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The enzyme activities of the major kinases found within the cytosolic and microsomal fractions of embryonic avian calvaria osteoblasts were assayed for their specificity for various noncollagenous extracellular matrix (ECM) proteins of bone. At least 6 proteins with M(r)'s of 66, 58, 50, 36, 30, and 22 kD out of more than 30 of the noncollagenous proteins of the bone ECM were phosphorylated by the kinase(s) found in both osteoblast cellular fractions. The purification and N-terminal sequence analysis of three of the above proteins, M(r)'s 66 and 58 kD (+50 kD), identified them as chicken bone sialoprotein (BSP) and osteopontin (OPN), respectively. Heparin, a specific inhibitor of factor-independent protein kinase (FIPK) activity, blocked the phosphorylation of all six ECM proteins by the microsomal kinase(s) but only inhibited the phosphorylation of the 66, 50, and 36 kD by the cytosolic enzyme(s). Casein kinase II (a known FIPK) showed a similar phosphorylation pattern of the same bone ECM proteins as the FIPK(s) found in osteoblast cell extracts, while purified cyclic adenosine monophosphate (cAMP)-dependent protein kinase did not phosphorylate any of the ECM proteins. Use of dephosphorylated casein showed that in comparison with casein kinase II, casein was a poor substrate for the FIPK found in the osteoblast cellular extracts. Further studies, using FIPK(s) of osteoblasts and purified chicken OPN or bacterially produced recombinant murine OPN as a substrate, showed that both species of OPN were excellent substrates for the FIPK(s) found in osteoblasts. The phosphorylation of the purified chicken and recombinant mouse OPNs were evaluated by quantitative analysis using commercially available protein kinases. cAMP-dependent kinase showed no phosphorylation of either protein, and cyclic guanodine monophosphate (cGMP)-dependent kinase and protein kinase C incorporated 1.2 and 0.5 mol phosphate/mol OPN, respectively. However, both chicken and mouse OPNs were significantly phosphorylated by casein kinase II (9.3 and 9.0 mol of phosphate/mol of OPN, respectively). These results demonstrate that the noncollagenous proteins of the bone ECM, and in particular OPN, are predominantly phosphorylated by FIPK(s), and this class of kinase is the major enzyme found within the microsomal fraction of osteoblasts.

The calcium-phosphate (CA-P) crystals formed in the extracellular organic matrix synthesized by chicken osteoblasts in cell culture were examined after 30, 40, and 60 days of culture by a number of physical and chemical techniques including chemical analyses, X-ray diffraction, transmission electron microscopy of isolated crystals, and resolution-enhanced Fourier transform infrared spectroscopy. The data reveal that the solid inorganic calcium-phosphate phase consists of a very poorly crystalline apatite, having a low carbonate content and containing acid phosphate groups. The chemical and structural characteristics are generally similar to the apatite crystals found in young newly synthesized bone but there were small but significant differences found. The major significant differences noted were the rate at which maturational changes occurred in the crystals formed in cell culture compared with those noted in vivo and in synthetic carbonate apatite crystals equilibrated with the same cell culture medium, and the persistence of labile groups, especially HPO4(-2) ions during a relatively long period of incubation. Despite extensive chemical efforts to degrade the organic constituents and to disperse the individual crystals isolated from the organic matrix constituents, a large proportion of the crystals were found to be organized in both loosely and densely packed relatively large roughly spherical aggregates. A few of the aggregates were organized in the form of fibrils with the crystals oriented with their c-axes roughly parallel to the long axes of the crystal aggregate. With briefer periods of chemical treatment, larger aggregates of crystals were occasionally observed in which there was a distinct axial periodicity of approximately 70 nm. In such collagen-crystal fragments, the crystals were well-oriented with their c-axis roughly parallel to the long axes of the aggregate similar to the organization and relationships between crystals and collagen fibrils in native bone. Isolated crystals were in the shape of thin plates. At the end of 30 days of culture, many of the crystals were clearly larger than those observed in native chick bone, except for those in the very youngest (7- to 8-day-old) embryos. At the end of 40 and 60 days of culture, the crystal habit remained as thin plates but the crystals were predominantly smaller, similar to those found in older embryo and postnatal chicken bone. The marked tendency of the crystals to form relatively large aggregates that resist dispersion by techniques that readily disperse the crystals of bone, and the presence of a significant number of larger crystals has also been observed in studies of calcified cartilage. Resolution enhanced FTIR spectroscopy revealed the presence of a high concentration of labile phosphate groups, especially after 30 days of culture and just after the plateau of mineralization is reached.

Bone sialoprotein is an extracellular noncollagenous acidic protein that plays a role in bone mineralization and remodeling. Its expression is restricted to mineralized tissues and is subjected to variety of posttranslational modifications including phosphorylation and glycosylation. We have expressed the full-length and half domains of bovine bone sialoprotein in a prokaryotic system and identified the phosphorylation sites of casein kinase II. The N-terminal automated solid-phase sequencing defined four phosphorylated peptides: residues 28-38 (LEDS(P)EENGVFK), 51-86 (FYPELKRFAVQSSS(P)DS(P)S(P)EENGNGDS(P)S(P)EEEEEEEETS(P)), 151-165 (EDES(P)DEEEEEEEEEE), and 295-305 (GRGYDS(P)YDGQD). Nine phosphoserines were identified within the four peptides. Seven of them were in the N-terminus (S31, S64, S66, S67, S75, S76, and S86) and two were in the C-terminus (S154 and S300) of the protein.

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF, which differ in their expression patterns as well as their biochemical and biological properties. We examined the expression levels of VEGF isoforms and their receptors in the medial and lateral meniscus of rabbits under normal physiologic conditions as well their expression levels after 8 and 24 h of systemic normobaric hypoxia (13%). VEGF121 is the most abundant VEGF isoform in the medial and lateral meniscus, followed by VEGF165, VEGF189, and VEGF183. While the soluble VEGF121 and VEGF165 are only upregulated at 8 h of hypoxia, the membrane-bound VEGF183 and VEGF189 are further increased at 24 h. VEGFR-2 is expressed at a much higher level than VEGFR-1 under normal conditions, and both receptors are upregulated under hypoxia. Differential expression levels under normoxia as well as a differential response to hypoxia may indicate different functions of VEGF isoforms in the meniscus.

