| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1989: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
The interesting proteins for biological mineralization in the noncollagenous proteins of bone are at present thought to be osteonectin, osteopontin, osteocalcin, alpha_2-HS glycoprotein and so on. There are many biochemical investigations of those proteins, but little attention has been paid to the morphological analysis of them. In this study, I report the extraction and purification of high molecular weight noncollagenous proteins which are possibly the Caーbinding proteins of the newborn rat calvaria, and demonstrate the histochemical detection of them.
Three noncollagenous proteins (Mr=90,000, 66,000, 49,000) were dissolved by sequential extractions and each had an affinity for calcium ions. They have been identified to be sialoprotein, osteopntin and the analogue of alpha_2-HS glycoprotein derived from serum by their amino acid analyses and Nterminal sequence analyses. Osteopontin and sialoprotein, but not alpha_2-HS glycoprotein, were stained blue with Stains all, which has been known to stain the Caーbinding proteins blue, on an electrophoretic gel. Then newborn rat calvaria were stained with Stains all histolog ically. Blue staining resulting from Stains all was observed in areas of the originally mineralized bone matrix of demineralized specimens, but osteoid and bone cells were not stained blue with Stains all. And no apparent blue staining was observed in unーdemineralized section except the areas where mineralization was in progress. Then I raised specific antiserum against the 90kD protein which is expected to bind more tightly to the matrix by ionic bond then the other two proteins. In immunohistochemistry, the reaction products were found in the bone matrix and the initiating site of mineralization. No reaction products were seen in osteoid, bone cells and cartilaginous matrix.
This specific antiserum against this noncollagenous protein from bone matrix will be very useful for the analysis of the biological mineralization.
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