Atomic structure of intracellular amorphous calcium phosphate deposits.

Betts, F.; Blumenthal, Norman C.; Posner, Aaron S.; Becker, Gerald L.; Lehninger, Albert L.

doi:10.1073/pnas.72.6.2088

Cited by 104 publications

(64 citation statements)

References 9 publications

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“…We interpret these findings to indicate that the hollow spheres are composed of a residual lipid (and/or detergent) in which are absorbed the ϳ1.0-nm clusters of Ca 2ϩ and HPO 4 2Ϫ /PO 4 3Ϫ ions. The ϳ1.0-nm diameter of the dot-like subunits is similar in size to the core structure of ACP measured by radial distribution function analysis (42,55).…”

Section: Discussionmentioning

confidence: 68%

Physicochemical Characterization of the Nucleational Core of Matrix Vesicles

Genge

Dunkelberger

et al. 1997

Journal of Biological Chemistry

139

154

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While previous studies revealed that matrix vesicles (MV) contain a nucleational core (NC) that converts to apatite when incubated with synthetic cartilage lymph, the initial mineral phase present in MV is not well characterized. This study explored the physicochemical nature of this Ca 2؉ and P i -rich NC. MV, isolated from growth plate cartilage, were analyzed directly by solidstate 31 P NMR, or incubated with hydrazine or NaOCl to remove organic constituents. Other samples of MV were subjected to sequential treatments with enzymes, salt solutions, and detergents to expose the NC. We examined the NC using transmission electron microscopy, energy-dispersive analysis with x-rays, and electron and x-ray diffraction, Fourier transform-infrared spectroscopy, high performance thin-layer chromatographic analysis, and SDS-polyacrylamide gel electrophoresis. We found that most of the MV proteins and lipids could be removed without destroying the NC; however, NaOCl treatment annihilated its activity. SDS-polyacrylamide gel electrophoresis showed that annexin V, a phosphatidylserine (PS)-dependent Ca 2؉ -binding protein, was the major protein in the NC; high performance thin-layer chromatographic analysis revealed that the detergents removed the majority of the polar lipids, but left significant free cholesterol and fatty acids, and small but critical amounts of PS. Transmission electron microscopy showed that the NC was composed of clusters of ϳ1.0 nm subunits, which energy-dispersive analysis with x-rays revealed contained Ca and P i with a Ca/P ratio of 1.06 ؎ 0.01. Electron diffraction, x-ray diffraction, and Fourier transform-infrared analysis all indicated that the NC was noncrystalline.

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Section: Discussionmentioning

confidence: 68%

Physicochemical Characterization of the Nucleational Core of Matrix Vesicles

Genge

Dunkelberger

et al. 1997

Journal of Biological Chemistry

139

154

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“…Since y-carboxyglutamic acid is stable under the conditions of alkaline hydrolysis of proteins (1, 2), but decarboxylates on acid hydrolysis, peptides containing residues 1-19, 20-38, and 39-42 were subjected to alkaline hydrolysis and amino acid analysis. This established the presence of one Gla and no Glu residues in peptide [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] and two Gla and 1 Glu residues in peptide 20-38. There were no Gla residues in peptide 39-42.…”

Section: Resultsmentioning

confidence: 99%

“…There were no Gla residues in peptide 39-42. Identification of Gla at position 17 is based also on the release of one Gla and no Glu residues upon exhaustive carboxypeptidase y digestion of peptide [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], and on the complete absence of any detectable spot in the Glu and Gln positions upon thin-layer chromatography of the phenylthiohydantoin derivative of residue 17 (8 9 and the synthetic phenylthiohydantoin derivative of 4-hydroxyproline have the same mass spectrum and both give 4-hydroxyproline upon hydrolysis in HCI (9). The previous report that this protein contains no hydroxyproline (1) was in error.…”

Section: Resultsmentioning

confidence: 99%

Primary structure of the gamma-carboxyglutamic acid-containing protein from bovine bone.

