Dabrowska R, Hinssen H, Gałazkiewicz B, Nowak E
Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Biochem J. 1996 May 1;315 ( Pt 3)(Pt 3):753-9. doi: 10.1042/bj3150753.
We have investigated the cumulative effects of three smooth-muscle actin-binding proteins, gelsolin, caldesmon and tropomyosin, on actin activation of myosin Mg(2+)-ATPase activity under low-ionic-strength conditions. A combination of tropomyosin (at a stoicheiometric ratio to actin) and gelsolin (at a molar ratio to actin of up to 1:100) showed essentially additive stimulatory effects that were counteracted by caldesmon. Suppression of the gelsolin-induced activation of the ATPase by caldesmon was higher in the presence of tropomyosin although it was not complete even at stoicheiometric amounts of both proteins to actin. Since activation of actin-activated ATPase activity of myosin by gelsolin is related to its severing action, it is concluded that caldesmon and tropomyosin cannot fully protect actin filaments against the severing activity of gelsolin. Direct analysis of the actin-severing activity of gelsolin by a fluorimetric assay using pyrene-labelled actin confirmed this conclusion. Tropomyosin and caldesmon in saturating amounts relative to actin inhibited the activity of gelsolin by between 21 and 40% and 25 and 48% respectively, depending on the molar ratio of gelsolin to actin. The inhibitory effect was increased with a combination of both (up to 67%) although it was evident that even under these conditions the actin filaments were not fully protected from being severed by gelsolin. These findings were corroborated by electron-microscopic investigation of actin filaments with or without tropomyosin and caldesmon after the addition of gelsolin.
我们研究了三种平滑肌肌动蛋白结合蛋白，凝溶胶蛋白、钙调蛋白和原肌球蛋白，在低离子强度条件下对肌球蛋白Mg(2+)-ATP酶活性的肌动蛋白激活的累积效应。原肌球蛋白（与肌动蛋白的化学计量比）和凝溶胶蛋白（与肌动蛋白的摩尔比高达1:100）的组合显示出基本的加性刺激作用，而这种作用被钙调蛋白抵消。在存在原肌球蛋白的情况下，钙调蛋白对凝溶胶蛋白诱导的ATP酶激活的抑制作用更高，尽管即使两种蛋白与肌动蛋白的化学计量相等时，这种抑制也不完全。由于凝溶胶蛋白对肌球蛋白的肌动蛋白激活的ATP酶活性的激活与其切断作用有关，因此得出结论，钙调蛋白和原肌球蛋白不能完全保护肌动蛋白丝免受凝溶胶蛋白的切断活性的影响。使用芘标记的肌动蛋白通过荧光测定法对凝溶胶蛋白的肌动蛋白切断活性进行直接分析证实了这一结论。相对于肌动蛋白饱和量的原肌球蛋白和钙调蛋白分别抑制凝溶胶蛋白的活性21%至40%和25%至48%，这取决于凝溶胶蛋白与肌动蛋白的摩尔比。两种蛋白联合使用时抑制作用增强（高达67%），尽管很明显即使在这些条件下肌动蛋白丝也不能完全免受凝溶胶蛋白的切断。在添加凝溶胶蛋白后，对有或没有原肌球蛋白和钙调蛋白的肌动蛋白丝进行电子显微镜研究证实了这些发现。
