Strzelecka-Golaszewska H, Wozniak A, Hult T, Lindberg U
Nencki Institute of Experimental Biology, Department of Muscle Biochemistry, Warsaw, Poland.
Biochem J. 1996 Jun 15;316 ( Pt 3)(Pt 3):713-21. doi: 10.1042/bj3160713.
F-actins containing either Ca2+ or Mg2+ at the single high-affinity site for a divalent cation differ in their dynamic properties [Carlier (1991) J. Biol. Chem. 266, 1-4]. In an attempt to obtain information on the structural basis of this difference, we probed the conformation of specific sites in the subunits of Mg- and Ca-F-actin with limited proteolysis by subtilisin and trypsin. The influence of the kind of polymerizing salt was also investigated. At high proteinase concentrations required for digestion of actin in the polymer form, subtilisin gives a complex fragmentation pattern. In addition to the earlier known cleavage between Met47 and Gly48 in the DNAse-I-binding loop, cleavage of F-actin between Ser234 and Ser235 in subdomain 4 has recently been reported [Vahdat, Miller, Phillips, Muhlrad and Reisler (1995) FEBS Lett. 365, 149-151]. Here we show that actually a larger segment, comprising residues 227-235, is removed and the bond between Leu67 and Lys68 in subdomain 2 is split in both G- and F-actin, and that the differences in the fragmentation patterns of the G- and F-forms are accounted for by the protection of the bond 47-48 in F-actin. The subtilisin and trypsin cleavage sites in segment 61-69, subtilisin sites in segment 227-235 and trypsin sites between Lys373 and Cys374 were less accessible in Mg-F-actin than in Ca-F-actin. These are intramolecular effects, as similar changes were observed on Ca2+/Mg2+ replacement in G-actin. The cation-dependent effects, in particular those on segment 61-69, were however less pronounced in F-actin than in G-actin. The results suggest that substitution of Mg2+ for Ca2+, and KCl-induced polymerization of CaATP-G-actin, bring about a similar change in the conformation of subdomain 2 of the monomer. The presence of Mg2+ at the high-affinity site also resulted in an increased protection of the bond 47-48. This latter appears to be an intermolecular effect because it is specific for F-actin. The susceptibility to subtilisin and trypsin was also strongly influenced by the kind and concentration of polymerizing salt. The digestion patterns suggest that the exposure and/or flexibility of the regions containing the cleavage sites diminish with enhancement of the ionic strength of the solution. The results are discussed in terms of the current models of F-actin.
在二价阳离子的单一高亲和力位点含有Ca2+或Mg2+的F-肌动蛋白，其动态特性有所不同[卡利尔（1991年）《生物化学杂志》266卷，第1 - 4页]。为了获取有关这种差异结构基础的信息，我们用枯草杆菌蛋白酶和胰蛋白酶进行有限蛋白水解，探究了Mg - F - 肌动蛋白和Ca - F - 肌动蛋白亚基中特定位点的构象。还研究了聚合盐种类的影响。在消化聚合形式的肌动蛋白所需的高蛋白酶浓度下，枯草杆菌蛋白酶产生复杂的片段化模式。除了早期已知的在DNA酶I结合环中Met47和Gly48之间的切割外，最近有报道称在亚结构域4中Ser234和Ser235之间的F - 肌动蛋白也会被切割[瓦赫达特、米勒、菲利普斯、穆尔拉德和赖斯勒（1995年）《欧洲生物化学学会联合会快报》365卷，第149 - 151页]。在此我们表明，实际上一个更大的片段，包含227 - 235位残基，会被去除，并且在亚结构域2中Leu67和Lys68之间的键在G - 肌动蛋白和F - 肌动蛋白中都会断裂，G - 形式和F - 形式片段化模式的差异是由于F - 肌动蛋白中47 - 48键受到保护。在Mg - F - 肌动蛋白中，61 - 69片段中的枯草杆菌蛋白酶和胰蛋白酶切割位点、227 - 235片段中的枯草杆菌蛋白酶位点以及Lys373和Cys374之间的胰蛋白酶位点，比在Ca - F - 肌动蛋白中更难接近。这些是分子内效应，因为在G - 肌动蛋白中Ca2+/Mg2+替换时观察到了类似的变化。然而，阳离子依赖性效应，特别是对61 - 69片段的效应，在F - 肌动蛋白中比在G - 肌动蛋白中不太明显。结果表明，用Mg2+替代Ca2+以及KCl诱导CaATP - G - 肌动蛋白聚合，会使单体亚结构域2的构象发生类似变化。在高亲和力位点存在Mg2+也导致47 - 48键的保护增加。后者似乎是一种分子间效应，因为它对F - 肌动蛋白具有特异性。枯草杆菌蛋白酶和胰蛋白酶的敏感性也受到聚合盐种类和浓度的强烈影响。消化模式表明，随着溶液离子强度的增加，含有切割位点区域的暴露和/或柔韧性会降低。根据当前的F - 肌动蛋白模型对结果进行了讨论。
