Novy R E, Sellers J R, Liu L F, Lin J J
Department of Biological Sciences, University of Iowa, Iowa City 52242.
Cell Motil Cytoskeleton. 1993;26(3):248-61. doi: 10.1002/cm.970260308.
At least eight tropomyosin isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsm alpha) are expressed from four distinct genes in human fibroblasts. In order to elucidate isoform properties, we have subcloned hTM3 and hTM5 full-length cDNAs, as well as their chimeric cDNAs into the bacterial expression pET8C system. Bacterially expressed tropomyosin isoforms (called PEThTM3, PEThTM5, PEThTM5/3, and PEThTM3/5) were purified and characterized. Under optimal binding conditions, the binding of PEThTM5 isoform to F-actin was stronger than the PEThTM3 isoform. However, analysis of actin-binding by the McGhee and von Hippel equation revealed that PEThTM3 exhibits higher cooperativity in binding than PEThTM5 does. Furthermore, the chimera PEThTM5/3 which possessed the N-terminal fragment of hTM5 fused to the C-terminal fragment of hTM3 had even stronger actin binding ability. The reverse chimera PEThTM3/5 which possessed the N-terminal fragment of hTM3 fused to the C-terminal fragment of hTM5 demonstrated greatly reduced affinity to actin filaments. In addition, both chimeras had different KCl requirements for optimal binding to F-actin than their parental tropomyosins. A bacterially made C-terminal fragment of human fibroblast caldesmon (PETCaD39) and native chicken gizzard caldesmon were both able to enhance the actin-binding of these bacterially expressed tropomyosins. However, PETCaD39's enhancement of binding to F-actin was greater for PEThTM5 than PEThTM3. Under 30 mM KCl and 4 mM MgCl2, the low M(r) isoform PEThTM4 appeared to be able to amplify the actin-activated HMM ATPase activity by 4.7 fold, while the high M(r) isoform PEThTM3 stimulated the activity only 1.5 fold. The higher enhancement of ATPase activity by PEThTM5 than by PEThTM3 suggested that the low M(r) isoform hTM5 may be more involved in modulating nonmuscle cell motility than hTM3. These results further suggested that different isoforms of tropomyosin might have finite differences in their specific functions (e.g., cytoskeletal vs. motile) inside the cell.
在人类成纤维细胞中，至少有8种原肌球蛋白异构体（hTM1、hTM2、hTM3、hTM4、hTM5、hTM5a、hTM5b和hTMsmα）由4个不同的基因表达。为了阐明异构体的特性，我们已将hTM3和hTM5全长cDNA及其嵌合cDNA亚克隆到细菌表达pET8C系统中。对细菌表达的原肌球蛋白异构体（称为PEThTM3、PEThTM5、PEThTM5/3和PEThTM3/5）进行了纯化和特性分析。在最佳结合条件下，PEThTM5异构体与F-肌动蛋白的结合比PEThTM3异构体更强。然而，用McGhee和von Hippel方程分析肌动蛋白结合情况发现，PEThTM3在结合时表现出比PEThTM5更高的协同性。此外，具有hTM5 N端片段与hTM3 C端片段融合的嵌合体PEThTM5/3具有更强的肌动蛋白结合能力。具有hTM3 N端片段与hTM5 C端片段融合的反向嵌合体PEThTM3/5对肌动蛋白丝的亲和力大大降低。此外，与它们的亲本原肌球蛋白相比，两种嵌合体在与F-肌动蛋白最佳结合时对KCl的需求不同。细菌制备的人类成纤维细胞钙调蛋白的C端片段（PETCaD39）和天然鸡胗钙调蛋白都能够增强这些细菌表达的原肌球蛋白的肌动蛋白结合能力。然而，PETCaD39对PEThTM5的F-肌动蛋白结合增强作用比对PEThTM3的更大。在30 mM KCl和4 mM MgCl2条件下，低分子量异构体PEThTM4似乎能够将肌动蛋白激活的HMM ATP酶活性放大4.7倍，而高分子量异构体PEThTM3仅刺激该活性1.5倍。PEThTM5对ATP酶活性的增强作用高于PEThTM3，这表明低分子量异构体hTM5可能比hTM3更参与调节非肌肉细胞的运动。这些结果进一步表明，原肌球蛋白的不同异构体在细胞内的特定功能（如细胞骨架功能与运动功能）可能存在有限的差异。
