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Functional analysis of phosphorylation/dephosphorylation of cytoskeletal proteins in differentiation and growth of hematopoieic stem cell.

造血干细胞分化和生长过程中细胞骨架蛋白磷酸化/去磷酸化的功能分析。

基本信息

批准号：
07671181
负责人：
HIGASHIHARA Masaaki
金额：
$ 1.41万
依托单位：
1st Department of Internal Medicine, Faculy of Medicine University of Thokyo.
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Among the four projects in these two years, only one project was successfully achieved. That is, we could clarify the functional domain of light meromyosin of smooth muscle myosin. Now we are going to determine the amino-acid sequences of antibody-recognizing sites. Monoclonal antibodies (McAbs) against light meromyosin (LMM) were produced and characteried. Anti-LMM antibodies, but not anti-S-1 or anti-S-2 antibody, almost completely inhibited filament formation in a equimolar concentration by the analyzes of turvidity and SDS-PAGE.McAbs against LMM showed no effects on Ca2+-ATPase and acto-ATPase activities of gizzard myoshi. These results suggest that LMM plays a crucial role in the formation of filament, but not ATPase activities.Immunoelectroscopical analysis showed that the centroal region is more important for the filamentformation than c-terminal regions. By the analyzes with immunoblotting and ultracentrifugation, McAbs against thiophosphorylated myosin also detected unphsophorylated myosin. McAbs against phosphorylated vimentin produced by Dr.M.Inagaki showed several cytochemical findings : phosphorylated vimentins were detected in cytoplasm, showing diffuse patterns. To be of interest, in mitotic phase more intense signals were detected in perinuclear regions. These findings suggst that phosphorylated vimentin plays an important role in mitotic phase. We regret that the experiments of mutant gene of myosin light and heavy chain could not be performed, although we already received such genes form Dr.M.Ikebe (USA).

在这两年的四个项目中，只有一个项目成功实现。也就是说，我们可以明确平滑肌肌球蛋白轻酶解肌球蛋白的功能域。现在我们要确定抗体识别位点的氨基酸序列。制备了抗轻酶解肌球蛋白（LMM）的单克隆抗体（McAb）。浊度分析和SDS-PAGE分析表明，抗LMM单抗在等摩尔浓度下几乎完全抑制肌丝的形成，而抗S-1和抗S-2单抗对肌丝的Ca ~（2+）-ATPase和acto-ATPase活性无影响。这些结果表明，LMM在纤维的形成中起重要作用，但对ATP酶活性无影响，免疫电镜分析表明，中心区比C端区对纤维的形成更重要。通过免疫印迹和超电泳分析，抗硫代磷酸化肌球蛋白的单克隆抗体也可检测到非磷酸化肌球蛋白。稻垣先生生产的抗磷酸化波形蛋白单克隆抗体显示了几种细胞化学结果：在细胞质中检测到磷酸化波形蛋白，呈弥漫性模式。感兴趣的是，在有丝分裂期，在核周区域检测到更强的信号。这些结果表明磷酸化的波形蛋白在有丝分裂期起重要作用。虽然我们已经从美国池部博士那里得到了肌球蛋白轻链和重链的突变基因，但我们遗憾地不能进行这些基因的实验。