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1.
An 18,000-dalton protein (pI = 5.1) shown previously to be modulated by 1,25-dihydroxyvitamin D3 was purified to allow its further characterization. This protein from embryonic chick intestine was shown to comigrate during two-dimensional electrophoresis with an abundant protein from the intestine of 4-week-old chickens. The protein was purified from 4-week chick intestine and analyzed for amino acid composition, and 28 amino acids of its N-terminal sequence were determined. The N-terminal amino acid sequence had significant homology to cellular retinol binding protein II, an intestinal protein that has been recently sequenced. The purified 18-kilodalton protein was shown to bind retinol by fluorescence spectrophotometry. This 18-kilodalton protein is dramatically changed by 1,25-dihydroxyvitamin D3 in the chick embryonic organ culture system. Therefore, further study of it may lead to a better understanding of vitamin A and D interaction and how 1,25-dihydroxyvitamin D3 acts through proteins to stimulate intestinal calcium and phosphate transport. 相似文献
2.
3.
H S Gill J M Londowski R A Corradino A R Zinsmeister R Kumar 《Journal of steroid biochemistry》1988,31(2):147-160
We synthesized 25-hydroxy-26,27-dimethylvitamin D3, 9, and 1,25-dihydroxy-26,27-dimethylvitamin D3, 14, from chol-5-enic acid-3 beta-ol and tested their biological activity in vivo and in vitro. 9 was found to be highly potent vitamin D analog with bioactivity similar to that of 25-hydroxyvitamin D3. 9 bound to rat plasma vitamin D binding protein with approximately one-third the affinity of 25-hydroxyvitamin D3. In a duodenal organ culture system and in a competitive binding assay with chick intestinal 1,25-dihydroxyvitamin D receptor, 9 was significantly more potent than 25-hydroxyvitamin D3. 1,25-Dihydroxy-26,27-dimethylvitamin D3, 14 was also highly active in vivo. At doses of 1000-5000 pmol/rat, its action was more sustained than that of 1,25-dihydroxyvitamin D3. 14 bound to vitamin D binding protein about 18 times less effectively than 1,25-dihydroxyvitamin D3. 14 bound to the chick intestinal cytosol receptor with an affinity one-half that of 1,25-dihydroxyvitamin D3. In a duodenal organ culture system, 14 was about half as active as 1,25-dihydroxyvitamin D3. Extension of the sterol side chain, at C-26 and C-27, by methylene groups, prolongs the bioactivity of a vitamin D sterol hydroxylated at C-1 and C-25; the corresponding sterol, hydroxylated only at C-25, does not show any alteration of its bioactivity in vivo. These newly synthesized analogs may potentially be of therapeutic use in various mineral disorders. 相似文献
4.
C W Bishop N C Kendrick M C Dame H F DeLuca 《The Journal of biological chemistry》1985,260(9):5209-5212
Two-dimensional electrophoresis together with radiolabeling experiments was used to examine cytosolic proteins of embryonic chick duodenum for responses to 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 caused a striking decrease in [3H]leucine content of an 18,000-dalton protein (approximate pI, 5.1) after a 10-min pulse with radioisotope followed by a 4-h chase. Decreased [14C]leucine content of the same protein was also observed at various times following 1,25-dihydroxyvitamin D3 addition to culture media; a significant decrease in radiolabel incorporation occurred within 30 min after addition of the hormone. The results argue that 1,25-dihydroxyvitamin D3 causes either a decreased synthesis rate or a post-translational modification of this protein. This change joins the biosynthesis of calcium-binding protein as an early event in the response of chick embryonic intestine to 1,25-dihydroxyvitamin D3. 相似文献
5.
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) induces de novo biosynthesis of a specific calcium-binding protein (CaBP) in embryonic chick duodenum in organ culture. Using a highly sensitive and specific, peroxidase-antiperoxidase immunocytochemical procedure, 1,25(OH)2D3-induced CaBP in the organ-cultured duodenum was found only in the cytoplasm of absorptive cells, corresponding to its localization in rachitic chick duodenal cells after a single injection of 1,25(OH)2D3 in vivo. This observation, along with evidence correlating CaBP with calcium transport, strongly supports the use of the embryonic chick duodenal organ culture system as a physiologically relevant model of the vitamin D-dependent calcium absorptive mechanism. 相似文献
6.
