Mechanoenzymatic characterization of human myosin Vb.
Mechanoenzymatic characterization of human myosin Vb.
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DOI：
10.1021/bi051682b
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发表时间：
2006-02
期刊：
影响因子：
2.9
通讯作者：
Shinya Watanabe;K. Mabuchi;R. Ikebe;M. Ikebe
中科院分区：
文献类型：
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作者：
Shinya Watanabe;K. Mabuchi;R. Ikebe;M. Ikebe
在哺乳动物中有三种V类肌球蛋白亚型。虽然肌球蛋白Va已被研究得很好，但对其他肌球蛋白V亚型（Vb和Vc）在分子水平上的功能知之甚少。本文首次报道了人肌球蛋白Vb（HuM 5 B）的机械酶功能。电镜观察显示，HuM 5 B具有长颈双头结构，与肌球蛋白Va相似。HuM 5 B肌动蛋白激活的ATP酶活性的V（max）和K（actin）分别为9.7 +/- 0.4 s（-）（1）和8.5 +/- 0.1 microM。肌动蛋白激活的ATP酶活性的K（actin）和K（ATP）显著高于肌球蛋白Va。ADP显著抑制ATP酶活性。ADP从acto-HuM 5 B的释放速率为12.2 +/- 0.5 s（-）（1），与肌动蛋白激活的ATP酶活性的V（max）相当。这些结果表明，ADP释放是肌动蛋白激活的ATP酶循环的限速步骤，因此，HuM 5 B是一个高占空比肌球蛋白。一致地，肌动蛋白滑动速度（0.22 +/- 0.03 microm/s）在低运动密度下保持恒定。肌动蛋白丝着陆试验表明，单个HuM 5 B分子足以连续移动肌动蛋白丝，表明HuM 5 b是一个进行性马达。
There are three isoforms of class V myosin in mammals. While myosin Va has been studied well, little is known about the function of other myosin V isoforms (Vb and Vc) at a molecular level. Here we report the mechanoenzymatic function of human myosin Vb (HuM5B) for the first time. Electron microscopic observation showed that HuM5B has a double-headed structure with a long neck like myosin Va. V(max) and K(actin) of the actin-activated ATPase activity of HuM5B were 9.7 +/- 0.4 s(-)(1) and 8.5 +/- 0.1 microM, respectively. K(actin) and K(ATP) of the actin-activated ATPase activity were significantly higher than those of myosin Va. ADP markedly inhibited the ATPase activity. The rate of release of ADP from acto-HuM5B was 12.2 +/- 0.5 s(-)(1), which was comparable to the V(max) of the actin-activated ATPase activity. These results suggest that ADP release is the rate-limiting step for the actin-activated ATPase cycle; thus, HuM5B is a high duty ratio myosin. Consistently, the actin gliding velocity (0.22 +/- 0.03 microm/s) remained constant at a low motor density. The actin filament landing assay revealed that a single HuM5B molecule is sufficient to move the actin filament continuously, indicating that HuM5b is a processive motor.