MACROPHAGE ALPHA-ACTININ IS NOT A CALCIUM-MODULATED ACTIN-BINDING PROTEIN
MACROPHAGE ALPHA-ACTININ IS NOT A CALCIUM-MODULATED ACTIN-BINDING PROTEIN
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DOI：
10.1021/bi00052a045
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发表时间：
1993-01-12
期刊：
影响因子：
2.9
通讯作者：
HARRICANE, MC
中科院分区：
文献类型：
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作者：
PACAUD, M;HARRICANE, MC
从兔巨噬细胞中纯化α-辅肌动蛋白，通过设计去除其他辅肌动蛋白结合蛋白的程序使其表观均一。当1个α-辅肌动蛋白分子与10-12个肌动蛋白单体相互作用时，形成大束的细丝。这一过程涉及两类具有不同亲和力的肌动蛋白结合位点的连续占据。α-辅肌动蛋白对F-肌动蛋白的表观K（d）不受加入25 μ M游离Ca ~（2+）的影响。通过低速沉降试验、低剪切粘度和电子显微镜分析增加Ca 2+浓度对α-辅肌动蛋白-F-肌动蛋白相互作用的影响，表明Ca 2+在大约50-1000 μ M的范围内具有小的抑制作用。此外，α-辅肌动蛋白组装成束的肌动蛋白丝的能力明显抑制只有在Ca 2+浓度，这也影响了单独的F-肌动蛋白的物理性质。固定在硝酸纤维素膜上的α-辅肌动蛋白不结合可检测量的Ca 2+。然而，Ca 2+或Mg 2+结合α-辅肌动蛋白诱导色氨酸和酪氨酸残基的荧光发射强度的小的下降。Mg 2+诱导的最大变化小于Ca 2+，但Ca 2+和Mg 2+的影响被取消，通过添加140 mM KCl。在接近物理学的离子条件下，不能检测到表观K（d）高于80-100 μ M的Ca 2+结合位点。α-辅肌动蛋白的功能和物理性质的结果是一致的假设，即Ca 2+减少α-辅肌动蛋白-F-肌动蛋白相互作用的肌动蛋白丝和交联分子。尽管该结论与普遍接受的观点相矛盾，即α A是Ca 2+调节的肌动蛋白结合蛋白，但它可以从α-肌动蛋白单体中两个EF-手样基序的一级序列中预测[Arimura等人（1988）Eur. J. Biochem. 177，649-6551基于最近通过钙调蛋白的Ca 2+结合位点中的点突变证明的一些Ca 2+结合残基的关键作用[Haiech等人（1991）J. Biol. Chem. 266，3427-3431]。这也与我们先前的发现一致，即Ca 2+不影响来自巨噬细胞胞质提取物的肌动蛋白凝胶网络中α-辅肌动蛋白的行为[Pacaud & Harricane（1987）J. Cell Sci. 88，81-94]。
Alpha-actinin was purified from rabbit macrophages to apparent homogeneity by a procedure designed to remove other actin-binding proteins. Large bundles of filaments were formed when 1 molecule of alpha-actinin interacted with 10-12 actin monomers. This process involved the successive occupancy of two classes of actin-binding sites with different affinities. The apparent K(d) of alpha-actinin for F-actin was unaffected by the addition of 25 muM free Ca2+. Analysis of the influence of increasing Ca2+ concentrations on alpha-actinin-F-actin interactions by low-speed sedimentation assays, low-shear viscosity, and electron microscopy indicated that Ca2+ had a small inhibitory effect in the approximate range of 50-1000 muM. Furthermore, the ability of alpha-actinin to assemble actin filaments into bundles was apparently inhibited only at Ca2+ concentrations which also affected the physical properties of F-actin alone. Alpha-actinin immobilized on a nitrocellulose membrane did not bind detectable amounts of Ca2+. Nevertheless, Ca2+ or Mg2+ binding to alpha-actinin induced small decreases in the fluorescence emission intensity of tryptophan and tyrosine residues. The maximal change induced by Mg2+ was smaller than that observed with Ca2+, but Ca2+ and Mg2+ effects were abolished by the addition of 140 mM KCl. Under near-physicological ionic conditions, Ca2+-binding sites with an apparent K(d) higher than 80-100 muM could not be detected. The results on the functional and physical properties of alpha-actinin are consistent with the hypothesis that Ca2+ decreases alpha-actinin-F-actin interactions by acting both on actin filaments and on cross-linking molecules. Although this conclusion is in contradiction with the generally accepted idea that alphaA is a Ca2+-regulated actin-binding protein, it could be predicted from the primary sequence of the two EF-hand-like motifs in the alpha-actinin monomer [Arimura et al. (1988) Eur. J. Biochem. 177, 649-6551 based on the crucial role of some Ca2+-binding residues as recently demonstrated by point mutations in Ca2+-binding sites of calmodulin [Haiech et al. (1991) J. Biol. Chem. 266, 3427-3431]. It is also in agreement with our previous finding that Ca2+ does not affect the behavior of alpha-actinin in actin gel networks from macrophage cytosolic extracts [Pacaud & Harricane (1987) J. Cell Sci. 88, 81-94].