|Budget Amount *help
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1990: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1989: ¥4,700,000 (Direct Cost: ¥4,700,000)
A monoclonal antibody was raised against a mineralized tissue-specific sialoprotein containing no phosphorus using partially purified non-collagenous bone matrix proteins from rats as antigen. Then the sialoprotein was purified by high performance liquid chromatography from rat mandibulae using the monoclinal antibody as a marker. The sialoprotein (59kDa bone sialoprotein ; 59kDa-BSP) with a molecular weight of 59,000 containging 1.4% sialic acid but no detectable phosphorus. Immunochemical studies with the antibody showed that the protein was specific to mineralized tissues such as bone and dentin, and present in osteoblasts, osteocytes and bone matrix. No other soft tissues, such as the cartilage, liver, kidney and periosteum, were stained. However, Western blot analysis showed that plasma contained immunoreactive 59kDa-BSP. The quantitative amino acid composition of 59kDa-BSP resembled that of human alpha_2-HS glycoprotein (A2HSG). Amino acid sequence analyses of the amino-terminal region and four peptide fragments of 59kDa-BSP revealed that about 50% of the amino acids were homologous with those of human A2HSG. We next examined biosynthesis of 59kDa-BSP. When newborn rat calvaria were incubated with radioactive leucine, immunoreactive-59kDa-BSP was detected in their conditioned medium by fluorography. The conditioned medium of primary cultures of osteoblast-rich cells and abult rat hepatocytes also contained radioactive material immunoprecipitated with anti-59kDa-BSP. These results indicate that both hepatocytes and osteoblasts synthesize 59kDa-BSP. Several characteristics, including the amino acid sequence, suggest that 59kDa-BSP may be the rat counterpart of human A2HSG. However, rat 59kDa-BSP is a single peptide, whereas human A2HSG is known to be a heterodimer which is synthesized by the liver, transported in the blood stream and incorporated into calcified tissues.