| Project/Area Number |
04670247
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
細菌学
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| Research Institution | Ehime University |
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Principal Investigator |
SHINOMIYA Hiroto Ehime University School of Medicine, Department of Microbiology, Assistant Professor, 医学部, 講師 (80162618)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1993: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1992: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | macrophage / lipopolysaccharide / phosphorylation / plastin / motiv sequence / protein kinase / 蛋白質リン酸化酵素 / 癌細胞 / LPS / 蛋白質リン酸化反応 / プロテインキナーゼ / カルシウム / アクチン |
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| Research Abstract |
We demonstrated that bacterial LPS induced a special set of phosphorylated protein (pp) in murine peritoneal macrophages, and purified the most heavily phosphorylated substrate protein with a molecular mass of 65-kDa(pp65) to homogeneity. Amino acid sequences of the two pp65-derived peptides were determined. According to the amino acid sequences, we synthesized oligonucleotide primer pools including DNA sequences complementary to all possible codons. The pp65 gene was amplified by the method based on the polymerase chain reaction technique employing the oligonucleotides and the macrophage cDNA.Sequence of the amplified gene was performed by Sanger's dideoxy chain termination method. It was surprising to us that the sequence has more than 90% of homology with that of human plastin ; a recently identified novel protein that transformation-dependently appears in neoplastic human cells.
We already clarified that serine residues on pp65 are exclusively phosphorylated, which means that serine kinases are activated in macrophages after LPS-stimulation. In order to determine what kind of serine kinases are involed in the pp65-phosphorylation, We isolated the ^<32>P-serine containig peptide from pp65 which had generated in the ^<32>P-labeled and LPS-stimulated macrophages and determined the sequence. The result indicated that pp65 having sequence could be phosphorylated by cAMP-dependent kinase, protein kinase C or casein kinase II.further investigations are currenly being done. It therefore seemed that precise analysis on the pp65 functions should lead to progress in our understanding of the macrophage activation at the molecular level.
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