NATIVE STRUCTURE AND PHYSICAL-PROPERTIES OF BOVINE BRAIN KINESIN AND IDENTIFICATION OF THE ATP-BINDING SUBUNIT POLYPEPTIDE
NATIVE STRUCTURE AND PHYSICAL-PROPERTIES OF BOVINE BRAIN KINESIN AND IDENTIFICATION OF THE ATP-BINDING SUBUNIT POLYPEPTIDE
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DOI：
10.1021/bi00409a043
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发表时间：
1988-05-03
期刊：
影响因子：
2.9
通讯作者：
BRADY, ST
中科院分区：
文献类型：
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作者：
BLOOM, GS;WAGNER, MC;BRADY, ST
在5“-腺苷酰亚胺二磷酸（AMP-PNP）存在下，通过微管结合步骤，然后通过凝胶过滤层析和蔗糖梯度离心，从牛脑胞质溶胶中广泛纯化驱动蛋白。产物始终含有124，000（124 K）和64，000（64 K）道尔顿多肽。这两种多肽似乎分别代表驱动蛋白的重链和轻链，因为它们在蔗糖梯度上共纯化至恒定和等摩尔化学计量，并且在AMP-PNP而不是ATP存在下稳定地结合至微管。124 K和64 K在十二烷基硫酸钠-聚丙烯酰胺凝胶中的迁移率在还原条件下与非还原条件下相同。扩散系数为（2.24 ±. 0.21）×10-7 cm 2 s-1，沉降系数为（9.56 ± 0.01）。0.34）×分别用凝胶过滤法和蔗糖梯度超离心法测定了天然驱动蛋白的10-13 s。这些值用于计算约379，000的天然分子量，并表明驱动蛋白具有约20的轴比。广泛纯化的驱动蛋白具有微管激活的ATP酶活性，在使用[32 P]ATP的光亲和标记实验中，只有124 K亚基掺入ATP。总的来说，这些数据有利于解释牛脑驱动蛋白是一种高度伸长的微管激活的ATP酶，包含两个亚基，每个亚基的道尔顿为124，000和64，000，亚基之间没有通过二硫键相互连接，重链是ATP结合亚基。
Kinesin was extensively purified from bovine brain cytosol by a microtubule-binding step in the presence of 5''-adenylyl imidodiphosphate (AMP-PNP), followed by gel filtration chromatography and sucrose gradient ultracentrifugation. The products consistently contained 124,000 (124K) and 64,000 (64K) dalton polypeptides. These two polypeptides appear to represent heavy and light chains of kinesin, respectively, because they copurified on sucrose gradients to a constant and equimolar stoichiometry and bound stably to microtubules in the presence of AMP-PNP but not ATP. The mobilities of 124K and 64K in sodium dodecyl sulfate-polyacrylamide gels under reducing conditions were the same as under nonreducing conditions. A diffusion coefficient of (2.24 .+-. 0.21) .times. 10-7 cm2 s-1 and a sedimentation coefficient of (9.56 .+-. 0.34) .times. 10-13 s were determined for native kinesin by gel fitration and sucrose gradient ultracentrifugation, respectively. These values were used to calculate a native molecular weight of about 379,000 and suggest that kinesin has an axial ratio of approximately 20. Extensively purified kinesin exhibited microtubule-activated ATPase activity, and only the 124K subunit incorporated ATP in photoaffinity labeling experiments using [32P]ATP. Collectively, these data favor the interpretation that bovine brain kinesin is a highly elongated, microtubule-activated ATPase comprising two subunits each of 124,000 and 64,000 daltons, that the subunits are not linked to one another by disulfide bonds, and that the heavy chains are the ATP-binding subunits.