Analysis of the plastin-pathway involved in macrophage activation.

我们证明了细菌内毒素诱导了小鼠腹腔巨噬细胞中一组特殊的磷酸化蛋白(Pp)，并纯化了最严重的磷酸化底物蛋白，其相对分子质量为65 kDa(Pp65)。测定了两个pp65衍生多肽的氨基酸序列。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库。采用聚合酶链式反应技术扩增pp65基因，扩增产物经Sanger‘s双脱氧链终止法测序。令我们惊讶的是，该序列与最近发现的人类肿瘤细胞中出现的一种新的蛋白质--人纤溶酶原蛋白的同源性超过90%。我们已经澄清，pp65上的丝氨酸残基是唯一的磷酸化的，这意味着在内毒素刺激后巨噬细胞中的丝氨酸激酶被激活。为了确定pp65-磷酸化涉及哪种丝氨酸激酶，我们从β&lt；32&gt；P标记的巨噬细胞和脂多糖刺激的巨噬细胞产生的pp65中分离出含有β&lt；32&gt；P-丝氨酸多肽并测定了其序列。结果表明，具有序列的pp65可以被cAMP依赖的激酶、蛋白激酶C或酪蛋白激酶II磷酸化，目前正在进行进一步的研究。因此，对pp65功能的精确分析似乎应该有助于我们在分子水平上理解巨噬细胞的激活。

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.