SITE-DIRECTED MUTAGENESIS OF NONMUSCLE MYOSIN HEAVY CHAINS

鸡脑非肌肉肌球蛋白重链编码基因的克隆 (MHC-B)显示在25-50个氨基酸的盒中存在 和50-20个KDA连接，位于MHC(高桥)的头部区域 等人，J.Biol.化学。267：17,864,1992)。这些插入物被放置在 靠近非肌肉肌球蛋白中的ATP和肌动蛋白结合部位 分别是重链。表达插入片段的异构体 盒式磁带仅限于神经系统中表达的肌球蛋白和 与未插入的异构体一起存在。后者也是 除脑和脊髓外，还在许多其他组织中表达 电源线。为了研究两种动物在生物活性上的差异 插入和非插入的异构体，我们开始了表达cDNA的研究 编码MHC-B非插入蛋白氨基末端1231个氨基酸 肌球蛋白轻链为20kD和17kD。嗯-Exp (含有150kD、20kD和17kD的多肽 化学计量比)在低离子强度下溶解，并与兔结合 骨骼肌肌动蛋白以一种依赖于ATP的方式。它有能力 在体外动力测定系统中推进肌动蛋白细丝的研究 被肌动蛋白激活以水解镁三磷酸腺苷。这两种生物 属性 肌球蛋白光对20kDA肌球蛋白轻链的必需磷酸化作用 链激酶型。表达的20kD肌球蛋白LIGH的胰酶肽谱 链，先前被肌球蛋白轻链激酶和蛋白质磷酸化 激酶C，类似于天然轻链产生的那些。自.以来 蛋白激酶C使乙酰化的丝氨酸残基(丝氨酸-1)磷酸化， 多肽图谱上的这种相似性表明昆虫细胞 翻译后以类似的方式处理表达的轻链 举止。最近，我们成功地表达了含有MHC的 在ATP结合位点附近插入了10个氨基酸的序列 (即在25-50 KDA交界处)。此插入内容还包含推定的 Cyclin-p34-cdc2蛋白和/或MAP II蛋白的磷酸化位点。 我们目前正在对这种表达的异构体进行鉴定。

Cloning of the CDNA encoding a chicken brain nonmuscle myosin heavy chain (MHC-B) revealed the presence of cassettes of amino acids at the 25-50 and 50-20 Kda junctions, located in the head region of the MHC (Takahashi et al., J. Biol. Chem. 267: 17,864, 1992). These inserts are located near to the ATP-binding and actin-binding sites in the nonmuscle myosin heavy chain, respectively. The isoforms that expressed the inserted cassettes were confined to myosin expressed in the nervous system and were present along with the noninserted isoform. The latter is also expressed in numerous other tissues in addition to the brain and spinal cord. To study the differences in biological activity between the inserted and noninserted isoforms, we initiated studies to express CDNA encoding the amino-terminal 1231 amino acids of the MHC-B noninserted isoform along with the 20 Kd and 17 Kd myosin light chains. HMM-exp (containing the 150 Kd, 20 Kd and 17 Kd polypeptides in the proper stoichiometry) was soluble at low ionic strength and bound to rabbit skeletal muscle actin in an ATP-dependent manner. It has the ability to propel actin filaments in an in vitro motility assay system and to be activated by actin to hydrolyze MgATP. Both of these biological properties required phosphorylation of the 20 Kda myosin light chain by myosin light chain kinase. Tryptic peptide maps of the expressed 20 Kd myosin light chain, previously phosphorylated by myosin light chain kinase and protein kinase C, resembled those generated from the native light chain. Since protein kinase C phosphorylates an acetylated serine residue (serine-1), this similarity in peptide maps suggests that insect cell posttranslationally processes the expressed light chain in a similar manner. Recently, we have succeeded in expressing the MHC that contains an inserted sequence of 10 amino acids near to the ATP-binding site (i.e., at the 25-50 Kda junction). This insert also contains a putative site for phosphorylation by cyclin-p34-cdc2 kinase and/or MAP II kinase. We are presently characterizing this expressed isoform.