Funatsu T, Asami Y, Ishiwata S
Department of Physics, School of Science and Engineering, Waseda University, Tokyo.
J Biochem. 1988 Jan;103(1):61-71. doi: 10.1093/oxfordjournals.jbchem.a122240.
We examined the function of beta-actinin as a pointed end capping protein of thin filaments in skeletal muscle. An improvement in preparing beta-actinin yielded purified beta-actinin which retained its activity for more than a week. Two-dimensional gel electrophoresis showed that the two subunits, beta I and beta II, of beta-actinin are, respectively, split into two to three components (isoforms) with different isoelectric points. Polyclonal antibody was raised by injecting such purified and undenatured chicken breast muscle beta-actinin composed of several components into a rabbit. Immuno-gold labeling examination with electron microscopy of an F-actin-beta-actinin complex decorated with HMM showed that 85% of bound gold particles was on the pointed end of actin filaments, while the remaining 15% was on the barbed end. This suggests that in beta-actinin preparation pointed end and barbed end capping proteins inevitably coexist. Immunofluorescence and immunoelectron microscopy directly showed that beta-actinin is located at the pointed end of thin filaments in myofibrils; it was also suggested that a capping protein having common antigenic determinants to beta-actinin is located at Z-line. Thus, the physiological function of beta-actinin as a pointed end capping protein was examined as follows: When beta-actinin was dissociated from the pointed end of thin filaments in an I-Z-I brush by using a high salt solution, thin filaments could be disassembled at the pointed ends at concentrations of exogenous actin lower than a critical value. At a physiological ionic strength, these salt-washed thin filaments gradually shortened at a constant rate of about 45 nm/h. Both the association and dissociation of monomeric actin at the pointed end were suppressed by the rebinding of exogenous beta-actinin. The main physiological role of beta-actinin is therefore to stabilize thin filaments in the sarcomere by preventing addition and removal of actin monomers at the pointed filament end.
我们研究了β-辅肌动蛋白作为骨骼肌细肌丝尖端部加帽蛋白的功能。β-辅肌动蛋白制备方法的改进得到了纯化的β-辅肌动蛋白，其活性可保持一周以上。二维凝胶电泳显示，β-辅肌动蛋白的两个亚基βI和βII分别被分成两到三个具有不同等电点的组分（同工型）。通过将由几种组分组成的纯化且未变性的鸡胸肌β-辅肌动蛋白注射到兔子体内，制备了多克隆抗体。用HMM修饰的F-肌动蛋白-β-辅肌动蛋白复合物进行电子显微镜免疫金标记检查，结果显示85%的结合金颗粒位于肌动蛋白丝的尖端，其余15%位于肌动蛋白丝的另一端。这表明在β-辅肌动蛋白的制备过程中，尖端部和另一端部加帽蛋白不可避免地共存。免疫荧光和免疫电子显微镜直接显示β-辅肌动蛋白位于肌原纤维细肌丝的尖端；还表明与β-辅肌动蛋白具有共同抗原决定簇的加帽蛋白位于Z线。因此，β-辅肌动蛋白作为尖端部加帽蛋白的生理功能如下：当使用高盐溶液使β-辅肌动蛋白从I-Z-I刷状结构中细肌丝的尖端解离时，在外源肌动蛋白浓度低于临界值的情况下，细肌丝可在尖端处解聚。在生理离子强度下，这些经盐洗的细肌丝以约45 nm/h的恒定速率逐渐缩短。外源β-辅肌动蛋白的重新结合抑制了肌动蛋白单体在尖端的结合和解离。因此，β-辅肌动蛋白的主要生理作用是通过防止肌动蛋白单体在细肌丝尖端的添加和去除来稳定肌节中的细肌丝。