J M Londowski L K Revelle S B Kost R A Corradino R Kumar 《Journal of steroid biochemistry》1985,23(1):81-85
We synthesized 3 beta-thiovitamin D3 from 7-dehydrocholesterol and tested its biological activity and protein binding properties. The thiovitamin was found to be a weak vitamin D agonist at high doses in vivo. It was poorly bound by both vitamin D-binding protein as well as by the intestinal cytosol receptor for 1,25-dihydroxyvitamin D. It did not increase the synthesis of calcium binding protein in the chick embryonic duodenum and did not block the activity of 1,25-dihydroxyvitamin D3 in this system. We conclude that 3 beta-thiovitamin D3 is a weak vitamin D agonist in vivo with no agonist activity or antagonist activity to 1,25-dihydroxyvitamin D3 in the chick embryonic duodenum. 相似文献
7.
R A Corradino C S Fullmer R H Wasserman 《Archives of biochemistry and biophysics》1976,174(2):738-743
Vitamin D3 or a potent metabolite, 1,25-dihydroxycholecalciferol, induces calcium-binding protein (CaBP) synthesis and stimulates transmucosal calcium transport in embryonic chick duodena maintained in novel organ culture apparatus. When added to the sterol-free culture medium, highly purified chick intestinal CaBP, similarly and specifically, stimulates calcium transport in the cultured duodena. These results clearly demonstrate the involvement of CaBP in intestinal calcium transport. 相似文献
8.
S Ameenuddin M L Sunde H F DeLuca N Ikekawa Y Kobayashi 《Archives of biochemistry and biophysics》1983,226(2):666-670
The provision of 1,25-dihydroxyvitamin D3 as the only source of dietary vitamin D3 to laying hens failed to support normal embryonic development in their fertile eggs. Significant (P less than .001) improvement in embryonic survival to hatching in these eggs resulted from injections of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or 24,24-difluoro-25-hydroxyvitamin D3 prior to incubation. Maximum embryonic survival with lowest embryonic mortality was observed when 0.20 micrograms/egg of 1,25-dihydroxyvitamin D3 or 0.60 micrograms/egg 25-hydroxyvitamin D3 was injected. These results indicate that several forms of vitamin D, two of which cannot be converted to 24,25-dihydroxyvitamin D3, can provide this activity; and of the vitamin D compounds tested, 1,25-dihydroxyvitamin D3 may be the most active in supporting embryonic survival in the chick when delivered directly by injection. 相似文献
9.
We synthesized 22-fluorovitamin D3 from (22S) cholest-5-ene-3 beta, 22-diol-3 beta-acetate 2. Compound 2 was treated with diethylaminosulfur trifluoride to give 22-fluorocholest-5-en-3 beta-acetate 3 and (E) 22-dehydrocholest-5-en-3 beta-acetate. Compound 3 was treated with N-bromosuccinimide to give a mixture of the respective 5,7- and 4,6-dienes. The 5,7-diene of 3 was separated from the 4,6-diene using the dienophile 4-phenyl-1,2,4-triazoline-3, 5-dione. 22-Fluoro-5 alpha,8 alpha-(3,5-dioxo-4-phenyl-1, 2,4-triazolino)-cholest-6-en-3 beta-acetate 4 was purified by flash chromatography and treated with lithium aluminum hydride to generate 22-fluorocholesta-5,7-dien-3 beta-ol 5. Photolysis of the diene 5, followed by thermal equilibration, resulted in the synthesis of 22-fluorovitamin D3 7. The vitamin 7 increased active intestinal calcium transport only at a dose of 50,000 pmol/rat, whereas vitamin D3 increased intestinal calcium transport at a dose of between 50 and 500 pmol/rat. 22-Fluorovitamin D3 7 did not mobilize bone and soft tissue calcium at a dose as high as 50,000 pmol/rat, whereas vitamin D3 was successful in doing so at a dose of 500 pmol/rat. When tested in the duodenal organ culture system, 22-fluorovitamin D3 7 had approximately 1/25th the potency of vitamin D3. It did not antagonize the activity of 1,25-dihydroxyvitamin D3. 22-Fluorovitamin D3 7 bound to the rat plasma vitamin D binding protein less avidly than vitamin D3. 22-Fluorovitamin D3 was bound very poorly to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. We conclude that the introduction of fluorine at the C-22 position results in a vitamin D sterol with decreased biologic activity when compared to vitamin D3. The presence of a fluorine group at C-22 position inhibits the binding of the vitamin to rat vitamin D binding protein when compared to the binding of its hydrogen analog, vitamin D3. 相似文献
10.