A new system for the local delivery of chemotherapy to malignant solid tumors has been developed based on calcium phosphate (CaP) nanoparticles. The adsorption of the anti-neoplastic drug cis-diamminedichloroplatinum (cisplatin) was characterized on three types of apatitic CaP (poorly and well crystallized hydroxyapatite, and carbonated apatite). Adsorption isotherms obtained in chloride-free phosphate solutions at pH = 7.4 (24 and 37 degrees C) indicate that cisplatin adsorption increases with temperature and increases with decreasing crystallinity. Release studies in phosphate buffer saline (containing the chloride ion essential for release) showed that while the cumulative amount of released drug was the same for all apatites at 20 days (approximately 70% of the total bound), the least crystalline material released the drug more slowly. The drug release rate increased slightly with temperature. Cytotoxicity testing was conducted in a K8 clonal murine osteosarcoma cell line to verify that drug activity was retained after adsorption onto the apatite crystals. K8 cells were plated onto dried films of the apatite/cisplatin conjugates and after 24 h, viability was measured with tritiated uridine. The apatite/cisplatin formulations exhibited cytotoxic effects with a dose dependent diminishment of cell viability.

Cytosolic and microsomal protein kinase preparations from cultured chicken osteoblasts were found to phosphorylate up to six major proteins with Mrs 66, 58, 50, 36, 32, and 22 kDa in chicken bone extract. Use of heparin led to the conclusion that these proteins were predominantly phosphorylated by factor-independent protein kinase (FIPK) present both in microsomal and cytosolic preparations. It was confirmed that microsomal preparation contained predominantly FIPK, whereas cytosolic preparation contained additional kinases, that can phosphorylate the bone proteins. Use of purified chicken bone osteopontin (OPN) (58 kDa) and recombinant OPN led to the same conclusions. The identify of the protein kinases was clearly established by using a series of synthetic peptide substrates. Quantitative analysis utilizing pure protein kinases and purified chicken bone OPN, recombinant mouse OPN, and bovine bone OPN and BSP led to introduction of approximately 9 moles of phosphate/mole of OPN and 6.6 moles phosphate/mole bovine bone sialoprotein (BSP) by casein kinase II. cGMP-dependent protein kinase and protein kinase C both introduced 0.5-1.2 moles phosphate/mole of OPN and BSP, whereas cAMP-dependent protein kinase led to no significant phosphorylation of OPN or BSP. Consistent with the above results, sites of phosphorylation identified for OPN (metabolically labeled) and BSP (labeled by casein kinase II) revealed that predominant phosphorylated sites have recognition sequences for FIPK.

Calcium phosphate crystals deposited in the organic matrix synthesized by chick bone osteoblasts in culture were studied by x-ray and electron diffraction, Fourier transform infrared spectroscopy and chemical composition. The amounts of mineral phase deposited with time and the extent of calcification (% of mineral phase in the tissue) were also determined as a function of time, as were the nature of the changes in the short range order of the crystals. The amount of mineral deposited and the extent of calcification increased with time; the tissue not only contained more crystals of apatite, but the extent of calcification also increased with time as it does in vivo. After 30 days of culture the extent of calcification in the cell culture matrix was similar to that in late chick embryonic and early postnatal chick tibiae. The nature of the CO3 and HPD4 environments were similar to those found in vivo although the concentrations of these ions and the changes in their concentrations with time appeared to develop more slowly in cell culture than they do in vivo. However, the general overall pathway of maturation was similar in cell culture to that observed in vivo.

Chicken bone powder, sequentially extracted with 4M guanidine HCl (NG), 0.3M HCl (H), 0.5 NaCl at neutral pH (NS), and 4M guanidine HCl pH 7.4 (G), preferentially solubilized osteocalcin (OC), osteopontin (OPN), alpha 2 HS-glycoprotein, bone sialoprotein (BSP), and osteonectin (ON). These studies provide the first amino acid sequences for a non-mammalian form of ON and both N-terminal and internal amino acid sequences obtained for ON demonstrated that the avian form of this protein is more than 80% conserved for all the peptides sequenced in this study. A novel approximately 60 kDa protein was identified with a unique N-terminal amino acid sequence and several internal amino acid sequences. Amino acid composition of this protein indicates that it is similar to other acidic glycosylated phosphoprotein of bone, and it is phosphorylated principally on serine residues, although a small amount of phosphorylated threonine residues were also detected. Solubility characteristics of this protein differed from those of ON, OPN and Decorin, and, it was not recognized by polyclonal antibodies to ON, BSP, OPN or by a monoclonal antibody to Decorin. This protein was not present in chicken serum and no comparable sequences were found in the GenBank or EMBL protein sequence database. These studies provide the first sequence for a non-mammalian form of ON and identify a novel acidic bone protein.

To understand the role of post-translational modifications on the structure and function of osteopontin, a secreted glycosylated phosphoprotein, we expressed mouse osteopontin in E. coli as a fusion protein with glutathione-S-transferase (GST). The purified fusion protein was cleaved by factor Xa generating GST (26 kDa) and recombinant osteopontin (60 kDa). The fusion protein was phosphorylated in vitro by cytosolic, microsomal, and casein kinase II fractions from mouse kidney homogenates. The fusion protein and recombinant osteopontin were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. The suitability of the fusion and recombinant proteins as model substrates for the study of the function(s) and post-translational modifications of osteopontin is discussed.

Affinity chromatography of “Enamelin Extracts” of developing bovine molar enamel on CNBr activated Sepharose 4B to which polyclonal antibodies of whole bovine serum and fetuin were cross-linked, revealed that at most, only 1–2% of the proteins in the extracts were not bound to the columns. The approximately 98% or more of the proteins in such extracts were bound to the resin and were eluted in the position of the serum proteins and fetuin. The small amount of protein which was not bound to the affinity column and which was eluted very early, was subjected to SDS-PAGE and immunostained with polyclonal antibodies to two non-amelogenin proteins isolated and identified previously (∼26kDa and 22kDa). The antibody to the ∼50kDa protein immunostained strongly with only one protein band of ∼26kDa. The antibody to the ∼22kDa band reacted strongly with one protein band of ∼22kDa, weakly with an ∼100kDa and very weakly with several lower molecular weight bands, suggesting either aggregation and degredation of the 22...

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.

Osteopontin is a noncollagenous, phosphorylated extracellular glycoprotein, expressed in mineralized and nonmineralized tissues, organs and body fluids. The protein contains an RGD tripeptide cell-binding motif, and is subjected to a variety of posttranslational modifications that play important roles in its multiple biological functions, such as bone remodeling and inhibition of pathological calcification. In this study, we have expressed bovine osteopontin in a prokaryotic system and identified the seven amino acid residues phosphorylated in vitro by CKII.

