary gland was achieved by a simple tissue culture technique. This binder was totally diluted out from the mammary explant in a time-dependent manner within 2 days of tissue culture, allowing conditions most suitable for 1,25-dihydroxyvitamin D3-receptor studies. In comparison with other methods to eliminate 6S plasma-generated vitamin D-binding protein the present technique is easy, simple and does not require the use of other vitamin D metabolites to study differential displacement of 1,25-dihydroxyvitamin D3 binding to the heterogeneous binding sites. 相似文献
18.
Endothelial cells (EC) under stress release paracrine mediators that facilitate accumulation of vascular smooth muscle cells (VSCM) at sites of vascular injury. We found that medium conditioned by serum-starved EC increase proliferation and migration of VSCM in vitro. Fractionation of the conditioned medium followed by mass spectral analysis identified one bioactive component as vitamin D-binding protein (DBP). DBP induced both proliferation and migration of VSMC in vitro in association with increased phosphorylation of ERK 1/2. PD 98059, a biochemical inhibitor of ERK 1/2, abrogated these proliferative and migratory responses in VSMC. DBP is an important carrier for the vitamin-D sterols, 25-hydroxyvitamin-D, and 1alpha,25-dihydroxyvitamin-D. Both sterols inhibited the activity of DBP on VSMC, suggesting that vitamin D binding sites are important for initiating the activities of DBP on VSMC. Release of DBP at sites of endothelial injury represents a novel pathway favoring accumulation of VSMC at sites of vascular injury. 相似文献
19.
One-dimensional polyacrylamide gel electrophoresis (1D PAGE) followed by immunoblotting revealed genetic polymorphism of GC protein in sheep (variants F, S, V) and mouflon (variants F and S, apparently identical to F and S of sheep). The frequency of Gcs allele ranged from 0.84 to 1.0 in the 12 breeds of sheep studied. GcV allele was observed only in Tsigai breed with a frequency of 0.017. 相似文献
20.
The vitamin D-binding protein, Gc, was purified from human serum and crystallized using the hanging-drop method. The best crystals were grown from 28% polyethylene glycol 400 in 50 mM-sodium acetate at pH 4.8. These crystals diffract to 3.4 A and the observed diffraction is consistent with orthorhombic space groups P4(1) and P4(3). The unit cell parameters were determined to be a = b = 135.5 A and c = 75.6 A. 相似文献
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