Human small intestine was found to contain a retinol-binding protein similar to the gut-specific cellular retinol-binding protein, type two [CRBP (II)], described in the rat. This newly detected human protein was immunochemically distinct from human cellular retinol binding protein previously described but immunochemically similar to rat CRBP (II). The partially purified protein bound retinol and exhibited fluorescence excitation and emission spectra distinct from those spectra for retinol bound to pure human CRBP but similar to the spectra for retinol bound to rat CRBP (II). Human CRBP (II) could be localized to the villus-associated enterocytes by immunohistochemistry, using antiserum against rat CRBP (II). The protein was abundant representing 0.4% of the total soluble protein in a jejunum mucosal extract. This protein may play an important role in the absorption and necessary intestinal metabolism of vitamin A. 相似文献
11.
1,25-Dihydroxyvitamin D2 has been prepared from 25-hydroxyvitamin D2 using rachitic chick kidney mitochondria. This metabolite was highly purified by Sephadex LH-20 chromatography and by preparative high-pressure liquid chromatography. Its purity was assessed by analytical high-pressure liquid chromatography which revealed no other 254-nm absorbing material and by mass spectrometry. The concentration of dilute solutions of 1,25-dihydroxyvitamin D2 was determined by high-pressure liquid chromatography and deflection of the 254-nm column monitor. The 1,25-dihydroxyvitamin D2 was then shown to be 1/5 to 1/10 as active as 1,25-dihydroxyvitamin D3 in the chick while it had previously been shown to be equal in activity in the rat. Thus, discrimination against the vitamin D2 side chain by the chick persists in the metabolically active 1,25-dihydroxyvitamin D compounds. 相似文献
12.
Unique rearrangement of ergocalciferol side chain in vitro: production of a biologically highly active homologue of 1,25-dihydroxyvitamin D3 总被引:1,自引:0,他引:1
In vitro incubation of 24-epi-25-hydroxyvitamin D2 with chicken kidney homogenate produced several compounds, one of which had an affinity equal to that of 1,25-dihydroxyvitamin D2 for the chick intestinal receptor. The affinity of 24-epi-1,25-dihydroxyvitamin D2 for the same receptor was found to be half that of 1,25-dihydroxyvitamin D2. The unknown compound was produced only when homogenate was prepared from pooled kidneys taken from both vitamin D deficient and replete chickens. The compound has been tentatively identified as 1,25-dihydroxy-22-dehydro-26-homovitamin D3 by ultraviolet absorption spectrophotometry and mass spectrometry. Chemical synthesis of 1,25-dihydroxy-22-dehydro-26-homovitamin D3 provided additional evidence for the structure. Administration of this 26-homologue of 1,25-dihydroxyvitamin D3 at the dose level of 650 pmol/rat stimulated bone calcium mobilization in the hypocalcemic rat equal to that of 1,25-dihydroxyvitamin D3. Thus, this paper demonstrates unique methyl migration on the side chain of 24-epi-1,25-dihydroxyvitamin D3 to form a more biologically potent analogue. 相似文献
13.
Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in chick duodenal mucosal cell. Relationship to its mechanism of action 总被引:11,自引:0,他引:11
Pretreatment of the D-deficient chick with 1,25-dihydroxyvitamin D3 increases de novo synthesis of phosphatidylcholine by a stimulation of CDP-choline: sn-1,2-diacylglycerol choline-phosphotransferase reaction. The time course of change in the incorporation of [3H]choline and [14C]ethanolamine into the brush border lipid fraction after 1,25-dihydroxyvitamin D3 treatment correlates closely with the time course of change in calcium uptake into the brush border membrane vesicles. Prior treatment with cycloheximide does not block this increase in phosphatidylcholine synthesis. In addition, 1,25-dihydroxyvitamin D3 administration increases the incorporation of [3H]arachidonic acid into the phosphatidylcholine fraction of the brush border to a great extent but does not increase the incorporation of [3H]palmitic acid into the phosphatidylcholine fraction. The incorporation of these 3H labeled fatty acids into diacylglycerol is not changed by 1,25-dihydroxyvitamin D3. These data indicate that 1,25-dihydroxyvitamin D3 enhances the synthesis of phosphatidylcholine independent of new protein synthesis, and also increases the incorporation of unsaturated fatty acids into phosphatidylcholine. From these results we suggest that changes in phospholipid metabolism in the enterocyte are the mechanisms by which 1,25-dihydroxyvitamin D3 acts to enhance calcium entry across the brush border membrane. 相似文献
14.