Embryonic chicken osteoblasts cultured over a 30 day period were used as a model system for studying the expression of bone phosphoproteins during cellular differentiation and the possible role of these proteins in extracellular matrix mineralization. Accumulation of total phosphoprotein in the cultures, as determined by O-phosphoserine (Ser-P) and O-phosphothreonine (Thr-P) amino acid analysis, revealed a greater than 10-fold increase over the 30 day period. Total phosphoprotein synthesis, as assessed by (32P)-, (3H)-Ser-P, and (14C)-Thr-P protein labeling, showed the highest levels concurrent with initial mineral deposition within the matrix. The major phosphoprotein present in chicken bones and synthesized by the cultured osteoblasts had a molecular weight of approximately 66 kDa. This 66 kDa bone phosphoprotein (66 kDa BPP) was purified to homogeneity and was used for antibody production. Application of this antibody in Western blot analysis revealed that 66 kDa BPP was present only in protein extracts of mineralizing cultured osteoblasts and was absent in cultures of non-mineralizing chondrocytes, myoblasts, and tendon fibroblasts. The 66 kDa BPP in vitro accumulated continuously in the extracellular matrix in a manner that paralleled both phosphoprotein synthesis and total phospho-amino acid production. A comparison of the results obtained in vitro to those from developing embryonic tibiae in vivo demonstrated a similar qualitative and temporal expression of phosphoprotein and a continual accumulation of 66 kDa BPP in the matrix with advancing mineralization and developmental age. Ultrastructural immunocytochemistry using the 66 kDa BPP antibody and the protein A-gold technique revealed specific immunolabeling over electron-dense regions of mineralization in the cultures that appeared identical to the distribution of labeling observed in vivo (McKee et al.: Connect. Tissue Res., 21:21-29, 1989; Anat. Rec., 228:77-92, 1990). These results demonstrate that this major 66 kDa BPP was expressed concurrently with other differentiated osteoblast functions and suggests that it may play a role in the initiation or regulation of mineralization.

The major enamelin protein component present in EDTA or EDTA/guanidine hydrochloride extracts of developing bovine enamel has a molecular mass of about 67 kDa; it has an amino acid composition similar to that of bovine serum albumin and reacts with polyclonal and monoclonal antibodies to albumin. Two-dimensional separation of the components in the enamelin extract by isoelectric focusing and SDS/PAGE reveal that the major approximately 67-kDa component and almost all of the minor Coomassie-staining protein components of approximately 67 kDa, as well as many of the other minor components with different molecular masses, also react with polyclonal and monoclonal antialbumin. The approximately 67-kDa band eluted after SDS/PAGE, as well as the major approximately 67-kDa spots eluted after two-dimensional separation, were found to have N-terminal amino acid sequences identical to that of bovine serum albumin. Albumin accounted for at least 70-80% of the total protein content of the enamelin extract and was essentially the only protein in the approximately 67-kDa component. The serum proteins alpha-2 HS glycoprotein, gamma-globulin and fetuin, and the proline-rich salivary protein termed P-B were also identified in the enamelin extract. The serum proteins and the salivary protein account for greater than 95% of the proteins in the enamelin extracts. Of the remaining very small amounts of non-serum or salivary protein isolated from the enamelin extracts, three minor components were isolated which had N-terminal amino acid sequences which were not similar to any known protein in the protein sequence data base and could therefore conceivably be true 'enamelins' synthesized by ameloblasts. One additional protein had the first five N-terminal amino acids and residue 8 of amelogenin, residues 6 and 7 being different from those of amelogenin. Two other very minor protein components had amino acid compositions distinct from the amelogenins and the serum proteins, but were N-terminally blocked on attempted sequencing. None of the components in the neutral soluble low-ionic-strength extract or in the 4 M guanidine hydrochloride extract, both of which consist principally of amelogenins, immunoreacted with anti-albumin or with any of the antibodies to other serum proteins and fetuin, despite the fact that the amelogenin extracts also contain non-amelogenin proteins. On the basis of the data presented, studies employing antibodies to the so-called enamelin proteins and hypotheses as to their molecular conformation, their roles as evolutionary markers, or their positive role in mineralization should be reconsidered and reviewed.

In order to understand the mechanism of the post-translational processing of bone phosphoproteins in embryonic bone, periosteal bone strips isolated from 12-day-embryonic-chick tibiae were cultured and the bone proteins labelled with Na2H32PO4. Of the total radiolabelled proteins recovered from the medium and bone extracts in the absence of SDS (‘medium’, ‘EDTA extract’ and ‘EDTA/guanidinium chloride extract’), nearly 80% of the radioactivity was found in the EDTA extract. The three major radiolabelled phosphoproteins in the EDTA extract of apparent Mr 68,000, 63,000 and 58,000 reacted with polyclonal as well as monoclonal antibodies raised against ‘32-kDa’ and ‘150-kDa’ bone phosphoproteins which were derived from 14-week-old chicken. Therefore these phosphorylated embryonic proteins are identified as chicken bone phosphoproteins. Judging from their common N-terminal sequences, differences in the patterns obtained by labelling them with several radioisotopes, and slightly different amino acid compositions, these components seem to have been derived from the same original protein by sequential proteolytic cleavage and other processing such as glycosylation and phosphorylation.

The vibrational circular dichroism (VCD) spectra of metal-free phosvitin are presented as a function of pH and analyzed both qualitatively and by using a factor analysis approach referenced to a protein data set. The qualitative pattern of both the IR and VCD changes is consistent with a coil-to-sheet transition occurring as pH is progressively decreased to values lower than 3. A similar transition was seen in commercial preparation of phosvitin which still contained metal ions, but there the transition was more gradual and occurred at somewhat different pH values. Such a gradual change is also evident in the solution phase absorption band profile but is made clearer using Fourier deconvolution. Based on VCD results, the low pH transition appears to occur with two distinct manifestations of the beta-sheet form. However, at the lowest pH values the sample may precipitate. These two forms are not distinguishable with Fourier transform infrared alone and may be due to a twist of the beta-sheet form or to aggregation.

Amino acid analyses of undecalcified samples of fossil crocodile and rhinoceros enamel and dentin from mature teeth revealed that the total protein content of these mineralized fossil tissues varied from ~0.01–0.007% by weight. Except in one instance, amino acid analyses of the enamel proteins revealed them to be free of collagen and to have an amino acid composition similar to the proteins obtained from the enamel of mature modern vertebrates. Molecular sieving of the acid soluble enamel proteins demonstrated that the components consisted principally of small peptides and free amino acids, as in the enamel of modern vertebrates.Based on the presence of hydroxyproline (hyp) and hydroxylysine (hyl), collagen was detected in undecalcified mature dentin of fossil rhinoceros, but not in undecalcified crocodile dentin. It was only by sequential extraction procedures that the presence of collagen in the dentin of fossil crocodile was established, emphasizing the utility and importance of analyzing the soluble components in specific extracts of the fossil. Based on these data and the concentrations of hyp and hyl in the material solubilized by the various solvents, the dentin of fossil rhinoceros contained considerably more collagen per weight and as a percentage of the total protein in the dentin than did the dentin of fossil crocodile.As with modern dentin, EDTA and dilute acid solubilize the noncollagenous proteins and peptides found in fossil enamel and dentin, some of which contain O-phosphoserine [Ser(P)], an amino acid unique to mineralized connective tissues. Similar to recent reported findings from fossil bone, less of the original content of the noncollagenous proteins, including those phosphorylated, is degraded and removed from the enamel and dentin during fossilization than the percentage of dentinal collagen and the nonphosphorylated domains of the enamel proteins which are removed. This selective resistance to degradation and removal of the noncollagenous proteins, including phosphoproteins, may reflect the strong interaction of these proteins with the mineral phase of fossilized tissues. The amount and close packing of the inorganic crystals may also inhibit the interaction of the proteins in the interior of the enamel and dentin with the geochemical environment.

