中国生物化学与分子生物学报
中國生物化學與分子生物學報
중국생물화학여분자생물학보
CHINESE JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY
2006年
12期
953-959
,共7页
dynamitin%ATP酶%动力蛋白激活蛋白%动力蛋白%微管
dynamitin%ATP酶%動力蛋白激活蛋白%動力蛋白%微管
dynamitin%ATP매%동력단백격활단백%동력단백%미관
dynamitin%ATPase%dynactin%dynein%microtubules
动力蛋白激活蛋白(dynactin)是一个与胞浆内动力蛋白的功能相关的多亚基复合物.动力蛋白(dynein)为向微管负端运输的马达蛋白,其多种功能包括细胞核迁移、有丝分裂纺锤体定位以及细胞间期和有丝分裂的细胞骨架再组装.Dynamitin,是一个50 kD的动力蛋白激活蛋白亚单位,对于稳定动力蛋白激活蛋白复合物是非常重要的.为研究这种稳定性机制,分析了dynamitin的序列,并揭示dynamitin的一些DNA序列与ATP酶的Walker A和Walker B序列具有同源性.纯化的谷胱甘肽巯基转移酶标签蛋白dynamitin和无此标签的蛋白dynamitin都特异性显示了ATP酶活性.DNA序列Walker A的失活突变可废除dynamitin蛋白的ATP酶活性,而Walker B序列无此作用.因此,突变实验进一步证实dynamitin蛋白的ATP酶活性.ATP酶活性的动力学研究结果表明Km为125.78μmol/L和kcat为7.4 min-1.
動力蛋白激活蛋白(dynactin)是一箇與胞漿內動力蛋白的功能相關的多亞基複閤物.動力蛋白(dynein)為嚮微管負耑運輸的馬達蛋白,其多種功能包括細胞覈遷移、有絲分裂紡錘體定位以及細胞間期和有絲分裂的細胞骨架再組裝.Dynamitin,是一箇50 kD的動力蛋白激活蛋白亞單位,對于穩定動力蛋白激活蛋白複閤物是非常重要的.為研究這種穩定性機製,分析瞭dynamitin的序列,併揭示dynamitin的一些DNA序列與ATP酶的Walker A和Walker B序列具有同源性.純化的穀胱甘肽巰基轉移酶標籤蛋白dynamitin和無此標籤的蛋白dynamitin都特異性顯示瞭ATP酶活性.DNA序列Walker A的失活突變可廢除dynamitin蛋白的ATP酶活性,而Walker B序列無此作用.因此,突變實驗進一步證實dynamitin蛋白的ATP酶活性.ATP酶活性的動力學研究結果錶明Km為125.78μmol/L和kcat為7.4 min-1.
동력단백격활단백(dynactin)시일개여포장내동력단백적공능상관적다아기복합물.동력단백(dynein)위향미관부단운수적마체단백,기다충공능포괄세포핵천이、유사분렬방추체정위이급세포간기화유사분렬적세포골가재조장.Dynamitin,시일개50 kD적동력단백격활단백아단위,대우은정동력단백격활단백복합물시비상중요적.위연구저충은정성궤제,분석료dynamitin적서렬,병게시dynamitin적일사DNA서렬여ATP매적Walker A화Walker B서렬구유동원성.순화적곡광감태구기전이매표첨단백dynamitin화무차표첨적단백dynamitin도특이성현시료ATP매활성.DNA서렬Walker A적실활돌변가폐제dynamitin단백적ATP매활성,이Walker B서렬무차작용.인차,돌변실험진일보증실dynamitin단백적ATP매활성.ATP매활성적동역학연구결과표명Km위125.78μmol/L화kcat위7.4 min-1.
Dynactin is a multi-subunit complex that has been implicated in the function of cytoplasmic dynein which is a minus end - directed microtubule motor protein with numerous functions including nuclear migration, mitotic spindle orientation, and cytoskeletal reorientation during interphase and mitosis. Dynamitin,the 50 kD subunit of dynactin, is important for stabilizing the dynactin complex. To gain more insight into the mechanism of stabilizing, we analyzed the sequence of dynamitin and revealed that domains of dynamitin is homology to the Walker A and Walker B ATPase motifs. The purified GST-dynamitin and GST-free dynamitin both showed ATPase activity specifically. An inactivating mutation in the Walker A, but not the Walker B ATPase motif abolished the ATPase activity of dynamitin. The mutational analysis studies further supported that dynamitin is an ATPase. Kinetic studies of the ATPase activity of dynamitin revealed a Km for ATP of 125.78μmol/L and a kcat of 7.4 min-1.
