A Specific High-Affinity Binding Macromolecule for 1,25-Dihydroxyvitamin D
            <sub>3</sub>
            in Fetal Bone

Kream, BE; Jose, Mimi J.L.; Yamada, Sachiko; DeLuca, H. F.

doi:10.1126/science.887939

Cited by 246 publications

(63 citation statements)

References 17 publications

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“…The hormone is then produced from vitamin D3 by sequential hydroxylation reactions in the liver and kidney (4). Established target organs for 1,25-(OH)2D3 include intestine (5, 6), bone (7,8), and kidney (9,10). In these organs, cytosolic receptor-like proteins with high affinity for 1,25-(OH)2D3 and a sedimentation coefficient of 3.0-3.7 S have been detected.…”

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Characterization of a receptor-like protein for 1,25-dihydroxyvitamin D3 in rat skin.

Simpson¹,

DeLuca²

1980

Proc. Natl. Acad. Sci. U.S.A.

103

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Isolated rat epidermis possesses a cytosolic 3.5 S receptor-like protein for 1,25-dihydroxyvitamin D3. This 3.5S binder has a high affinity (Kd = 1.4 X 10-'0 M) for 1,25-dihydroxyvitamin D3 and is present in low concentrations (31 fmol of binding sites per mg of cytosol protein). Analog competition for receptor binding revealed the following potency order: 1,25-dihydroxyvitamin D3 >> 25-hydroxyvitamin D3 > la-hydroxyvitamin D3 > 24(R),25-dihydroxyvitamin D3 >> vitamin D3. The receptor has a molecular weight of 60,000, has affinity for DNA-celtulose, and aggregates in the presence of lowtassium concentrations. The 1,25-dihydroxyvitamin D3 binder is stabilized by sodium molybdate (10 mM). Addition of phenylmethylsulfonyl fluoride (3 mM) was found to yield more reproducible receptor preparations. The presence of a 1,25-dihydroxyvitamin D3 binding macromolecule with properties similar to those of the bone and intestinal receptor suggests that skin is an additional target organ for this hormone.Evidence indicates that la,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is the hormonally active form of vitamin D3 responsible for maintenance of plasma phosphate and calcium concentrations (1). Vitamin D3 has been shown to be derived from 7-dehydrocholesterol via a photolysis reaction that occurs in the skin (2, 3). The hormone is then produced from vitamin D3 by sequential hydroxylation reactions in the liver and kidney (4). Established target organs for 1,25-(OH)2D3 include intestine (5, 6), bone (7,8), and kidney (9,10). In these organs, cytosolic receptor-like proteins with high affinity for 1,25-(OH)2D3 and a sedimentation coefficient of 3.0-3.7 S have been detected. The existence of similar 1,25-(OH)2D3 binders in the parathyroid (11) and pituitary (12) suggest that these organs also may be sites for 1,25-(OH)2D3 activities. A revealing study by Stumpf et al. (13) has presented autoradiographic evidence for nuclear localization of 1,25-(OH)2D3 in a number of novel organs including rat skin; this suggests that skin may also be a target tissue for 1,25-(OH)2D3. In support of this contention, 1,25-(OH)2D3 has recently been shown to stimulate the synthesis of a specific calcium-binding protein in skin (14).In the present communication, we identify and characterize a receptor-like protein for 1,25-(OH)2D3 in isolated rat epidermal tissue; the use of isolated epidermis yielded a cytosol less contaminated with the serum 25-hydroxyvitamin D3 (25-OH-D3) transport protein and permitted extensive characterization of the rat skin 1,25-(OH)2D3 binder. The data strongly suggest that this receptor-like macromolecule is similar to the species that exists in the intestine, bone, and kidney. Furthermore, the data corroborate other lines of evidence that skin is a target organ for 1,25-(OH)2D3. Preparation of Epidermis Cytosol. One-day-old male rats from Holtzman (Madison, WI) were cooled to 5-10°C for 30 min in order to decrease blood perfusion of the skin. The animals were sacrificed and the skin was removed. Loose connective tissu...

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confidence: 99%

Characterization of a receptor-like protein for 1,25-dihydroxyvitamin D3 in rat skin.

Simpson¹,

DeLuca²

1980

Proc. Natl. Acad. Sci. U.S.A.

103

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“…This receptor remains the most extensively studied, despite its identification recently in a number of other tissues including parathyroid gland (11), bone (12), pancreas (13,14), and kidney (13,14). The intestinal receptor sediments in high-salt sucrose gradients as a 3-3.7S macromolecule, has a molecular weight (estimated by agarose gel filtration) of 47,000, and displays all the characteristics typical of true steroid hormone receptors, including specificity, high affinity, and low capacity (2).…”

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confidence: 99%

Purification of chicken intestinal receptor for 1,25-dihydroxyvitamin D.

Pike¹,

Haussler²

1979

Proc. Natl. Acad. Sci. U.S.A.