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF. Those isoforms differ in biochemical and biological properties, and it has been reported that their expression patterns are tissue and age specific as well. We investigated the expression levels of VEGF isoforms (VEGF121, VEGF165, VEGF183, VEGF189) and its receptors (VEGFR-1, flt-1 and VEGFR-2, flk-1/KDR) in the anterior cruciate ligament (ACL) of 2- to 3-week-, 2-month-, and 18-month-old New Zealand White rabbits using Sybr green Real-Time RT-PCR. VEGF isoforms and both receptors were expressed in the ACL at all investigated ages. VEGF121 was found to be the most abundant isoform at the ages under investigation, followed by VEGF165, VEGF189 and VEGF183. All isoforms showed decreased expression levels with age, however the larger membrane bound isoforms, VEGF183 and VEGF189, showed the most striking age-associated decrease in expression level. VEGFR-1 expression levels increased with age, while the expression level of VEGFR-2 expression was highest at 2–3 weeks and was significantly lower at 2 and 18 months of age. Distinct age-associated differences in the expression level of VEGF isoforms as well as their receptors suggest differential physiological functions during development, maturation and ageing of the ACL.

Genetic mutations that disrupt osteoblast function can result in skeletal dysmorphogenesis or, more rarely, in increased postnatal bone formation. Here we show that Schnurri-3 (Shn3), a mammalian homolog of the Drosophila zinc finger adapter protein Shn, is an essential regulator of adult bone formation. Mice lacking Shn3 display adult-onset osteosclerosis with increased bone mass due to augmented osteoblast activity. Shn3 was found to control protein levels of Runx2, the principal transcriptional regulator of osteoblast differentiation, by promoting its degradation through recruitment of the E3 ubiquitin ligase WWP1 to Runx2. By this means, Runx2-mediated extracellular matrix mineralization was antagonized, revealing an essential role for Shn3 as a central regulator of postnatal bone mass.

Previous 31P cross-polarization and differential cross-polarization magic angle spinning (CP/MAS and DCP/MAS) solid-state NMR spectroscopy studies of native bone and of the isolated crystals of the calcified matrix synthesized by osteoblasts in cell culture identified and characterized the major PO(-3)(4) phosphate components of the mineral phase. The isotropic and anisotropic chemical shift parameters of the minor HPO(-2)(4) component in bone mineral and in mineral deposited in osteoblast cell cultures were found to differ significantly from those of brushite, octacalcium phosphate, and other synthetic calcium phosphates. However, because of in vivo and in vitro evidence that phosphoproteins may play a significant role in the nucleation of the solid mineral phase of calcium phosphate in bone and other vertebrate calcified tissues, the focus of the current solid-state 31P NMR experiments was to detect the possible presence of and characterize the phosphoryl groups of phosphoproteins in bone at the very earliest stages of bone mineralization, as well as the possible presence of calcium-phosphoprotein complexes. The present study demonstrates that by far the major phosphate components identified by solid-state 31P NMR in the very earliest stages of mineralization are protein phosphoryl groups which are not complexed with calcium. However, very small amounts of calcium-complexed protein phosphoryl groups as well as even smaller, trace amounts of apatite crystals were also present at the earliest phases of mineralization. These data support the hypothesis that phosphoproteins complexed with calcium play a significant role in the initiation of bone calcification.

Ameloblastic tissue samples from unerupted bone molars were used to prepare subcellular enamel protein kinase preparations, nuclear + plasma membrane, cytosolic and microsomal, and used in in vitro phosphorylation of purified 20 kDa bovine amelogenin in the presence of 32P-ATP. Both cytosolic and microsomal preparations can phosphorylate purified native amelogenins, the addition of Ca2+ slightly increased the microsomal enzyme activity or at least did not inhibit the activity, whereas the presence of Ca2+ substantially decreased the cytosolic kinase activity towards phosphorylation of amelogenins. A comparative analysis using the enamel microsomal kinase against osteopontin, dephosphorylated casein and bone sialoprotein showed no phosphorylation of the first two proteins, and only minor phosphorylation of the bone sialoprotein. Overall, the present work demonstrates for the first time that the protein kinase responsible for the phosphorylation of amelogenins is a novel kinase, which is not inhibited by Ca2+, unlike the microsomal protein kinase (casein kinase type-II) of bone which phosphorylates secretory proteins osteopontin and bone sialoprotein and is strongly CaZ+ inhibited. The direct phosphoserine analysis on the purified bovine 20 kDa amelogenin indicated the presence of 0.8 moles of phosphoserine/mole protein naturally occurring, consistent with the quantitative analysis of 14C-radiolabeling of phosphoserines by conversion to dehydroalanine and in situ reaction with the thiol agent, 14C-mercaptoethanol, 0.64 moles 14C-incorporated/mole 20 kDa amelogenin. The purified low Mramelogenins 5.3 kDa E4 (TRAP) and 7.2 kDa E3 (LRAP), were also derivatized by 14C-mercaptoethanol, providing 0.46 and 0.88 moles 14C-incorporated/mole respectively. Further studies of the 14C-radiolabeled E4 amelogenin by sequence analysis confirmed one site of label to be at position 16 from the N-terminal and hence provided a direct evidence for the naturally occurring phosphoserine residue at this position.

