Isolation and characterization of the N-terminal 23-kilodalton fragment of myosin subfragment 1.
Isolation and characterization of the N-terminal 23-kilodalton fragment of myosin subfragment 1.
复制标题
肌球蛋白亚片段 1 N 端 23 千道尔顿片段的分离和表征。
DOI：
10.1021/bi00435a056
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Muhlrad,A
中科院分区：
文献类型：
--
作者：
Muhlrad,A
（S1）。重链片段解离盐酸胍有限的胰蛋白酶解后，和23 kDa的片段通过凝胶过滤和离子交换色谱分离。最后，通过去除变性剂使片段复性。复性片段的圆二色性谱显示了有序结构的存在。当加入盐酸胍时，该片段的色氨酸荧光发射光谱显著地向红色移动，这表明色氨酸位于相对疏水的环境中。这两个23-kDatryp-tophans，不像其余的Sl-色氨酸，是完全接近丙烯酰胺，如荧光淬灭所示。分离的23-kDa片段与F-肌动蛋白在超电泳中共沉淀，并显着增加溶液中肌动蛋白的光散射，这表明肌动蛋白结合。结合是相当紧密的（Kd= 0.1 μ M）和离子强度依赖性的（随着离子强度的增加而降低）。ATP、焦磷酸盐和ADP使23-kDa-肌动蛋白复合物解离，但效力降低。分离的23-kDAF片段不具有ATP酶活性;然而，它可能通过与Sl竞争肌动蛋白上的结合位点来抑制Sl的肌动蛋白激活的ATP酶活性。F-肌动蛋白与固定在硝酸纤维素膜上的23-kDa片段结合。该片段被进一步切割，并且发现含有130-204段残基的所得肽之一结合硝酸纤维素膜上的肌动蛋白，表明23-kDa片段的该区域参与形成肌动蛋白结合位点。
(Sl). The heavy-chain fragments were dissociated by guanidine hydrochloride following limited trypsinolysis, and the 23-kDa fragment was isolated by gel filtration and ion-exchange chromatography. Finally, the fragment was renatured by removing the denaturants. The CD spectrum of the renatured fragment shows the presence of ordered structure. The tryptophan fluorescence emission spectrum of the fragment is considerably shifted to the red upon adding guanidine hydrochloride which indicates that the tryptophans are located in relatively hydrophobic environments. The two 23-kDatryp-tophans, unlike the rest of the Sl tryptophans, are fully accessible to acrylamide as indicated by fluorescence quenching. The isolated 23-kDa fragment cosediments with F-actin inthe ultracentrifuge and significantly increases the light scattering of actin in solution which indicates actin binding. The binding is rather tight (Kd= 0.1 juM) and ionic strength dependent (decreasing with increasing ionic strength). ATP, pyrophosphate, and ADP dissociate the 23-kDa-actin complex with decreasing effectiveness. The isolated 23-kDafragment does not have ATPase activity; however, it inhibits the actin-activated ATPase activity of Sl by competing presumably with Sl for binding sites on actin. F-Actin binds to the 23-kDa fragmentimmobilized on the nitrocellulose membrane. The fragment was further cleaved, and one of the resulting peptides, containing the 130-204 stretch of residues, was found to bind actin on the nitrocellulose membrane, indicating that this region of the 23-kDa fragment participates in forming an actin binding site.