Tohru Takagi

Complete amino acid sequence of amelogenin in developing bovine enamel

Pure amelogenin protein in developing bovine incisor enamel was isolated and its primary structure was investigated by sequencing the peptides obtained after clostripain and chymotrypsin digestions and CNBr degradation with an automated Edman sequencer. The enamel protein was found to be composed of 170 amino acid residues with one phosphate having a molecular weight of 19,350 and its complete amino acid sequence was elucidated. This protein has no sequence homology with any other tissue or secretory protein of known structure.

Cyclical changes in pH in bovine developing enamel as sequential bands

Developing enamel was stained with pH indicators and a banded colour pattern exhibiting alternate acidic (5.8-6.0) and neutral (7.0-7.2) staining was clearly visualized. The pH values of the enamel samples scraped from respective bands were confirmed by measuring them in suspensions with a pH meter. Neutral bands corresponded to red stripes of glyoxal bis(2-hydroxyanil) staining and acidic bands to unstained ones, suggested a correlation of the acidic and neutral stripes with the zones of ruffle-ended and smooth-ended ameloblasts.

Systematic purification of free and matrix-bound phosphophoryns of bovine dentin: Presence of matrix-bound phosphophoryn as a distinct molecular entity

SummaryFree and matrix-bound phosphophoryns, both highly phosphorylated proteins in dentin, were prepared from EDTA extract and CNBr-digests of bovine dentin. The two components were purified by DEAE-cellulose, SP-Sephadex, and gel filtration chromatography. The matrix-bound component was eluted as a distinct peak from the free component in the above chromatographic systems. Amino acid composition of the purified matrixbound component indicated that this component consisted of phosphophoryn and collagen in the ratio of 2:3 based on the number of the residues. The matrix-bound component could not be reconstituted by mixing phosphophoryn with collagen CNBr peptides. Artificial crosslink products of free phosphophoryn and collagen CNBr-peptides by the carbodiimide method showed similar properties to the physiological matrix-bound phosphophoryn. The bond between phosphophoryn and collagen of the matrix-bound component is assumed to be a covalent crosslink.

Basic Biological Sciences Comparative Collagen Biochemistry of Bovine Periodontium, Gingiva, and Dental Pulp

Cross-linking patterns of the collagen from bovine gingiva, periodontium, and dental pulp were analyzed chromatographically. The ratios of two main cross-links, dihydroxylysinonorleucine to hydroxylysinonorleucine, were 0.18, 0.31, and 0.49 for the bovine gingiva, periodontium, and dental pulp collagen, respectively. These ratios are similar to that of skin collagen rather than that of bone and dentin collagen.Cross-linking patterns of the collagen from bovine gingiva, periodontium, and dental pulp were analyzed chromatographically. The ratios of two main cross-links, dihydroxylysinonorleucine to hydroxylysinonorlecuine, were 0.18, 0.31, and 0.49 for the bovine gingiva, periodontium, and dental pulp collagen, respectively. These ratios are similar to that of skin collagen rather than that of bone and dentin collagen.

Amelogenin Degradation by an Enzyme Having Acidic pH Optimum and the Presence of Acidic Zones in Developing Bovine Enamel

Protein content in immature enamel decreases dramatically during development. Disappearance of high-molecular weight proteins and relative increase in low molecular weight fractions are observed at the later stage of the development. This fact suggests degradation of matrix components during the process of enamel development and maturation. We purified and characterized an enzyme responsible for amelogenin degradation in developing enamel. Interestingly, the optimum pH of the enzyme was found to be in acidic pH, approximately at 6. This result suggested importance of acidic conditions in degradation process of the amelogenin matrix during enamel maturation. The authors attempted to stain developing teeth with pH indicators and found the presence of alternating acidic and neutral zones in developing enamel.

In vitro mineralization in bovine tooth germ cell cultured with sintered hydroxyapatite

Unerupted teeth were extracted from the mandibles of calves, and the enamel surfaces containing ameloblasts and cells of papilla layer were scraped off. Cells migrated from the fragments were transferred into culture flasks containing small bars of sintered hydroxyapatite, and the cultures were incubated long-term. Most of the cells showed a fibroblastic morphology, but some of the cells formed an epithelial cell nest. The fibroblastic cells grew steadily, but the epithelial cells showed poor growth. The fibroblastic cells formed multiple cell layers and were embedded in collagenous matrix containing type I trimer collagen. By 3 months, fibrous bands appeared on the surface of the cell layers and surrounded the bars of sintered hydroxyapatite. Such bands increased in size with incubation time. Mineral deposition was observed in the well-developed bands. The deposited crystals were found by the X-ray powder diffraction method to be hydroxyapatite; the Ca/P molar ratio of 1.49 was determined by the ICP method. These results indicated that cells of papilla layer have the capacity to mineralize in vitro in the presence of sintered hydroxyapatite.

Location of an Intermolecular Crosslink in Bovine Bone Collagen

A peptide containing 59 amino acid residues with a stoichiometric amount of dihydroxylysinonorleucine (0.7 mole) and hydroxylysinonorleucine (0.2 mole) was isolated from reduced 3H labelled bovine bone collagen sequentially cleaved with CNBr and trypsin. Further cleavage of the isolated crosslinked peptide with periodate yielded a radioactive peptide of 45 residues and a non-radioactive peptide of 16 residues. From the characteristic amino acid composition of these peptides it was deduced that the peptide was derived from an intermolecularly crosslinked region between lysyl or hydroxylysl residues in the carboxy-terminal extension of alpha 1-CB6 (17C residue) and alpha 1-CB5 (87th residue). This finding supports the observation that the alpha 1-CB6 peak was prominent on carboxymethyl cellulose chromatography of the CNBr digest of bone collagen only after limited pepsin digestion, and is consistent with the results obtained from a smaller crosslinked peptide previously isolated from calf bone collagen.

Two new in vitro calcification systems showing the higher calcifiability of enamel proteins than dentin and bone matrices.

One of the difficulties in simulating in vivo calcification by in vitro experiments is how to prepare and apply a suitable calcifying solution. We have previously developed an entirely new model system consisting of 40% acrylamide gel blocks that contains matrix proteins and is immersed in fetal calf serum at 37 degrees C. (40% gel system) for 18 hr (Connect. Tissue Res., 33, 185, 1995). The gels were analyzed for immobilized calcium. In this system bovine enamel proteins (0.1% in the gel) showed the highest calcifiability among the tested matrices, followed by insoluble bovine dentin, bone and skin collagens. The 40% gel system provides a barrier for high molecular weight inhibitor molecules in the body fluid. The new calcifying system developed in this study consists of the matrix protein sealed in dialysis tubing within a glass chromatography column that was eluted with a calcifying solution. In this system (dialysis tubing system), again the enamel protein showed higher calcifiability than dentin, bone and skin collagens. It was also shown that enamel proteins became not only a reversible opaque gel, but also a relatively-irreversible coagulant, if the solution contained calcium and phosphate ions at concentration below saturation (1 mM calcium and 1 mM phosphate). With both systems combined, deposition and crystal growth of minerals in enamel proteins will be better understood than with previous methods.