Osteopontin (OPN) is one of the major secretory phosphoproteins in both calcifying and non-calcifying tissues. Evidence has accumulated for the biological importance of the phosphoproteins and, in particular, the phosphate groups in bone formation, resorption, and calcification. The precise locations of the phosphate groups in the OPN molecule were determined by metabolically labeling OPN with32P in cultured chicken osteoblasts, followed by purification to homogeneity. N-terminal sequencing showed a single sequence of WPVSKRQHAISA, consistent with that deduced from both cDNA, and previous amino acid sequencing of the protein isolated from chicken bone. Three 32P-labeled peptides were isolated by reverse-phase high performance liquid chromatography of thrombin-digested, 32P-labeled OPN. The N-terminal sequencing of each of these thrombin fragments gave single sequences as follows: WPVSKSRQHAIS, SHHTHRYHQDHVD, and ASKLRKAARKL, with approximate molecular masses of 5, 30, and 20 kDa. These data demonstrate that32P was incorporated throughout the N- to C-terminal sequence of the protein. Thrombin specifically cleaved chicken OPN at two sites: between Arg-22 and Ser-23, which generated the 5-kDa N-terminal end fragment, and another between Lys-138 and Ala-139, which generated the 30- and 20-kDa fragments. To further define the exact locations of the phosphorylated amino acids and the surrounding amino acid sequences, OPN was digested with trypsin, which generated seven major 32P-labeled peptides whose amino acid sequences were determined. The phosphorylated peptide regions of osteopontin were identified as amino acids 8–18 (QHAIS*AS*S*EEK), 39–54 (LASQQTHYS*S*EENAD), 150–171 (LIEDDAT*AEVGDSQLAGLWLPK), 179–191 (ELAQHQSVENDSR), 194–205 (FDS*PEVGGDSK), 214–219 (ES*LASR), and 239–248 (HSIENNEVTR). The phosphorylated amino acid sites are followed by an asterisk (*). Of the seven identified phosphorylated peptide regions, three were localized on the N-terminal end of the osteopontin molecule (with five phosphorylated serines) and contained the sequence motifs that were phosphorylated by casein kinase II type(s), whereas the remaining four peptides are concentrated toward the C-terminal half of the molecule (with five phosphorylated residues) and contained recognition motifs for other kinases as well as casein kinase II.

The large number of covalently bound phosphates on the extracellular phosphoproteins osteopontin (OPN) and bone sialoprotein (BSP) have been implicated in biological functions such as mineral deposition and osteoclast binding. In the present study the state of phosphorylation of BSP and OPN was evaluated by in vitro 32P labeling using a series of protein kinases and quantification. Both the purified bovine BSP and OPN were radiolabeled by [32P]ATP and factor-independent protein kinase. Quantification of 32P radioactivity incorporated on dephosphorylated BSP and OPN provided 6.6 and 8.9 mol of phosphate incorporated/mol, respectively. Native OPN incorporated 1.07 and BSP 2.46 mol of phosphate/mol by factor-independent protein kinase. These data led to calculations that OPN and BSP, respectively, contain 7.83 and 4.14 mol of phosphate/mol in their natural state. Thrombin digests of 32P-labeled BSP showed radioactivity to be associated with fragment of approximately molecular mass values 30 kDa (N-terminal half), with no observable radioactivity associated with the 40-kDa fragment (C-terminal half). Similar experiments with 32P-labeled OPN provided two radiolabeled thrombin fragments, with molecular mass 30 kDa (N-terminal half) and 20 kDa (C-terminal half), both were radioactive. The major phosphorylation was associated with the N-terminal half containing 7.0 mol of phosphate, and 1.9 mol of phosphate were associated with the C-terminal half. Additional experiments of in vitro phosphorylation of OPN and BSP by several other known protein kinases were carried out. cAMP-dependent protein kinase showed no phosphorylation of OPN or BSP, while protein kinase C and cGMP-dependent protein kinase led to minor phosphorylation, each of the latter introduced about 1 mol of phosphate/mol of OPN and BSP molecule.

Aqueous tissue processing and demineralization procedures may adversely affect the inorganic mineral phase of a calcified sample and, where mineral and organic constituents interact, may consequently also indirectly alter organic matrix ultrastructure and distribution. In the present work, the effects of demineralization have been investigated on the retention in chicken bone of two phosphoamino acids, O-phosphoserine and O-phosphothreonine, found in bone phosphoproteins proposed to be important in vertebrate mineralization and, more specifically, on the retention and distribution of a 66 kD bone phosphoprotein (66 kD BPP, osteopontin) also implicated in the calcification process. In tibiae fixed initially with 1% glutaraldehyde and then demineralized in 0.5 N HCl, 0.5 N acetic acid, or 0.1 M EDTA (all containing 1% glutaraldehyde), amino acid analyses and quantitative immunocytochemistry revealed that the phosphoamino acid content and the distribution of the 66 kD BPP were essentially the same as in fixed undemineralized controls. However, demineralization slightly altered the ultrastructural appearance of immunolabeled, electron-dense patches of organic material in the bone matrix. In unfixed bone demineralized with any of these acids, there was a substantial loss of phosphoamino acids and the 66 kD BPP from the bone matrix. The relative ability of these acids to extract phosphoproteins from unfixed bone was found to decrease in the order EDTA greater than HCl greater than acetic acid. These results emphasize the differential effects on structural components of various demineralization and extraction procedures for biochemical and immunocytochemical studies of biologic tissues. Furthermore, they demonstrate that initial fixation with glutaraldehyde retains phosphoproteins in bone, with or without demineralization, while being adequate for immunocytochemical localization of certain bone matrix proteins and that an understanding of the action of specimen preparation on organic constituents (as well as inorganic components) is essential for accurately describing ultrastructural matrix-mineral relationships.

Two phosphorylated proteins of approximately 66 kDa and approximately 60 kDa mass with different DEAE-Sephacel elution patterns were isolated from chicken bone and were shown to be genetically distinct by both biochemical and immunological analysis. A tryptic peptide from the 60 kDa protein was identified that was similar to a sequence of the rat bone sialoprotein II. Both proteins showed RGD inhibited cell-attachment with the MG-63 osteosarcoma cell, and the approximately 66 kDa phosphoprotein appeared to promote cell adhesion better than human vitronectin. The two phosphoproteins appear to share functional and biochemical characteristics and to be homologous to the mammalian bone phosphoproteins, osteopontin and bone sialoprotein II.

We have detected a protein kinase which phosphorylates bone phosphoproteins (BPPs) in the detergent extract of the membranous fractions in the periosteal bone strips of 12-day-embryonic-chick tibia. This enzyme, tentatively named BPP kinase, has a catalytic subunit of Mr approximately 39,000, utilizes GTP as well as ATP as a phospho-group donor, is inhibited by 2,3-bisphosphoglycerate and heparin, and is therefore similar to casein kinase II. The enzyme can phosphorylate dephosphorylated proteins such as casein, phosvitin and chicken BPPs, but the last-named are preferred substrates. The in vitro-phosphorylation-assay products of this enzyme in the extract were indistinguishable on an SDS/polyacrylamide gel from the major [32P]phosphoproteins metabolically labelled in the embryonic-chick bone tissue. The regulatory mechanisms of the phosphorylation process of BPPs by BPP kinase as well as the potential role of this enzyme in mineralization are discussed.

