RESUMO
The objectives of this study were to measure pH in developing enamel at progressively older (more mature) stages of amelogenesis in vivo, and then to formulate synthetic enamel fluid mixtures that approximated these pH values for in vitro studies. The ultimate goal was to characterize the molecular weights of proteinases visualized by enzymograms incubated in synthetic enamel fluid using gelatin and casein as substrates. For most experiments, the proteinases were extracted en masse from small freeze-dried enamel strips directly into a non-reducing sample preparation buffer. In some experiments, we pre-treated the enamel strips with acetic acid to determine if this common method for demineralization and protein extraction caused any changes in the activity levels of the enamel proteinases. In other experiments, we first soaked enamel strips in synthetic enamel fluid to determine solubility of the proteinases within an aqueous phase. The results indicated that the pH of developing enamel remained fairly constant near pH 7.23 across the secretory stage, but it was generally more acidic (6.93) and fluctuated in focal areas between mildly acidic (6.2-6.8) and near-neutral (7.2) conditions across the maturation stage. The pH then slowly rose to near 7.35 when the enamel was almost mature (hard). The acidic conditions were generally inhibitory to most enamel proteinases, but there were some caseinase activities in mid-maturation-stage enamel near 23-30 kDa which appeared to be activated by weakly acidic conditions (pH 6.28). Pre-treatment of enamel samples with 0.5 M acetic acid markedly altered the overall profile of enamel proteinases, causing activation of some latent proteinase activities and permanent inhibition of other activities. Most proteinases in whole homogenates were insoluble in synthetic enamel fluid. This suggests that they may be tightly bound, directly or indirectly, to matrix proteins or mineral components in situ.
Assuntos
Amelogênese/fisiologia , Proteínas do Esmalte Dentário/metabolismo , Esmalte Dentário/química , Esmalte Dentário/enzimologia , Endopeptidases/metabolismo , Ácido Acético/metabolismo , Animais , Proteínas do Esmalte Dentário/análise , Solubilidade do Esmalte Dentário , Órgão do Esmalte/citologia , Órgão do Esmalte/enzimologia , Indução Enzimática , Matriz Extracelular/enzimologia , Gelatinases/química , Gelatinases/metabolismo , Concentração de Íons de Hidrogênio , Modelos Lineares , Masculino , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Peso Molecular , Ratos , Ratos Wistar , Especificidade por SubstratoRESUMO
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 reticulum to Golgi apparatus, where they became sulfated. The results indicated that enamel organ cells (ameloblasts) rapidly incorporated 35SO4 into a major approximately 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 approximately 49 kDa and approximately 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 approximately 13-42 kDa, which appeared to originate from extracellular processing and/or degradation of the parent approximately 65 kDa sulfated enamel protein or its major approximately 49 kDa and approximately 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 approximately 65 kDa sulfated enamel protein.(ABSTRACT TRUNCATED AT 250 WORDS)