MAPPING OF THE MICROVILLAR 110K-CALMODULIN COMPLEX - CALMODULIN-ASSOCIATED OR CALMODULIN-FREE FRAGMENT OF THE 110-KD POLYPEPTIDE BIND F-ACTIN AND RETAIN ATPASE ACTIVITY
MAPPING OF THE MICROVILLAR 110K-CALMODULIN COMPLEX - CALMODULIN-ASSOCIATED OR CALMODULIN-FREE FRAGMENT OF THE 110-KD POLYPEPTIDE BIND F-ACTIN AND RETAIN ATPASE ACTIVITY
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DOI：
10.1083/jcb.106.2.367
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发表时间：
1988-02-01
影响因子：
7.8
通讯作者：
BRETSCHER, A
中科院分区：
文献类型：
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作者：
COLUCCIO, LM;BRETSCHER, A
从小肠微绒毛中分离的110 K-钙调素复合物是由一条110 kD多肽链与3 ~ 4个钙调素分子结合而成的ATP酶。这种复合物可能是微绒毛核心中的肌动蛋白丝与周围细胞膜之间的联系。为了研究其结构区域，我们用α-钙调蛋白部分切割了110 K-钙调蛋白复合物。胰凝乳蛋白酶;钙调蛋白在所用条件下基本保持完整。如125 I-钙调素覆盖，离子交换色谱法，和肌动蛋白结合测定，EGTA中产生的90 kD消化片段仍然与钙调素。90 K-钙调素复合物结合肌动蛋白在ATP可逆的方式和装饰肌动蛋白丝与箭头外观类似的F-肌动蛋白与母体复合物孵育后发现，结合发生在钙或EGTA的缓冲液。90 kD消化物的ATP酶活性与母体复合物非常相似。在钙中，消化混合物含有78 kD的片段，一组三个在λ40 kD，并且用α-葡聚糖酶产生32-kD片段（78-kD消化混合物）。在较长的孵育时间内，凝乳蛋白旋转;没有观察到这些片段与钙调蛋白的结合。地高辛和溴化氰裂解的时间过程表明，78-kD片段来自90-kD肽。78 kD混合物也能水解ATP。此外，通过离子交换色谱法从该78 kD混合物中去除钙调蛋白对消化物的ATP酶活性没有影响，表明ATP酶活性存在于110 kD多肽上。78 kD，三个片段中的两个在. apprx。40 kD，32 kD片段与F-肌动蛋白以ATP可逆的方式结合。电子显微镜下的肌动蛋白丝孵育后的78 kD消化混合物揭示了涂层丝，虽然没有观察到突出的箭头外观特征的父复合物。这些数据表明，钙调素是不需要的ATP酶活性或ATP-可逆结合的110 K-钙调素复合物F-肌动蛋白。此外，由于所有与F-肌动蛋白结合的片段都是以ATP可逆的方式结合的，因此F-肌动蛋白结合和ATP可逆性所需的位点可能就在附近。
The 110K-calmodulin complex isolated from intestinal microvilli is an ATPase consisting of one polypeptide chain of 110 kD in association with three to four calmodulin molecules. This complex is presumably the link between the actin filaments in the microvillar core and the surrounding cell membrane. To study its structural regions, we have partially cleaved the 110K-calmodulin complex with .alpha.-chymotrypsin; calmodulin remains essentially intact under the conditions used. As determined by 125I-calmodulin overlays, ion exchange chromatography, and actin-binding assays, a 90-kD digest fragment generated in EGTA remains associated with calmodulin. The 90K-calmodulin complex binds actin in an ATP-reversible manner and decorates actin filaments with an arrow-head appearance similar to that found after incubation of F-actin with the parent complex; binding occurs in either calcium- or EGTA-containing buffers. ATPase activity of the 90-kD digest closely resembles the parent complex. In calcium a digest mixture containing fragments of 78 kD, a group of three at .apprx. 40 kD, and a 32-kD fragment (78-kD digest mixture) is generated with .alpha.-chymotryspin at a longer incubation time; no association of these fragments with calmodulin is observed. Time courses of digestions and cyanogen bromide cleavage indicate that the 78-kD fragment derives from the 90-kD peptide. The 78-kD mixture can also hydrolyze ATP. Furthermore, removal of the calmodulin by ion exchange chromatography from this 78-kD mixture had no effect on the ATPase activity of the digest, indicating that the ATPase activity resides on the 110-kD polypeptide. The 78 kD, two of the three fragments at .apprx. 40 kD, and the 32-kD fragments associate with F-actin in an ATP-reversible manner. Electron microscopy of actin filaments after incubation with the 78-kD digest mixture reveals coated filaments, although the prominent arrowhead appearance characteristic of the parent complex is not observed. These data indicate that calmodulin is not required either for the ATPase activity or the ATP-reversible binding of the 110K-calmodulin complex to F-actin. In addition, since all the fragments that bind F-actin do so in an ATP-reversible manner, the sites required for F-actin binding and ATP reversibility likely reside nearby.