IMMUNOCYTOCHEMICAL AND IMMUNOCHEMICAL STUDY OF ENAMELINS, USING ANTIBODIES AGAINST PORCINE 89-KDA ENAMELIN AND ITS N-TERMINAL SYNTHETIC PEPTIDE, IN PORCINE TOOTH GERMS

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytoc hemically using two antibodies: a polyclonal antibody raised against t he porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptid e antibody against the porcine enamelin amino-terminus (EN). Immunoche mical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies u sed. In contrast, the middle and inner enamel layer mainly contained l ower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mine ralized collagen fibrils of dentin, while EN stained both enamel matri x islands and stippled material. At the matrix formation stage, both a ntibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneousl y, while EN primarily stained the prism sheath. The intense immunoreac tion over the surface layer of enamel matrix at the matrix formation s tage, following staining with 89 E and EN, disappeared by the end of t he transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the lat e differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin con tinues from late differentiation to the transition stage and the cleav age of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during ename l maturation.