Radioautographic Visualization of the Deposition of a Phosphoprotein at the Mineralization Front in the Dentin of the Rat Incisor

Abstract

A peptide that is rich in organically bound phosphorus and contains abundant serine residues has been identified in the dentin of man (1), fetal bovine (2, 3), and rat (4). This phosphoprotein may play a role in mineralization (5-9). Butler et al. (4) reported that the phosphoprotein of rat incisor dentin constituted 10.8% of the proteinaceous material recovered from decalcified incisor teeth while collagen comprised 84%. Since the phosphorus content of the phosphoprotein was estimated at 3.29% and that of collagen at 0.19% (4), much of the phosphorus taken up in organic form by the incisor would likely be present as phosphoprotein. With this in mind, it was decided to inject [~3P]phosphate into rats and examine the demineralized incisor teeth by radioautography in the hope of tracing phosphoprotein formation.The organic phosphorus of dentin phosphoprotein is believed to be attached to serine residues (6). In the rat incisor dentin, this amino acid comprises 35 residues per cent of the phosphoprotein and only four residues per cent of the cyanogen bromide peptides of collagen (4). Hence serine also appeared to be a suitable amino acid precursor to trace phosphoprotein formation by radioautography.Finally, the radioautographic pattern of the deposition of labeled phosphorus and serine was compared to that of [3H]proline. Proline may be used as a precursor to trace collagen, since it makes up 22.0 residues per cent of dentin collagen and only 2.4 residues per cent of the phosphoprotein (4). The results indicated that the pattern of phosphoprotein deposition into the dentin matrix is strikingly different from that of collagen. The fixative consisted of 2.5% glutaraldehyde in 0.05 M S6rensen's phosphate buffer with the addition of 0.1% sucrose and 0.5% dextrose. In the experiments using L-[2,~-gH]proline, the fixative employed was 3% formaldehyde (TAAB Laboratories, Emmer Green, Reading, England) in 0.1 M S6ren-sen's phosphate buffer with 0.1% sucrose added. The final pH of either fixative was 7.2-7.3. After perfusion for 15 min at room temperature the maxillary incisor teeth were immersed in fresh fixative for 2-3 h and demineralized in EDTA for 2 wk at 4°C (10). Although a 2-wk period is sufficient for exhaustive demineralization, some teeth were denfineralized for 3 or 4 wk in the experiments conducted with [3~P]phosphate. Specimens were washed overnight in 0.15 M S6rensen's buffer, sliced transversely with razor blades into 1-mm thick sections, postfixed for 1-2 h in 1% OsO4 in 0.1 IV[ S6rensen's buffer, dehy-838 MATERIALS AND METHODS Sherman

Isolation from embryonic bovine dental enamel of a polypeptide (E3) containing as its only phosphorylated sequence, Glu—O‐phosphoserine—Leu

“…The findings may also be relevant to the role of phosphorylated components in mineralization of bone and dentin [26][27][28][29][30][31][32], since soluble and collagen-bound phosphorylated peptides of dentin and bone are rich in aspartic and glutamic acids [32][33][34][35] and calcium binding studies have indicated that some of the calcium ions are probably bound to collagen as a complex between carboxyl and phosphate groups [36].…”

Radioautographic visualization and biochemical identification of O-phosphoserine- and O-phosphothreonine-containing phosphoproteins in mineralizing embryonic chick bone.

“…The first indication that phosphoproteins were indeed synthesized in mineralized tissues came from the elegant radioautographic studies of Weinstock and Leblond (10) vestigators followed the localization of 33P first in the odontoblasts and later at the mineralization front of the extracellular matrix ofthe dentin of rats injected with "Pi (inorganic orthophosphate).' Although the visualized radiolabel was not identified biochemically as being an integral component of a protein, the data were consistent with subsequent studies of Munksgaard et al (11), who demonstrated active biosynthesis of phosphoprotein by dentinal tissue in organ culture, and the in vivo biosynthesis studies of Dimuzio and Veis (12).…”

Testosterone and 6-N,2'-O-dibutyryladenosine 3′:5′-cyclic monophosphate stimulate protein and lysosomal enzyme secretion in rat seminal vesicle

“…After 4h of incubation in the presence of testosterone and dibutyryl cyclic AMP, 66 and 71 % of the tricWloroacetic acid-insoluble 32P appeared in the medium, the total secretion amounting to' 246 and 206 % respectively of the 370C controls ( (Singhal &; labelled components, as shown by the fact that 55 % 'Sutherland, 1975 We infer from the findings in the control slices that 32p1 must have been incorporated into secretory constituents shortly before the latter were packaged in secretory granules or lysosomes or both and released into the incubation medium. The phosphorylation of phosphoproteins of milk (the caseins) (Bingham et al, 1972) and tooth dentin (Weinstock & Leblond, 1973) is known to occur in the Golgi apparatus. It is therefore likely that the phosphorylation of secretory constituents in the seminal vesicle also takes place in the Golgi apparatus.…”