The mechanisms by which bone resorbing osteoclasts form and are activated by hormones are poorly understood. We show here that the generation of oxygen-derived free radicals in cultured bone is associated with the formation of new osteoclasts and enhanced bone resorption, identical to the effects seen when bones are treated with hormones such as parathyroid hormone (PTH) and interleukin 1 (IL-1). When free oxygen radicals were generated adjacent to bone surfaces in vivo, osteoclasts were also formed. PTH and IL-1-stimulated bone resorption was inhibited by both natural and recombinant superoxide dismutase, an enzyme that depletes tissues of superoxide anions. We used the marker nitroblue tetrazolium (NBT) to identify the cells that were responsible for free radical production in resorbing bones. NBT staining was detected only in osteoclasts in cultures of resorbing bones. NBT staining in osteoclasts was decreased in bones coincubated with calcitonin, an inhibitor of bone resorption. We also found that isolated avian osteoclasts stained positively for NBT. NBT staining in isolated osteoclasts was increased when the cells were incubated with bone particles, to which they attach. We confirmed the formation of superoxide anion in isolated avian osteoclasts using ferricytochrome c reduction as a method of detection. The reduction of ferricytochrome c in isolated osteoclasts was inhibited by superoxide dismutase.Our results "suggest that oxygen-derived free radicals, and particularly the superoxide anion, are intermediaries in the formation and activation of osteoclasts. (J. Clin. Invest. 1990.

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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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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