The synthesis and secretion of enamel proteins (EPs) in rat incisors was examined using cytochemical and biochemical methods. Radioautography after injection of 3H-methionine showed that ameloblasts in the presecretory, secretory, and maturation stages of amelogenesis actively synthesized and secreted proteins. Immunocytochemistry with an antibody to mouse amelogenins revealed the presence of EPs in the protein synthetic and secretory organelles of these cells at all three stages. Labeling was also found in elements of the endosomal/lysosomal compartment. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining of proteins extracted from enamel and enamel organ showed several protein bands. However, transfer to nitrocellulose paper and immunoblotting revealed that most of the proteins recognized by the antibody were situated between approximately 14 and 32 kDa. EPs were further characterized by using lectins to examine their carbohydrate content. Lectin-gold cytochemistry on sections showed the binding of wheat germ agglutinin and Helix pomatia lectin to secretory stage enamel. Lectin blotting indicated that the amelogenins were heterogeneously glycosylated and contained the sugars N-acetyl-glucosamine/N-acetyl-neuraminic acid and N-acetyl-D-galactosamine. Fluorography at 6 and 10 min and 1 h after injection of 35S-methionine revealed four labeled bands in the main amelogenin group near 22, 28, 30, and 32 kDa. A short-lived protein of approximately 58 kDa was also observed primarily in cells. The appearance of labeled proteins in enamel was paralleled by their disappearance from cells and the intensity of the radiolabeled protein bands, both, in enamel and in cells, decreased towards the maturation stage. These data are consistent with the concept that ameloblasts produce multiple amelogenins throughout amelogenesis.

The pattern and timing of the breakdown and loss of matrix proteins were studied in developing rat incisor enamel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fluorography, radioautography, and in vitro incubations of proteins isolated from freshly dissected, crushed pieces of enamel. For biochemical studies, the technique of Robinson et al. (1974, 1977, 1983) was used to transect the enamel organ and enamel into a series of strips at 1 mm intervals along the length of the tooth. The proteins in each strip were extracted and either quantified by Lowry analysis or applied to 12% slab (enamel) or 5-15% continuous gradient (enamel organ) SDS-polyacrylamide gels and separated by electrophoresis. The biochemical studies indicated that the amount of protein contained within an enamel strip increased gradually by volume across the secretory stage, reached a peak early during the maturation stage, and then declined rapidly thereafter. The distribution of enamel proteins on SDS-polyacrylamide gels changed markedly throughout this period. These changes included increases and decreases in the intensity of staining of proteins at certain molecular weights (e.g., 18 kDa) and the appearance and disappearance of some proteins not seen clearly near the start of the secretory stage of amelogenesis (e.g., 32 and 10 kDa). Labeling studies with 35S-methionine suggested that the "stacked" arrangement of proteins typical of forming enamel (secretory stage) actually represented a very dynamic association of proteins, with new ones being added at the top of the stack and then breaking down with time to become those seen at lower molecular weights. Across the secretory stage, new proteins were always added to the top of the stack, but during early maturation this activity slowed dramatically, allowing the breakdown of aging proteins to be visualized more clearly. Radioautographic studies with 3H-methionine indicated that the breakdown of newly secreted proteins also was correlated with a movement of label from the site of secretion into deeper, previously unlabeled, areas of forming enamel. In vitro studies revealed that the rate and degree of breakdown of enamel proteins varied markedly, depending on the stage of amelogenesis from which the proteins were extracted. Secretory stage enamel proteins showed slow in vitro degradation with accumulation of proteins near 18 kDa. Early maturation stage enamel proteins showed more rapid breakdown with little accumulation of proteins near 18 kDa, whereas late maturation stage enamel proteins showed complete degradation by 2 days of incubation in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

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