The major phosphoprotein synthesized by cultured chicken embryo osteoblasts had a molecular mass of approximately 66 kDa. The 32P label on the protein was cleaved by acid phosphatase treatment and O-[32P]phosphoserine and O-[32P]phosphothreonine could be identified after partial acid hydrolysis. The phosphoprotein contributed approximately 2.0% of the total protein synthesized by osteoblasts and was shown to be secreted, as shown by its presence in the culture media. Glycosylation was demonstrated by the fact that it could be labelled with [3H]galactosamine. The major ∼ 66-kDa phosphoprotein was resolved by isoelectric focusing into three major variants with pI values ranging over 3.7–3.9; all three forms appear to be the result of variation in the extent of protein phosphorylation. An identical ∼ 66-kDa phosphoprotein could be extracted from chicken bones which had both the same range of pI values and an identical elution position following DEAE-Sephacel chromatography. Analysis of the protein isolated from bone demonstrated the presence of sialic acid and, while amino-terminal sequence analysis and internal tryptic fragment sequence analysis of about 25% of the protein revealed little similarity to the rat phosphoprotein osteopontin, a conserved nine-residue sequence spanning the Arg-Gly-Asp cell-binding site of the rat protein osteopontin, was identified in the ∼ 66-kDa chicken protein. Peptide mapping with Staphylococcus aureus V8 protease of the in vivo protein compared to the in vitro synthesized protein demonstrated identical peptide fingerprints. The two proteins also had comparable amino acid compositions. Several smaller-molecular-mass phosphoproteins ranging in size over about 55–29 kDa were also observed in the HC1 extracts of bone. Peptide mapping of these species demonstrated that the ∼ 66-kDa, ∼ 55-kDa, and ∼ 45-kDa species had a common core of peptide fragments. Pulse/chase experiments in culture revealed no evidence for a defined pathway of intracellular proteolysis associated with the ∼ 66-kDa species since this phosphoprotein remained the prevalent species after a 24-h chase. Because of the predominant association of all the smaller-molecular-mass forms with the cell layer and an absence of a quantitative conversion to any of the smaller forms over a 24-h chase, these results suggested that the lower-molecular-mass species were not the result of proteolytic processing during synthesis or secretion, but rather represent proteolysis of the ∼ 66-kDa component in the extracellular matrix. In summary, these results demonstrate that cultured osteoblasts synthesize a major ∼ 66-kDa phosphoprotein that is identical to the major HCl-extractable phosphoprotein present in postnatal chick bone.

The content of osteocalcin and protein bound gamma-carboxyglutamic acid (Gla) was studied as a function of bone maturation and mineralization in normal and vitamin D-deficient, rachitic chickens. The Gla/Ca2+ ratio was elevated in rachitic bone, particularly in the most undermineralized regions. For example, there is a 10- to 20-fold elevation in Gla/Ca2+ in the newly synthesized, least mineralized rachitic bone fraction, which progressively decreases to a 1.5-fold elevation in the most highly mineralized areas of rachitic tissue. Osteocalcin, which is the principal Gla-containing protein of mature bone, was quantitated by radioimmunoassay using specific antiserum to the 5670-dalton chicken protein. Surprisingly, the osteocalcin concentration is decreased 50% in vitamin D-deficient bone. From this we infer that accumulated Gla-containing protein in vitamin D-deficient and poorly mineralized bone may possibly represent a precursor of osteocalcin.

O-Phosphoserine was positively identified as the phosphorylated moiety in human plasma fibronectin by 31P-NMR of intact peptides. These data correlated completely with chemical analyses which demonstrated the presence of O-phosphoserine at a concentration of 2 residues/molecule. Neither O-phosphothreonine nor O-phosphotyrosine was detected in partial acid and partial alkaline hydrolysates, respectively.

Ethylenediaminetetraacetic acid and HCl extracts of calcified chicken bone were fractionated by a variety of techniques, including molecular sieving in guanidinium chloride, ion-exchange chromatography on DEAE-cellulose, high-performance liquid chromatography (HPLC), reverse-phase HPLC, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using several different experimental schemas, we isolated 14 apparently homogeneous components varying in molecular weight from approximately 150K to approximately 4K-5K. The compositions of all of the phosphoproteins were characterized by high concentrations of Asp, Glu, Ser, Gly, and Ala. Seven of the components which were analyzed contained concentrations of carbohydrate varying from approximately 4% to approximately 17%. Three of the components containing O-phosphoserine which behaved as single bands on SDS-PAGE with molecular weights of approximately 150K, approximately 90K, and approximately 70K contained Hyp and Hyl or Hyl alone and may represent covalently bonded or strongly associated collagen-phosphoprotein complexes or hydroxylated Pro and/or Lys residues of the phosphoproteins. The findings that the amino acid compositions of several of the components were very similar and that N-terminal partial amino acid sequences of the approximately 90- and approximately 60-kilodalton (kDa) and of the approximately 150- and approximately 32-kDa components, respectively, were identical make it clear that some of the lower molecular weight components are derived by proteolysis from higher molecular weight species. In addition to proteolysis, we speculate that it is possible, from the N-terminal amino acid sequence data and preliminary cross-reaction studies of antibodies to four of the phosphoproteins, that the heterogeneity observed in the phosphoprotein components may also be due in part to there being more than one independent gene product for chicken bone phosphoproteins.

The hydroxylation of lysine in embryonic chick long bone and mandibular collagen was found to be approximately 3-fold greater than that of the collagens of adult animals. In contrast, no significant difference was found in extent of lysine hydroxylation of the collagens of frontal bones of embryos and postnatal animals. Both histochemical and biochemical evidence established that full thickness diaphyseal bone samples contained cartilage and, consequently, type II collagen which undoubtedly contributed to the higher hydroxylysine contents of young postnatal animals reported previously. DEAE ion exchange chromatography of the alpha 1(I) chains of lathyritic long bone and mandibular collagens isolated by carboxymethyl-cellulose ion exchange chromatography showed considerable heterogeneity, whereas the alpha 1(I) chains obtained from lathyritic frontal bone collagen did not. Three fractions of alpha 1(I) chains of long bones and mandibular collagen were isolated which differed significantly in their hydroxylysine contents. The relative proportion of the three peaks changed as a function of embryonic age and maturation: more of the alpha 1(I) chains with the highest hydroxylysine content was present in the collagen synthesized earliest during embryonic development. This is consistent with results which demonstrated that the collagens synthesized earliest during embryonic and postnatal development had the highest hydroxylysine contents.