Price¹,

Poser²,

Raman³

1976

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

210

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The amino-acid sequence of the y-carboxyglutamic acid-containing protein of bovine bone is presented. The sequence of 43 of the 49 residues was determined automatically on the intact protein and on a tryptic peptide. The remainder was determined by conventional procedures on tryptic and chymotryptic peptides. Residue 9 in the sequence has been identified as 4-hydroxyproline. The rotein contains three y-carboxyglutamic acid residues, which are located at positions 17,21, and 24. The role of these unusual amino acids in the binding interaction between the protein and hydroxyapatite crystals is discussed.We described previously the isolation from calf bone of a 5700 molecular weight protein that contains three y-carboxyglutamic acid (Gla) residues (1). This protein (Gla protein) was extracted from calf cortical bone upon demineralization in EDTA and accounts for 1-2% of the total protein in calf bone. The same Gla protein was also found in calf cancellous bone and tooth dentine, and a similar protein was isolated from swordfish vertebrae and human tibia (1). A Gla-containing protein also has been isolated from chicken tibia (2). The abundance and wide distribution of this unusual bone protein suggest that it has an important function in calcified tissues. We have described two important properties of the Gla protein: its strong binding to hydroxyapatite crystals and not to amorphous calcium phosphate, and its inhibition of the initial formation of hydroxyapatite crystal nuclei (1). To obtain further insight into the function of this protein, we have undertaken the determination of its amino acid sequence. MATERIALS AND METHODSPurified calf bone Gla protein was isolated from calf cortical bone as described (1), and was reduced and S-aminoethylated (3) prior to use. Automatic sequence analysis was performed with the Beckman Sequencer (model 890 B). Residues were identified by gas chromatography of the phenylthiohydantoin derivatives (4) and by amino acid analysis of the residues released from phenylthiohydantoin derivatives by hydrolysis in HI (5). The Gla protein was subjected to tryptic or chymotryptic hydrolysis. Peptides were isolated by gel filtration on Sephadex G-25, by ion exchange chromatography, and by high voltage electrophoresis. The sequences of small tryptic and chymotryptic peptides were determined by manual Edman degradation and subsequent dansylation techniques (6). Carboxypeptidase y (EC 3.4.12.1) digestion was carried out with 0.3% carboxypeptidase y for 3-15 hr at 25°in 0.1 M pyridine acetate buffer, pH 5.5. Amino acid analyses were performed with a Beckman amino acid analyzer (model 119 B) on acid and alkaline hydrolysates (1). Amide side chains were identified by thin-layer chromatography. RESULTSThe amino acid sequence of the Gla protein from calf bone is shown in Fig. 1. A sequencer analysis of the intact S-aminoethylated protein established the sequence of the first 20 residues and also identified additional residues in positions up to residue 35. Three peptides were isolated from a t...

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“…The atomic positions of the structure of OCP are very close to HA structure, which is the precondition for possible epitaxial growth and formation of interlayered structures, important for explanation of the process of biomineralization. A special position holds the amorphous calcium phosphate (ACP) which structure is built of Ca 9 (PO 4 ) 6 , so called Posner's clusters, where Ca 2+ and PO 4 3-ions are arranged in a hexagonal dense packing (Betts et al, 1975;Blumenthal et al, 1977). The existing symmetry relations between these structures ensure the easier phase transformations.…”

Section: Structuresmentioning

confidence: 99%

“…It is known that the fast mixing, the high supersaturation and the presence of Mg 2+ and CO 3 2-ions provoke the precipitation of an amorphous calcium-deficient product (Sinyaev et al, 2001;Combes & Rey, 2010). The continuous co-precipitation was carried out by mixing Solution 1 and Solution 2 (Table 4) at a rate of 3 ml/min to precipitate in glycine buffer (Sykora, 1976) (Betts et al, 1975;Blumenthal et al, 1977) of the complex formula Ca w Mg x Zn y Na z K u (PO 4 ) v (CO 3 ) 6-v (w+x+y+z+u ≤ 9) are the first particles formed in the studied complex electrolyte SBFCaCl 2 -MgCl 2 /ZnCl 2 -KOH -H 2 O system. A modifying ion, whose ionic radius and electrical charge are closer to those of the Ca 2+ ions, will be more readily incorporated into the Pozner's clusters.…”

Section: Biomimetic Precipitation Of Ion Modified Precursorsmentioning

confidence: 99%