Copurification of kinesin polypeptides with microtubule-stimulated Mg-ATPase activity and kinetic analysis of enzymatic properties.
Copurification of kinesin polypeptides with microtubule-stimulated Mg-ATPase activity and kinetic analysis of enzymatic properties.
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驱动蛋白多肽与微管刺激的 Mg-ATP 酶活性的共纯化以及酶特性的动力学分析。
DOI：
10.1002/cm.970120403
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发表时间：
1989
影响因子：
--
通讯作者：
Brady,ST
中科院分区：
文献类型：
--
作者：
Wagner,MC;Pfister,KK;Bloom,GS;Brady,ST
驱动蛋白三磷酸腺苷酶 (ATPase) 是一种拟议的膜细胞器运输马达，测定其动力学特性需要足够量的驱动蛋白和一致的酶活性水平。详细介绍了从 800 g 牛脑中生产约 2 mg 纯度高达 96% 的驱动蛋白的纯化程序。该方案包括使用 5'-腺苷酰亚胺二磷酸 (AMP-PNP) 的微管亲和步骤；然后进行凝胶过滤、离子交换和羟基磷灰石层析；然后进行蔗糖密度梯度离心。在整个纯化过程中，微管激活的驱动蛋白 ATP 酶活性与驱动蛋白多肽共洗脱。在微管存在下，高度纯化的驱动蛋白的 Vmax 为 0.31 μmol/min/mg，Kmfor ATP 为 0.20 mM。这些研究中获得的动力学常数与 ATP 和微管的生理水平相当。发现测定缓冲液条件的变化会显着影响 ATP 酶活性。对驱动蛋白利用多种阳离子-ATP 复合物能力的研究表明，驱动蛋白是一种微管刺激的 Mg-ATP 酶，但在微管存在的情况下，驱动蛋白能够水解 Ca-ATP、Mn-ATP、Co-ATP 以及 Mg-ATP。在没有微管的情况下，Ca-ATP 似乎是最好的底物。对几种 ATP 酶抑制剂的研究确定，钒酸盐在最低浓度的抑制剂下抑制驱动蛋白 ATP 酶，但亚毫摩尔浓度的 AMP-PNP 也会对 ATP 酶产生显着抑制。其他驱动蛋白抑制剂包括 N-乙基马来酰亚胺、二磷酸腺苷 (ADP)、焦磷酸盐和三聚磷酸盐。驱动蛋白 ATP 酶动力学特性的进一步表征对于理解膜细胞器沿微管运输的分子机制非常重要。
Determination of kinetic properties for kinesin adenosine triphosphatase (ATPase), a proposed motor for transport of membranous organelles, requires adequate amounts of kinesin with a consistent level of enzymatic activity. A purification procedure is detailed that produces approximately 2 mg of kinesin at up to 96% purity from 800 g of bovine brain. This protocol consists of a microtubule affinity step using 5′‐adenylylimidodiphosphate (AMP‐PNP); followed by gel filtration, ion exchange, and hydroxylapatite chromatography; and then sucrose density gradient centrifugation. The microtubule‐activated ATPase activity of kinesin coeluted with kinesin polypeptides throughout the purification. Highly purified kinesin had a Vmaxof 0.31 μmol/min/mg in the presence of microtubules, with a Kmfor ATP of 0.20 mM. The kinetic constants obtained in these studies compare favorably with physiological levels of ATP and microtubules. Variations in buffer conditions for the assay were found to affect ATPase activity significantly. A study of the ability of kinesin to utilize a variety of cation‐ATP complexes indicated that kinesin is a microtubule‐stimulated Mg‐ATPase, but kinesin is able to hydrolyze Ca‐ATP, Mn‐ATP, and Co‐ATP as well as Mg‐ATP in the presence of microtubules. In the absence of microtubules, Ca‐ATP appears to be the best substrate. Studies with several inhibitors of ATPases determined that vanadate inhibited kinesin ATPase at the lowest concentrations of inhibitor, but significant inhibition of the ATPase also occurred with submillimolar concentrations of AMP‐PNP. Other inhibitors of kinesin include N‐ethylmaleimide, adenosine diphosphate (ADP), pyrophosphate, and tripolyphosphate. Further characterization of the kinetic properties of the kinesin ATPase is important for understanding the molecular mechanisms for transport of membranous organelles along microtubules.