The extraction and characterization of soluble anionic phosphoproteins from bone

Spector, Alan; Glimcher, Melvin J.

doi:10.1016/0005-2795(72)90040-2

Cited by 76 publications

(44 citation statements)

References 28 publications

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“…We believe it may be concluded that the "P-labeled components visualized in this study by radioautography represent principally the phosphoproteins isolated and characterized previously (14,(21)(22)(23). The localization of silver grains over endoplasmic reticulum and Golgi apparatus of osteoblasts provides further strong evidence that the phosphoproteins are synthesized principally by these cells, a conclusion consistent with the results recently obtained in cell culture by Gotoh et al (15).…”

Section: Discussionsupporting

confidence: 92%

Radioautographic visualization and biochemical identification of O-phosphoserine- and O-phosphothreonine-containing phosphoproteins in mineralizing embryonic chick bone.

Landis¹,

Sanzone²,

Brickley-Parsons³

et al. 1984

The Journal of Cell Biology

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We injected NaH 233P04 into normal 14-d-old embryonic chicks and examined the long bones by both radioautography and biochemical analyses from 10 to 240 min after the injection was completed . At 30 min, determination of the radiographic grain density revealed that 33p was concentrated principally in fibroblasts, preosteoblasts, and osteoblasts . With time, there was a progressive increase in the density of silver grains located over both the osteogenic cells and the regions of uncalcified (osteoid) and calcified extracellular organic matrices. Biochemical analyses identified 33p-O_phosphoserine as the major 33p component in glutaraldehyde-treated whole demineralized bone tissue and in EDTA-soluble, nondiffusible proteins extracted from the bones, both at the same time periods that 33P-induced silver grains were visualized by radioautography. 33p-O_phosphothreonine was also identified in experiments using a dosage of 10 mCi per embryo . The results provide the first combined direct biochemical and radioautographic identification that phosphoproteins are synthesized in bone and are located morphologically at the sites of mineralization . The data provide further evidence that phosphoproteins play a critical role in the biological calcification of vertebrate tissues.

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Section: Discussionsupporting

confidence: 92%

Radioautographic visualization and biochemical identification of O-phosphoserine- and O-phosphothreonine-containing phosphoproteins in mineralizing embryonic chick bone.

Landis¹,

Sanzone²,

Brickley-Parsons³

et al. 1984

The Journal of Cell Biology

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“…A similar Gla-containing protein component has also been recently isolated from chicken bone (9). The low molecular weight and the high electrophoretic mobility of these Gla proteins indicate that they are probably the major component of the anionic protein fraction previously isolated from EDTA extracts of fetal calf and chicken bones (14). We …”

Section: Resultsmentioning

confidence: 77%

Characterization of a gamma-carboxyglutamic acid-containing protein from bone.

Price¹,

Otsuka²,

Poser³

et al. 1976

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

708

351

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1447 on chicken bone a presumptive Gla fraction was isolated by ion exchange chromatography from alkaline hydrolysates of the chicken bone and identified as Gla on the basis of both its conversion to glutamic acid upon heating in acid and its coelution with a similar putative Gla fraction in alkaline hydrolysates of prothrombin (7). In the present study we have used Gla synthesized by the procedure of Morris et al. (9) to show further that the mass spectra of the presumptive Gla from bovine bone and synthetic Gla are identical. Studies with synthetic Gla have also enabled us to show that Gla is indeed completely stable under the conditions of alkaline protein hydrolysis, and to establish its ninhydrin color factor for quantitative estimation of Gla content in proteins.In the present work we report the chemical composition of the bovine Gla protein and the sequence of its first 15 residues, which establish that the bovine Gla protein is not a fragment of the bovine blood clotting factors. We also present evidence of a similar Gla-containing protein in most other bovine calcified tissues and in a variety of other vertebrates. The low molecular weight and high electrophoretic mobility of the main Gla protein in chicken bone (7) indicate that it is probably another member of this class of proteins. We find that the bovine Gla protein binds strongly to the mineral phase of bone, with an average stoichiometry of one Gla protein per crystal, and that the bovine Gla protein is a potent inhibitor of hydroxyapatite crystallization. MATERIALS AND METHODSCortical bone was obtained from the central section of the femur of freshly slaughtered calves or cows. Bone samples were freed of marrow and connective tissue, ground to a particle size that passed through a 210-,gm sieve, and washed with several changes of water for 24 hr at 4°. The bone was then dialyzed against several changes of 0.5 M EDTA, pH 8, at 40 for 8-10 days. The soluble fraction inside the dialysis sack was collected by centrifugation, dialyzed exhaustively against 5 mM NH4HCO3, and lyophilized. After gel filtration on Sephadex G-100 (Fig. 1), peak fractions containing the Gla protein were lyophilized and then chromatographed on a 2 X 50 cm column of DEAE-Sephadex A25 at 25°with a linear gradient in 0.1 M Tris-HCI, pH 8.0, from 0 to 0.75 M NaCl. The procedure for the isolation of Gla protein from swordfish and human bone and bovine dentine was altered only in the use of 50 mM rather than 5 mM NH4HCO3 to suppress Gla protein precipitation. Hydroxyapatite crystals were made by mixing calcium chloride and sodium phosphate solutions at 5 mM concentrations, pH 7.4, and 250. Hydroxyapatite also was purchased from Clarkson Chemical Co. Amorphous calcium phosphate was prepared by mixing saturated solutions of calcium chloride and sodium phosphate at pH 7.4 and 250.

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“…Thus it is tempting to speculate that the Gla-containing protein may serve as the nucleation substrate, although an equally plausible argument can be advanced that they function in just the opposite way. Similar considerations (16) have evolved concerning the possible role of the phosphoproteins in the initiation of calcification in enamel (15), bone (20), and dentin (21).…”

Section: Methodsmentioning

confidence: 68%

The presence of protein-bound gamma-carboxyglutamic acid in calcium-containing renal calculi.

Lian¹,

Prien²,

Glimcher³

et al. 1977

J. Clin. Invest.

152

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The extraction and characterization of soluble anionic phosphoproteins from bone

Cited by 76 publications

References 28 publications

Radioautographic visualization and biochemical identification of O-phosphoserine- and O-phosphothreonine-containing phosphoproteins in mineralizing embryonic chick bone.

Radioautographic visualization and biochemical identification of O-phosphoserine- and O-phosphothreonine-containing phosphoproteins in mineralizing embryonic chick bone.

Characterization of a gamma-carboxyglutamic acid-containing protein from bone.

The presence of protein-bound gamma-carboxyglutamic acid in calcium-containing renal calculi.

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