101

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The 3.3S chicken intestinal receptor for 1,25-dihydroxyvitamin D [1,25-0H)2D] has been purified approximately 86,000-fold from the cytosolic fraction. The receptor was selectively precipitated with Polymin P from high-speed supernatants derived from 800 g of intestinal mucosa and then sequentially chromatographed on DNA-cellulose, Sephacryl, blue dextran-Sepharose, DNA-cellulose, and heparin-Sepharose. Polyacrylamide gel electrophoresis of [8][9][10] ] on the chicken intestinal mucosal cell, the hormone first undergoes an obligatory coupling within the cytoplasm to a specific high-affinity receptor molecule (1, 2), and then rapidly accumulates within the nucleus as a functionally active complex bound to chromatin. This model is strongly substantiated by both biochemical observations (3, 4) and recent autoradiographic localization studies (5, 6). Although details of the events that follow remain obscure, most evidence suggests that the complex specifically induces the biosynthesis of nucleic acids that are ultimately modified, transported, and then translated in the cytoplasm into biologically active components of mineral transport (7-10). Thus, vitamin D, through its hormonal metabolite 1,25-(OH)2D, promotes the enhanced absorption of calcium and phosphate across the intestinal epithelium, thereby contributing to the maintenance of mineral homeostasis. A specific receptor for 1,25-(OH)2D was first observed in the intestines of vitamin D-deficient (rachitic) chickens. This receptor remains the most extensively studied, despite its identification recently in a number of other tissues including parathyroid gland (11), bone (12), pancreas (13,14), and kidney (13,14). The intestinal receptor sediments in high-salt sucrose gradients as a 3-3.7S macromolecule, has a molecular weight (estimated by agarose gel filtration) of 47,000, and displays all the characteristics typical of true steroid hormone receptors, including specificity, high affinity, and low capacity (2). Moderate purifications of this protein have been achieved recently by using ion exchange and blue dextran-SepharoseThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 5485 chromatography (15). However, efforts have been generally hampered by its lack of abundance within the cell and also by its extreme lability as purification proceeds. Nevertheless, we report here a major purification of the 1,25-(OH)2D receptor from rachitic chicken intestine achieved by utilizing selective precipitation, group selective (pseudo) affinity chromatography, and gel filtration. MATERIALS AND METHODSAnimals and Materials. White Leghorn chickens were raised on a vitamin D-deficient diet (16) for 4 weeks before sacrifice. 3H-Labeled 25-hydroxyvitamin D3 (110 Ci/mmol; 1 Ci = 3.7 X 10'0 becquerels) was obtained from Amersham, converted biologically into 1,25-(OH)2[3H]D3 (110 Ci/mmol) as described (17), and then u...

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“…Furthermore, conversion of 25(OH)D3 to 24,25(OH)2D3 (8) and to 1,25(OH)2D3 (9) has been suggested to take place in bone cells. However, most of these studies have been performed in isolated bone cells, often from fetal rats (2,3,7,8) or from vitamin D-deficient animals (1). The handling of the different vitamin D metabolites by the bone of normal, adult vitamin D-repleted animals has not been studied.…”

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Extraction of Vitamin D Metabolites by Bones of Normal Adult Dogs

Ølgaard¹,

Schwartz²,

Finco³

et al. 1982

J. Clin. Invest.

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A B S T R A C T Using the isolated30±3% in a DBP free medium and no inhibition of the extraction was demonstrated after addition of DBP (720 ng/ml of perfusate). However, addition of DBP in a concentration of 14.4 jig/ml of perfusate reduced the extraction of 1,25(OH)2D3 to 8±2%, a value still significantly higher than that seen after addition of 20 times less DBP to perfusions with 25(OH)D3 and 24,25(OH)2D3. It is concluded that the isolated perfused bone of normal dogs can extract significant amounts of 25(OH)D3, 1,25(OH)2D3, and 24,25(OH)2D3. Small concentrations of DBP (720 ng/ml) in the perfusate significantly inhibited the extraction of 25(OH)D3 and 24,25(OH)2D3. A carrier role for DBP is suggested and it is proposed that the levels of free vitamin D are important for extraction of the metabolites by bone. Therefore, due to the different affinities of DBP for the various me-

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A Specific High-Affinity Binding Macromolecule for 1,25-Dihydroxyvitamin D ₃ in Fetal Bone

Cited by 246 publications

References 17 publications

Characterization of a receptor-like protein for 1,25-dihydroxyvitamin D3 in rat skin.

Characterization of a receptor-like protein for 1,25-dihydroxyvitamin D3 in rat skin.

Purification of chicken intestinal receptor for 1,25-dihydroxyvitamin D.

Extraction of Vitamin D Metabolites by Bones of Normal Adult Dogs

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A Specific High-Affinity Binding Macromolecule for 1,25-Dihydroxyvitamin D 3 in Fetal Bone

Cited by 246 publications

References 17 publications

Characterization of a receptor-like protein for 1,25-dihydroxyvitamin D3 in rat skin.

Characterization of a receptor-like protein for 1,25-dihydroxyvitamin D3 in rat skin.

Purification of chicken intestinal receptor for 1,25-dihydroxyvitamin D.

Extraction of Vitamin D Metabolites by Bones of Normal Adult Dogs

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A Specific High-Affinity Binding Macromolecule for 1,25-Dihydroxyvitamin D ₃ in Fetal Bone