We synthesized a novel vitamin D analog, 22-hydroxyvitamin D3 9 and tested its biologic activity (and antivitamin properties) in vivo in vitamin D-deficient rats, and in vitro in the chick embryonic duodenum. We examined its ability to bind to the sterol carrier protein, vitamin D binding protein and the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. The new vitamin 9 was synthesized from 3 beta-hydroxy-22,23-dinorcholenic acid 1 in 12 steps. The vitamin 9 displayed no vitamin D agonist activity in the intestine or in bone in vivo and did not block the activity of vitamin D3 or 25-hydroxyvitamin D3. It was a weak vitamin D3 agonist in the chick embryonal duodenum in vitro. It did not antagonize the activity of 1,25-dihydroxyvitamin D3. Vitamin 9 bound to the chick intestinal cytosol receptor with low affinity. 22-Hydroxyvitamin D3 and various vitamin D sterols were bound to vitamin D binding protein in the following order: 25-hydroxyvitamin D3. (24R)-24,25-dihydroxyvitamin D3, and (25S)-25,26-dihydroxyvitamin D3 greater than 22-hydroxyvitamin D3 greater than 11 alpha-hydroxyvitamin D3 greater than 1,25-dihydroxyvitamin D3 greater than vitamin D3. We conclude that the introduction of a hydroxyl group at C-22 in the side chain of the vitamin D3 molecule decreases its biological activity. 相似文献
15.
Y Tanaka H F DeLuca Y Kobayashi N Ikekawa 《Archives of biochemistry and biophysics》1984,229(1):348-354
A new fluoro analog of 1,25-dihydroxyvitamin D3, i.e., 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3, has been compared with the native hormone, 1,25-dihydroxyvitamin D3, in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D3. This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism. 相似文献
16.
Characterization of rat cellular retinol-binding protein II expressed in Escherichia coli 总被引:3,自引:0,他引:3
E Li B Locke N C Yang D E Ong J I Gordon 《The Journal of biological chemistry》1987,262(28):13773-13779
Rat cellular retinol-binding protein II (CRBP II) is a small (15.6 kDa) intracellular protein that binds all-trans-retinol. In the adult rat, expression of the CRBP II gene is essentially limited to the small intestinal lining cells (enterocytes), suggesting that CRBP II may be uniquely adapted for intestinal metabolism of newly absorbed retinol. Functional and structural analysis of this protein has been hampered by difficulties in freeing rat intestinal CRBP II from its ligand without denaturation. To circumvent this problem, we have obtained efficient expression of rat apoCRBP II in Escherichia coli. The purified E. coli-derived apoprotein, when complexed with all-trans-retinol, demonstrates fluorescence excitation-emission spectra and absorption spectra indistinguishable from that of CRBP II-retinol isolated from rat intestine. Quantitative ligand binding studies were performed by monitoring either the fluorescence of bound retinol or the quenching of protein fluorescence. They revealed that E. coli-derived CRBP II binds retinol tightly (the apparent dissociation constant is estimated to be 10(-7)-10(-8) M), with a stoichiometry of 1:1. Fluorescence quenching studies used acrylamide as a probe for the exposure of the 4 tryptophan residues to solvent. The results indicate that although there is heterogeneity in the exposure of these 4 tryptophan residues to solvent, they are situated in a relatively nonpolar environment. These studies suggest that E. coli-derived apoCRBP II will serve as a useful model for studying retinol-protein interactions. 相似文献
17.
