Enamel matrix protein turnover during amelogenesis: Basic biochemical properties of short-lived sulfated enamel proteins

The formation and turnover of sulfated enamel proteins was investigated by SDS-PAGE, fluorography, and TCA-precipitations using freeze-dried incisors of rats injected intravenously with 35S-sulfate (35SO4) and processed at various intervals from 1.6 minutes to 4 hours thereafter. Some rats were injected first with 35SO4 followed 5 minutes later by 0.3 mg of cycloheximide. This was done to terminate protein translation and allow events related to extracellular processing and degradation of the sulfated enamel proteins to be visualized more distinctly. Other rats were injected with cycloheximide followed at 0 minutes (simultaneous injection) to 30 minutes later by 35SO4. This was done to characterize the time required for proteins to travel from endoplasmic reticuium to Golgi apparatus, where they became sulfated. The results indicated that enamel organ cells (ameloblasts) rapidly incorporated 35SO4 into a major ∼65 kDa protein that was secreted into the enamel within 6–7.5 minutes. This parent protein appeared to be processed extracellularly within 15 minutes into major ∼49 kDa and ∼25 kDa fragments which themselves had apparent half-lives of about 1 and 2 hours, respectively. There were also many minor sulfated fragments varying in molecular weight (Mr) from ∼13–42 kDa, which appeared to originate from extracellular processing and/or degradation of the parent ∼65 kDa sulfated enamel protein or its major ∼49 kDa and ∼25 kDa fragments. Experiments with glycosidases further suggested that the majority of sulfate groups were attached to sugars N-linked by asparagine to the core of the ∼65 kDa sulfated enamel protein. The sulfated enamel proteins resemble acidic glycoproteins previously classified as “enamelins”. Unlike the enamelins, however, they are short-lived and do not appear to survive in enamel as it matures.