When chicken embryonic progenitor cells were selected and grown in culture as previously described, by 30 days cellular differentiation could be demonstrated by expression of in vivo levels of osteocalcin, alkaline phosphatase (APase), type I collagen and phosphoproteins (PP). Ultrastructural analysis revealed that the cultures were similar morphologically to young osteoid in vivo with structural features including well-developed, orthogonally arranged collagen fibrils with 64-70 nm periodicity and electron opaque areas consisting of very poorly crystalline hydroxyapatite. Analysis of collagen synthesis versus collagen accumulation in the matrix indicated a temporal inconsistency between the time of synthesis and accumulation, suggesting that accumulation was largely controlled at the level of fibril formation. Analysis of PP accumulation demonstrated a 10-fold increase in total phosphoamino acid content over the 30 day time course. PP synthesis analyzed by [3H]-Ser(P) and [14C]-Thr(P) incorporation showed an induction similar to that seen for APase. Experiments undertaken to characterize the nature of PP synthesized by the cultures identified a unique 66 kD protein. This protein was purified from chick tibial and calvarial bone and a polyclonal antibody was raised in rabbits. Ultrastructural immunocytochemistry using this antibody and the protein A-gold technique revealed specific immunolabelling over regions of mineralizing matrix in vitro, a reaction identical to that observed for the distribution of this 66 kD PP in vivo during embryonic tibial bone development in the chicken.

The biosynthesis in vivo of the two reducible aldimine crosslinks of immature rabbit articular collagen, hydroxylysinohydroxynorleucine and hydroxylysinonorleucine, is demonstrated. The peak amount of crosslink was detected 1–2 weeks following labeling of the cartilage with [ 14 C]lysine. The subsequent diminution which occurred was due primarily to a decrease in the amount of hydroxylysinohydroxynorleucine. Natural reduction of the aldimine crosslinks in vivo did not occur. Glucosylgalactosyl hydroxylysine and galactosylhydroxylysine, in a 1.45 1.00 ratio, were synthesized. Seventy-three percent of the hydroxylysine residues were glycosylated. [ 3 H]NaBH 4 reduction of non- 14 C-labeled cartilage showed diminished amounts of reducible crosslink with time and the presence of hexosyl lysines and hexosyl hydroxylysines in mature articular cartilage.

The amount of non-collagenous proteins is increased greatly during the pathological calcification of rat skin experimentally induced by dihydrotachysterol (DHT) and Ovalbumin (topical cutaneous calciphylaxis). This is accompanied by an increase in the total amount and concentrations of protein-bound serine phosphate [Ser(P)], threonine phosphate [Thr(P)] and gamma-carboxyglutamic acid (Gla), almost all of which can be extracted from the tissue and can be dissociated from collagen in 0.5M EDTA. The EDTA-soluble, non-collagenous proteins are rich in aspartic and glutamic acids, similar to the non-collagenous, EDTA-soluble proteins of bone, cementum and calcified cartilage, and quite distinct from those of dentin and enamel.

Twelve-day-old embryonic chick mandibles were cultured in vitro for 6 days. Measurements of the weights of the explants, their mineral and protein components, and the EDTA-extractable proteins established that bone tissue synthesizes O-phosphoserine- and O-phosphothreonine-containing phosphoproteins which are similar to those present in embryonic and postnatal chicken bone matrix. The synthesis of the phosphoproteins was further confirmed by the demonstration that radioactively labeled O-phosphoserine and O-phosphothreonine were identified in bone and in the EDTA-extractable phosphoproteins after pulse-labeling chick mandibles in vitro with radioactively labeled serine and threonine, respectively.

Phosvitin, a highly phosphorylated glycoprotein, represents the major fraction of hen egg yolk phosphoproteins. Circular dichroism, Fourier transform infrared spectroscopy, and Fourier transform infrared photoacoustic and fluorescence spectroscopic methods were employed to determine the secondary structure of the protein in both the solid and solution phases. This was supplemented by a Chou-Fasman type of predictive algorithm for the first 25 residues at the N terminus of the dephosphorylated protein. A three-compartment model consisting of alpha-helical, beta-sheet, and beta-turn components with beta-turns occurring at the interface between alpha-helical and beta-sheet regions in the proximity of O-phosphoserine residues is suggested from the combined analyses. Beta-sheets appear to be the dominant secondary structural component in phosvitin in the solid and solution phases. The suggested model bears many similarities to other phosphoproteins reported in the literature. The secondary structure of phosvitin is observed to be sensitive to environmental factors as previously reported although the present studies differ in some respects from earlier results. Preliminary results suggest that Ca2+ ions trigger a decrease in beta-sheet structure at pH 2.

Amino acid analyses of carefully dissected samples of the articular cartilage of postnatal chickens indicates the presence of significant amounts of a collagen with a low hydroxylysine content. It was found that 9 M LiCl solutions preferentially extract the components of a collagen whose components when identified by ion exchange chromatography and amino-acid analyses, established that they were derived from Type I collagen. Type II collagen was essentially insoluble in solutions of 9 M LiCl, but could be solubilized in 4 M CaCl2 after prior treatment of the cartilage with pepsin. The αl (II) components were then isolated and characterized by their behavior during ion exchange chromatography and by their amino acid composition. The findings establish that the articular cartilage of postnatal chickens contains substantial amounts of Type I collagen and suggest that the nature and stability of the crosslinkages in Type I collagen of cartilage differs significantly from those of Type II collagen of cartilage. Moreover, it is also postulated that the nature of the major intermolecular cross-linkages in the Type I collagen of cartilage are similar to those present in Type I collagen of bone rather than those present in the Type I collabens of skin and other connective tissues.

Purified components of chicken bone collagen contain approximately 4 atoms of organic phosphorus per mol of collagen, located principally in the alpha 2 chains. Previous analyses have demonstrated the absence of O-phosphoserine, O-phosphothreonine, and other phosphorylated hydroxy amino acids, phosphoamidated amino acids, and phosphorylated sugars. In the present report we establish that chicken bone collagen contains gamma-glutamyl phosphate. This was accomplished by the isolation of tritiated alpha-amino-delta-hydroxyvaleric acid after reductive cleavage with NaB[3H]H4 of the gamma components, the alpha 2 chains, and peptides enriched in organic phosphorus that were derived from the alpha 2 chains. Tritiated alpha-amino-delta-hydroxyvaleric acid was not detected in any of the following unphosphorylated proteins after cleavage with NaB[3H]H4:albumin and lysozyme, the alpha 2 chains of several unmineralized tissues, and, most importantly, dephosphorylated alpha 2 chains of chicken bone collagen. The alpha 2 chain of chicken bone collagen is the first structural protein found to contain an acyl phosphate.

The major, non-amelogenin protein component (enamelin) present in EDTA or EDTA-GU HCl extracts of developing bovine enamel has a molecular weight of approximately 67 kD and an amino acid composition rich in asp, glu, ala, leuc and lys. Elution of this component from 1.5 and 3.0 mm thick strips SDS-PAGE gels and subsequent analyses show that the component selectively reacts with polyclonal antibody to bovine serum albumin. Absolute identification of this major enamelin component as serum albumin is established by an amino acid sequence of the first 40 N-Terminal amino acids which was found to be identical to bovine serum albumin. In addition to albumin, alpha-2 HS glycoprotein was also identified in the same extracts by Western blotting against a monospecific polyclonal antibody against human alpha-2 HS glycoprotein.

