Articular cartilage has the ability to retard the invasiveness of inflammatory cells such as leucocytes. This resistance is in part due to the avascular nature, organization, and composition of the extracellular matrix. Extractable components (anti-invasion factors, [AIF]) within the matrix have been shown to be responsible for protecting the tissue against proteases produced by invading leucocytes. In the present study, AIF is examined for leucocyte elastase inhibitory activity using both analytical and preparative electrophoretic and chromatographic techniques. A specific leucocyte elastase inhibitor was isolated and identified as a 15,000 dalton protein on SDS-PAGE. The inhibitor is distinct from aprotinin (Trasylol) in size, electrophoretic pattern, and comparative peptide maps. The inhibitor exhibited a highly cationic (pI greater than 9.5) as well as a strong hydrophobic character. These properties of primary structure were confirmed by amino acid composition and sequence of the amino-terminal residues. The amino-terminal sequence showed no homologies to other proteins or known protease inhibitors. This matrix-derived protease inhibitor may be an important mediator in the regulation of and protection against cartilage destruction.

Animals↗

PubMed1985

The biosynthesis of dentin phosphophoryns by rat incisor odontoblasts in organ culture.

An in vitro system consisting of rat incisor fragments was used to study the process of dentinogenesis. In order to establish the usefulness of the organ culture, the biosynthesis and deposition of the major noncollagenous components of dentin, the phosphophoryns, were followed for specific lengths of time in culture. Three criteria were satisfied: (1) the synthesis of proteins which appeared to be chemically identical to the native proteins of dentin, (2) the accumulation of the phosphophoryns within the matrix or time, and (3) the association of the secreted proteins with the mineral phase of dentin. The synthesis of phosphophoryns was determined by using both (3H)-serine and (32P)-inorganic phosphate as precursors for synthesis of protein and post-translational modification of serine to phosphoserine. In vitro synthesized phosphophoryns were characterized by 1) their accumulation and EDTA extractability from within dentin, 2) calcium chloride precipitability, 3) elution on anion-exchange columns (DEAE cellulose and AGMP50), and 4) Mr's on SDS-PAGE and Sepharose CL-6B columns. This novel system of studying dentinogenesis provides a model with which to study the regulation of extracellular matrix protein synthesis and may be useful for revealing the effect of other agents which influence tooth development and mineralized tissue metabolism in general.

Animals↗

PubMed1983

The biosynthesis of dentine gamma-carboxyglutamic acid-containing proteins by rat incisor odontoblasts in organ culture.

Rat odontoblasts were shown to synthesize and secrete gamma-carboxyglutamic acid(Gla)-containing proteins into dentine after organ culture in the presence of radiolabelled amino acid precursors. Purified dentine Gla-containing protein from rat incisors was used as antigen to prepare rabbit antisera as a probe of dentine Gla-containing-protein biosynthesis in organ cultures of dentine (rat incisor) and bone (rat calvaria). Use of the antiserum also pointed out the cross-reactivity of a high-M, glycoprotein present within the dentine matrix. The present results are significant in identifying dentine gla-containing protein as endogenous to mineralizing dentine and may relate to the commonality between calcifying connective tissues in general.

1-Carboxyglutamic Acid↗

PubMed1983

Multiple forms of rat dentin phosphoproteins.

Previous studies have shown that the phosphoprotein from rat dentin is heterogenous and can be partially separated into two fractions by ion-exchange chromatography. These proteins were further characterized by polyacrylamide gel electrophoresis, gel chromatography, and amino acid and phosphate analysis, after chromatographic separations on ion-exchange columns. On 5-15% gradient gels, the phosphoproteins extracted from rat dentin and precipitated by CaCl2 gave three Alcian blue-staining bands with apparent molecular weights in the 90-95,000 range. The two slower-moving bands corresponded to highly phosphorylated proteins (HP) that had phosphoserine contents of greater than 400 residues per thousand and contained little or no valine, leucine, phenylalanine, or arginine. The faster-moving band corresponded to a moderately phosphorylated protein that contained about 250 residues per thousand of phosphoserine and greater quantities of glutamic acid, proline, and several other amino acids than HP. The nature of the phosphoproteins in HP was further studied after total removal of the phosphate with an insoluble form of bovine intestinal alkaline phosphatase. The dephosphorylated product (dP-HP) gave a single major band on gel electrophoresis but showed evidence for two closely related NH2-terminal sequences, Asp-Asp-Asp-Asn and Asp-Asp-Pro-Asn. The dephosphorylated material was separated into two components (dP-HP1 and dP-HP2) by chromatography on QAE-Sephadex A-25. The amino acid compositions of the two components showed that they differed in their primary structures. This conclusion was verified by the finding of the proline-containing sequence in dP-HP2. In addition to these two groups of phosphoproteins, a third class, LP, contains low levels of phosphoserine and high amounts of glutamic acid (W.T. Butler, M. Bhown, M.T. DiMuzio, and A. Linde, (1981) Coll. Res. 1, 187-199).

Amino Acids↗