The time course of calcium-binding protein induction by 1,25-dihydroxyvitamin D3 was examined in embryonic chick duodena by single-label autoradiography. Duodena were excised from 19-day-old embryos, cultured for 24 h in defined medium, and exposed to 1,25-dihydroxyvitamin D3 at 1/2, 1, 1 1/2, 2, 2 1/2, 4, 6, and 20 h before the end of the culture period. Control duodena were identically handled but were not exposed to the hormone. All duodena were pulsed with [14C]leucine during the final 30 min of incubation. Cytosolic proteins, extracted from each radiolabeled tissue, were resolved by two-dimensional electrophoresis and autoradiographs were prepared from the resulting gels. Calcium-binding protein was detectable by autoradiography in duodena cultured with hormone for 1 h or more but not in the control duodena. The autoradiographs were converted to digital images by a scanning densitometer and entered into the Man-computer Interactive Data Access System. The total radioactivity incorporated into calcium-binding protein during the 30-min pulse was determined by computer analysis of the digitized autoradiographs, and the rate of calcium-binding protein biosynthesis was calculated. Duodena cultured with hormone for 1-20 h synthesized calcium-binding protein at rates of 129 +/- 17 to 8800 +/- 110 pg/mg of cytosolic protein/30 min. These rates demonstrate that calcium-binding protein is induced within the first hour of exposure to 1,25-dihydroxyvitamin D3, but in amounts too small to be detected by commonly used immunoassays. 相似文献
18.
M F Holick L A Baxter P K Schraufrogel T E Tavela H F DeLuca 《The Journal of biological chemistry》1976,251(2):397-402
24R,24,25-Dihydroxyvitamin D3 is capable of inducing a minimal intestinal calcium transport response in chicks when compared to an equal amount of 25-hydroxyvitamin D3. 1,24,25-Trihydroxyvitamin D3 is also less active than 1,25-dihydroxyvitamin D3, and its activity is much shorter lived than that of 1,25-dihydroxyvitamin D3. A comparison of the metabolism of 25-hydroxy[26,27-3H]vitamin D3 and 24,25-dihydroxy[26,27-3H]vitamin D3 in the rat and chick shows that 24,25-dihydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3 disappear at least 10 times more rapidly from the blood and intestine of chicks. Furthermore, examination of the excretory products from both of these species demonstrates that chicks receiving a single dose of 24,25-dihydroxy[26,27-3H]vitamin D3 excrete 66% of the total radioactivity by 48 hours, whereas rats receiving the same dose excrete less than one-half that amount. These results demonstrate that 24,25-dihydroxyvitamin D3 is considerably less biologically active in the chick than in the rat, probably due to more rapid metabolism and excretion. 相似文献
19.
Reduction of retinaldehyde bound to cellular retinol-binding protein (type II) by microsomes from rat small intestine 总被引:2,自引:0,他引:2
Cellular retinol-binding protein, type II (CRBP (II], an abundant protein of the rat small intestine, has recently been shown to be able to bind retinaldehyde in addition to retinol (MacDonald, P.N., and Ong, D. E. (1987) J. Biol. Chem. 262, 10550-10556). Retinaldehyde is produced in the intestine by oxidative cleavage of beta-carotene. The next step in the intestinal metabolism of vitamin A is the reduction of retinaldehyde to retinol which is then esterified for incorporation into chylomicrons. In the present study retinaldehyde bound to CRBP(II) was found to be available for reduction by microsomal preparations from rat small intestinal mucosa. The microsomal activity was about 8 times greater than the activity observed for an equal amount of cytosolic protein. Retinaldehyde reduction utilized either NADH or NADPH as cofactor, with NADH being slightly more effective. The apparent Km for retinaldehyde-CRBP(II) was 0.5 microM, and the Vmax was approximately 300 pmol/min/mg protein, a rate more than sufficient for the needs of the animal. The product retinol remained complexed to CRBP(II). The microsomal enzyme activity reduced free and bound retinaldehyde to approximately the same extent, although the aldehyde function of retinaldehyde bound to CRBP(II) was less accessible to chemical reducing agents than that of free retinaldehyde. Retinol bound to CRBP(II) could not be oxidized by the microsomal activity in the presence of NAD+, while free retinol or retinol bound to bovine serum albumin was oxidized to retinaldehyde. The more favorable reduction versus oxidation of retinoid bound to CRBP(II) consequently favored the reaction known to be required for the ultimate conversion of beta-carotene to retinyl esters for export from the gut. 相似文献
20.
Vitamin D stimulates absorption of spd-glucose in chick jejunum and ileum by a specific action on the maximal velocity of Na+-gradient driven spd-glucose transport across the brush-border membrane of intestinal cells. Induction of spd-glucose transport by either vitamin D-3 or 1,25-dihydroxyvitamin D-3 in embryonic intestine can be blocked by inhibitors of RNA and protein synthesis. 相似文献