INTERACTION OF RAT-LIVER LYSOSOMAL MEMBRANES WITH ACTIN
INTERACTION OF RAT-LIVER LYSOSOMAL MEMBRANES WITH ACTIN
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DOI：
10.1083/jcb.99.2.680
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发表时间：
1984-01-01
影响因子：
7.8
通讯作者：
ROME, LH
中科院分区：
文献类型：
--
作者：
MEHRABIAN, M;BAME, KJ;ROME, LH
通过在不连续的甲泛葡胺梯度中离心，从纯化80倍的溶酶体制备膜。当盐洗膜与兔肌肉肌动蛋白结合时，粘度的增加可以使用落球粘度计测量。溶酶体膜-肌动蛋白相互作用是肌动蛋白和膜浓度依赖性的，并且在假定的生理条件下（2 mM MgCl 2、1 mM MgATP、中性pH和游离Ca浓度< 10-8 M）似乎是最佳的。膜的肌动蛋白交联活性在pH 6.4时最佳。10-7和10-9 M游离Ca离子之间的相互作用是最大的，并受到抑制。在Ca浓度> 0.5 × 10 - 6时为60%。10-7 M.如果用链霉蛋白酶预处理膜，或者如果在不存在蛋白酶抑制剂的情况下纯化膜，则肌动蛋白-溶酶体膜相互作用被破坏。如果用高盐洗涤或用KCl和尿素萃取膜，则相互作用不会被破坏。此外，还进行了肌动蛋白-溶酶体膜相互作用的沉降试验以证实粘度测定数据。结果表明存在一个完整的溶酶体膜肌动蛋白结合蛋白。
Membranes were prepared from lysosomes purified 80-fold by centrifugation in a discontinuous metrizamide gradient. When salt-washed membranes were combined with rabbit muscle actin, an increase in viscosity could be measured using a falling ball viscometer. The lysosomal membrane-actin interaction was actin- and membrane-concentration dependent and appeared to be optimal under presumed physiological conditions (2 mM MgCl2, 1 mM MgATP, neutral pH, and free Ca concentration < 10-8 M). The actin crosslinking activity of the membrane was optimal at pH 6.4. The interaction was maximal between 10-7 and 10-9 M free Ca ions and inhibited by .apprx. 60% at concentrations of Ca > 0.5 .times. 10-7 M. The actin-lysosomal membrane interaction was destroyed if the membranes were pretreated with Pronase, or if the membranes were purified in the absence of protease inhibitors. The interaction was not destroyed if membranes were washed with high salt or extracted with KCl and urea. In addition, a sedimentation assay for the actin-lysosomal membrane interaction was also performed to corroborate the viscometry data. Results suggest the existence of an integral lysosomal membrane actin-binding protein.