Metal-binding studies for a de novo designed calcium-binding protein

Undegraded polyribosome preparations may be obtained from chick intestinal mucosa if ribonuclease activity is strictly controlled. This is best achieved by homogenization of the mucosa directly in rat liver cell-sap. 2. The extent of amino acid incorporation by chick intestinal polyribosomes is greatly influenced by the source of the cell-sap. Sephadex-treated intestinal cell-sap caused impaired incorporation and release of completed polypeptide chains, whereas Sephadex-treated rat liver cell-sap promoted the polymerization of up to 90 amino acids per ribosome. Under optimum conditions 30–35% of the nascent polypeptide chains are completed and released. 3. The preparation of an antiserum against the calcium-binding protein formed in response to vitamin D is described. It is shown that the antiserum is highly specific for calcium-binding protein. 4. This antiserum was used to investigate the ability of chick intestinal polyribosomes to synthesize calciumbinding protein. Only polyribosomes from chicks receiving vitamin D have the ability to synthesize calcium-binding protein. Moreover, the product formed in vitro has the same electrophoretic mobility as calcium-binding protein synthesized in vivo. 5. It is concluded that one of the main functions of vitamin D in the small intestine is to induce the synthesis de novo of calcium-binding protein.

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1H nuclear magnetic resonance studies of ytterbium-substituted porcine intestinal calcium-binding protein

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o85-123 ◽

1985 ◽

Vol 63 (9) ◽

pp. 992-997 ◽

Cited By ~ 2

Author(s):

Judith G. Shelling ◽

Theo Hofmann ◽

Brian D. Sykes

Keyword(s):

Nuclear Magnetic Resonance ◽

Magnetic Resonance ◽

Metal Binding ◽

Calcium Binding ◽

Chemical Exchange ◽

Binding Protein ◽

Calcium Binding Protein ◽

Line Broadening ◽

1H Nuclear Magnetic Resonance ◽

Nuclear Magnetic

The addition of ytterbium to calcium-saturated porcine intestinal calcium-binding protein resulted in the appearance of broad lanthanide-shifted resonances well outside the normally observed region of the 1H nuclear magnetic resonance spectrum of the calcium form of the protein. Variation of the salt concentration and temperature have led us to conclude that aggregation and chemical exchange do not contribute to the line widths of these resonances. Assuming that the line broadening of these lanthanide-shifted resonances arises from the contribution of the susceptibility line-broadening mechanism for protein residues proximal to the bound Yb3+ ion, we have calculated Yb3+–proton distances for nuclei in the metal-binding site. These lanthanide-shifted resonances provide very sensitive probes of the structure of the protein in solution.

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1,25-Dihydroxyvitamin D3-induced calcium-binding protein: localization in organ-cultured embryonic chick duodenum.

Journal of Histochemistry & Cytochemistry ◽

10.1177/33.5.2580884 ◽

1985 ◽

Vol 33 (5) ◽

pp. 477-479 ◽

Cited By ~ 2

Author(s):

R A Corradino ◽

A N Taylor

Keyword(s):

Organ Culture ◽

Calcium Binding ◽

De Novo ◽

Binding Protein ◽

Protein Localization ◽

Calcium Binding Protein ◽

Embryonic Chick ◽

Dihydroxyvitamin D3 ◽

1,25 Dihydroxyvitamin D3

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.

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