Wenegieme E F, Naren A P, Bobich J A
Department of Chemistry, Texas Christian University, Fort Worth 76129, USA.
Biometals. 1996 Jul;9(3):259-65. doi: 10.1007/BF00817925.
We examined the effects of changing KCl concentration on the secondary structures of alpha-actinins using circular dichroism (CD), 1,1'-bis(4-anilino) naphthalene-5,5'-disulfonic acid (bisANS) fluorescence and proteolysis experiments. Under near-physiological conditions, divalent cations also were added and changes in conformation were investigated. In 25 mM KH2PO4, pH 7.5, increasing KCl from 0 to 120 mM led to decreases in alpha-helix conformation for brain, platelet and heart alpha-actinins (40.5-30.2%, 65.5-37.8% and 37.5-27.8%, respectively). In buffered 120 mM KCl, 0.65 mM calcium produced small changes in the CD spectra of both brain and platelet alpha-actinin, but had no effect on heart alpha-actinin. bisANS fluorescence of all three alpha-actinins also showed significant changes in conformation with increasing KCl. However, in buffered 120 mM KCl increasing concentrations of Ca2+ or Mg2+ did not have significant effects on the bisANS fluorescence of any alpha-actinin. Digestion of brain, platelet and heart alpha-actinins with alpha-chymotrypsin showed an increase of proteolytic susceptibility in 120 mM KCl. These experiments also showed that increasing the concentration of Ca2+ or Mg2+ led to greater changes in digestion fragment patterns in the absence of KCl than in the presence of 120 mM KCl. The results suggest that alpha-actinins exist in different conformations depending on the ionic strength of the medium, which could explain the differences in calcium and F-actin binding results obtained from different alpha-actinins.
我们使用圆二色性（CD）、1,1'-双（4-苯胺基）萘-5,5'-二磺酸（bisANS）荧光和蛋白水解实验，研究了改变氯化钾（KCl）浓度对α-辅肌动蛋白二级结构的影响。在接近生理条件下，还添加了二价阳离子，并研究了构象变化。在25 mM磷酸二氢钾（KH2PO4），pH 7.5的条件下，将KCl浓度从0增加到120 mM会导致脑、血小板和心脏α-辅肌动蛋白的α-螺旋构象减少（分别为40.5 - 30.2%、65.5 - 37.8%和37.5 - 27.8%）。在含有120 mM KCl的缓冲液中，0.65 mM钙对脑和血小板α-辅肌动蛋白的CD光谱产生了微小变化，但对心脏α-辅肌动蛋白没有影响。随着KCl浓度增加，所有三种α-辅肌动蛋白的bisANS荧光也显示出显著的构象变化。然而，在含有120 mM KCl的缓冲液中，增加Ca2+或Mg2+的浓度对任何α-辅肌动蛋白的bisANS荧光都没有显著影响。用α-胰凝乳蛋白酶消化脑、血小板和心脏α-辅肌动蛋白表明，在120 mM KCl中蛋白水解敏感性增加。这些实验还表明，在不存在KCl的情况下，增加Ca2+或Mg2+的浓度比在存在120 mM KCl时导致消化片段模式的变化更大。结果表明，α-辅肌动蛋白根据介质的离子强度以不同构象存在，这可以解释从不同α-辅肌动蛋白获得的钙和F-肌动蛋白结合结果的差异。
