权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Analysis of the plastin-pathway involved in macrophage activation.

分析参与巨噬细胞激活的塑性蛋白途径。

基本信息

批准号：
04670247
负责人：
SHINOMIYA Hiroto
金额：
$ 1.28万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

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.

我们证明，细菌LPS诱导一组特殊的磷酸化蛋白（PP）在小鼠腹腔巨噬细胞，并纯化最严重的磷酸化底物蛋白的分子量为65 kDa（pp 65）的同质性。测定了两种pp 65衍生肽的氨基酸序列。根据氨基酸序列，我们合成了包含与所有可能密码子互补的DNA序列的寡核苷酸引物池。采用聚合酶链反应技术，利用寡核苷酸和巨噬细胞cDNA扩增pp 65基因，用桑格双脱氧链终止法进行序列测定。令人惊讶的是，该序列与人plastin的同源性超过90%，plastin是最近发现的一种新蛋白质，其转化依赖性地出现在肿瘤性人类细胞中。我们已经阐明，pp 65上的丝氨酸残基仅被磷酸化，这意味着丝氨酸激酶在LPS刺激后在巨噬细胞中被激活。为了确定参与pp 65磷酸化的丝氨酸激酶种类，我们从经β-P标记和LPS刺激<32>的巨噬细胞产生的pp 65中分离出β-P-丝氨酸肽<32>，并测定了其序列。结果表明，具有序列的pp 65可被cAMP依赖性激酶、蛋白激酶C或酪蛋白激酶II磷酸化。因此，似乎pp 65功能的精确分析应该导致我们在分子水平上理解巨噬细胞活化的进展。