Ohtsuka Y, Nakae H, Abe H, Obinata T
Department of Biology, Faculty of Science, Graduate School of Science and Technology, Chiba University, Japan.
Biochim Biophys Acta. 1998 Apr 2;1383(2):219-31. doi: 10.1016/s0167-4838(97)00211-2.
The body wall of the ascidian is composed of unusual multi-nucleated smooth muscle cells enriched with thin actin filaments containing troponin-tropomyosin which run along the longitudinal cell axis without being organized into striated structures. We purified an actin-binding protein of 80 kDa, tentatively termed 80K protein, from the body wall muscle of ascidian, Halocynthia roretzi, and characterized the functional properties and molecular structures. In the presence of Ca2+, the 80K protein accelerated the initial phase of actin polymerization, namely the nucleation process, decreased the level of polymerization at the steady state, caused marked reduction in viscosity of an F-actin solution, and fragmented F-actin filaments, while in the absence of Ca2+, it remained associated with F-actin without severing the filaments. The interaction of the 80K protein with actin was inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). When actin was polymerized in the presence of acrosome actin bundles from horseshoe crab sperm, the 80K protein inhibited the growth of actin filaments at the barbed end but not at the pointed end, indicating that the 80K protein functions as a barbed-end capping protein. In order to characterize the molecular structure of the 80K protein, cDNAs encoding this protein were isolated from the lambda gt11 cDNA library of the ascidian muscle by using a monoclonal antibody (AS23) specific for this protein and the entire sequence was determined. The deduced peptide sequence showed about 44% homology in amino acid residues with the human gelsolin sequence, and in addition, 6 repeating segments were observed in the sequence of the 80K protein as has been described in the gelsolin sequence. These results indicate strongly that the 80K protein belongs to the gelsolin family.
海鞘的体壁由异常的多核平滑肌细胞组成，这些细胞富含细肌动蛋白丝，其中含有肌钙蛋白 - 原肌球蛋白，它们沿细胞纵轴排列，未形成横纹结构。我们从海鞘柄海鞘的体壁肌肉中纯化了一种80 kDa的肌动蛋白结合蛋白，暂称为80K蛋白，并对其功能特性和分子结构进行了表征。在Ca2+存在的情况下，80K蛋白加速了肌动蛋白聚合的初始阶段，即成核过程，降低了稳态时的聚合水平，导致F - 肌动蛋白溶液的粘度显著降低，并使F - 肌动蛋白丝断裂，而在没有Ca2+的情况下，它与F - 肌动蛋白结合但不切断肌动蛋白丝。80K蛋白与肌动蛋白的相互作用受到磷脂酰肌醇4,5 - 二磷酸（PIP2）的抑制。当肌动蛋白在鲎精子顶体肌动蛋白束存在的情况下聚合时，80K蛋白抑制肌动蛋白丝在带刺末端的生长，但不抑制在尖端的生长，表明80K蛋白作为一种带刺末端封端蛋白发挥作用。为了表征80K蛋白的分子结构，使用针对该蛋白的单克隆抗体（AS23）从海鞘肌肉的λgtll cDNA文库中分离出编码该蛋白的cDNA，并确定了其完整序列。推导的肽序列在氨基酸残基上与人凝溶胶蛋白序列显示出约44%的同源性，此外，在80K蛋白的序列中观察到6个重复片段，如在凝溶胶蛋白序列中所描述的那样。这些结果有力地表明80K蛋白属于凝溶胶蛋白家族。
