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 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)
Smith
C E
CE
Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.
Chen
W Y
WY
Issid
M
M
Fazel
A
A
D000566:Amelogenesis / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D001345:Autoradiography; D003513:Cycloheximide / Q000494:pharmacology; D003743:Dental Enamel / Q000187:drug effects / Q000378:metabolism*; D003746:Dental Enamel Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007180:Incisor; D007700:Kinetics; D008297:Male; D008715:Methionine / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes
D003746:Dental Enamel Proteins; D006023:Glycoproteins; D011500:Protein Synthesis Inhibitors; D013431:Sulfates; D013462:Sulfur Radioisotopes; D003513:Cycloheximide; D008715:Methionine