O-phosphoserine [Ser(P)], O-phosphothreonine [Thr(P)], and gamma-carboxyglutamic acid (Gla) have been identified in native, calcified cementum and in non-collagenous proteins which can be extracted from the tissue in EDTA at neutral pH. The concentrations of Ser(P) and Thr(P) and the amino acid composition of the EDTA extractable proteins are more similar to those found in bone than in dentin or enamel. The concentration of Gla in cementum is lower than it is in bone and higher than it is in enamel, which contains essentially no Gla. Based on the contents of Gla in these mineralized tissues and the distribution of alkaline and acid phosphatases in these tissues, it is speculated that Gla may be part of these or other proenzymes rather than being involved directly and structurally with the deposition of the mineral phase.

High-voltage paper electrophoresis and analytical and preparative amino acid ion-exchange chromatography were used to investigate the products resulting from strong acid treatment and strong acid treatment and subsequent alkalinization of alpha-amino-delta-hydroxyvaleric acid. After strong acid treatment, alpha-amino-delta-hydroxyvaleric acid and proline, as well as several unidentified components, presumably lactones, were recovered. No definitive identification of the putative unstable lactones was accomplished either by physical-chemical means or by ion-exchange chromatography or high-voltage paper electrophoresis. Nevertheless, after acid treatment and alkalinization, only alpha-amino-delta-hydroxyvaleric acid and proline were recovered, in approximately equal amounts. The formation of such large amounts of proline from the acid treatment and alkalinization of alpha-amino-delta-hydroxyvaleric acid is therefore an important consideration in quantitating the number of gamma-glutamyl phosphate residues present in various proteins.

Secondary structural features of bovine amelogenin, a hydrophobic protein of developing enamel implicated in ename mineralization, are derived using 2D NMR spectroscopy in solution and molecular mechanics-dynamics studies. A β-turn: β-sheet model with some “unordered” segments was previously proposed from circular dichroism, Fourier-transform infrared and Raman spectroscopy augmented by Chou-Fasman predictive algorithm. The proposed structure contains a repetitive β-turn segment, “β-spiral” between Gln112 and Leu138 residues containing a (Pro, Leu, Gln) ricn segment. The β-spiral structure offers a probable site for interaction of Ca++ ions. Assignment of proton resonances using 2D COSY spectroscopy is presently in progress. Preliminary 2D NOESY spectra have revealed the presence of Tyr residues (TRAP segment) on the surface of amelogenin molecule and clusters of cross peaks reminiscent of β-turns and sheets which are consistent with the primary structure and proposed secondary structures of amelogenin. T...

γ-Carboxyglutamic acid (Gla) is identified in the proteins associated with several types of ectopic calcifications in which hydroxyapatite is the major mineral component. These included the calcified skin and subcutaneous plaques from a patient with dermatomyositis, the calcium containing material extruded from the skin of a patient with scleroderma, and the calcified, atheromatous plaques from aorta. Alkaline hydrolysis of biopsy material from these and from normal tissue revealed the presence of Gla only in the plaque specimens. Since a γ-carboxyglutamic acid-containing protein is normally present in bone and absent in unmineralized tissues, the presence of Gla in soft tissue calcifications is a potentially significant finding, especially in view of its known calcium and phospholipid binding properties.

The peptides tyrosyltyrosine and Asp,(Gly) 2 ,Tyr,Arg have been isolated respectively from tryptic digests and chymotryptic digests of chicken bone collagen solubilized in denaturing solvents. By homology to the structure of calf skin collagen, the peptides appear to derive from the carboxyterminus of the α1 chain. To confirm this, α1 and α2 chains were isolated from bone gelatin and tyrosyltyrosine was recovered from a tryptic digest of α1 but not from α2. Since tyrosine is not present at the carboxyterminus of the α1 chain of neutral salt-soluble and acid-soluble bone collagen of lathyritic chicks, it is concluded that a carboxyterminal sequence is missing from α1 chains of such extracts.

The solubility of decalcified bone collagen was studied in various salts and nonelectrolytes. It was found that collagen was extracted as gelatin at low temperature and at near neutral pH by a number of salts known to promote the conversion of collagen to gelatin. Successive extractions by such reagents dissolved essentially all the bone collagen. The extracted gelatins representing ∼ 80% of the total amount of collagen present in the bone, consisted predominantly of a component having the characteristics of the single stranded α-chains. It was concluded that the stability to dissolution of the collagen in the native bone tissue in solvents such as cold NaCl or dilute acetic acid is due primarily to the way in which the collagen macromolecules interact in the fibrils. In contrast to the protein mediated solvent—solvent interactions which are thought to play a major role in stabilizing the three polypeptide chains in the collagen helical configuration, the intermolecular interactions are influenced primarily by stereochemical factors that control direct protein—protein interactions.

Embryonic bovine jaw and femur bone, and 14-week-old chicken metatarsal bone were demineralized in 0.2 M EDTA. Analysis of the extracts revealed proteins rich in the acidic amino acids, aspartic and glutamic acids, and containing from 0.75% organic phosphorus in the femur extracts to 0.54% organic phosphorus in the chicken bone extracts. In addition, hexose, hexosamine, uronic acid and sialic acid were also present. Further purification of the peptides solubilized in EDTA was achieved by Sephadex G-100 molecular sieve filtration and Whatman DE-52 ion exchange chromatography. From these procedures a component was isolated that appeared to be homogeneous by acrylamide disc gel electrophoresis and ion exchange chromatography. Its amino acid composition revealed a high content of anionic amino acids and the presence of hydroxyproline in both bovine jaw bone and femur bone, but not in chicken bone. Organic phosphorus and serine were present in almost equimolar amounts. A possible role for these anionic phosphorus-containing proteins in facilitating the initiation of mineralization is discussed.

Mouse bone collagenase, a specific tissue collagenase, was isolated from the tissue culture media of 5 day old mouse tibiae. By a combination of (NH4)2SO4 precipitation, molecular sieve filtration and ion exchange chromatography a fraction was obtained with a yield of approximately 11% which had a specific enzyme activity 120-fold greater than the original crude extract. The molecular weight of the fraction containing the enzyme activity was approximately 41000 as determined by calibrated molecular sieve filtration studies. There were at least two distinct major fractions and one minor fraction possessing collagenase activity which had distinct isoelectric points. It is not clear whether these fractions represent isoenzymes from bone or from bone and cartilage, or are derived from a single enzyme which has been minimally degraded by the extraction and purification techniques used. Collagenase activity was inhibited by EDTA, cysteine and horse serum, but not by phenylmethylsulfonylfluoride, epsilon amino caproic acid or soybean trypsin inhibitor.