Altered phosphorylation of rat dentine phosphoproteins by fluoride in vivo

Dentine phosphoproteins have been proposed to have an important role in min eralization. This study focused on the influence of fluoride on the biochem ical composition of dentine phosphoproteins and attempts to relate changes to the altered mineralization witnessed during fluorosis. Wistar rats were rendered fluorotic by the administration of 20 ppm sodium fluoride in their drinking water nd libitum, a nonfluorotic group received double-distilled, deionized water only. After 17 weeks, the teeth showed signs of fluorosis. The incisors were removed, split longitudinally, and the pulps were remove d. Teeth were powdered and demineralized in 10% EDTA with protease inhibito rs, after which the organic matrix was extracted with 4 M guanidinium chlor ide. Phosphoproteins were selectively precipitated from the soluble extract by the addition of 1.0 M calcium chloride and further purified by anion ex change chromatography. SDS-PAGE revealed two protein bands with molecular w eights of 130 kDa and 66 kDa in the nonfluorotic fraction and 116 kDa and 6 6 kDa in the fluorotic fraction. Western blotting analysis identified the 6 6 kDa band as alpha(2)-HS glycoprotein which co-precipitated with phosphopr oteins. Electroelution of the protein bands was performed with subsequent b iochemical analyses. Phosphate content was determined for each protein band and was detectable in the 116 kDa and 130 kDa bands from the fluorotic and nonfluorotic samples, respectively, with a decreased level noted in the 11 6 kDa band. The presence of phosphate and the amino acid analysis of these bands suggested their identity to be dentine phosphoproteins. No changes in the ratio of amino acids was detected in fluorotic samples. The fluoride-i nduced alterations to the biochemical structure of dentine phosphoproteins would appear to influence the phosphorylation of these macromolecules only, possibly affecting posttranslational events. Such alterations may play a r ole in disrupting the patterns of mineralization seen during fluorosis.