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首页> 外文期刊>Biochemistry >FUNCTIONAL EXPRESSION OF MAMMALIAN MYOSIN I-BETA - ANALYSIS OF ITS MOTOR ACTIVITY
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FUNCTIONAL EXPRESSION OF MAMMALIAN MYOSIN I-BETA - ANALYSIS OF ITS MOTOR ACTIVITY

机译：哺乳动物肌球蛋白I-BET的功能表达-其运动活性分析

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The motor function of vertebrate unconventional myosins is not well understood. In this study, we initiated the baculovirus expression system to characterize a novel myosin I from bovine adrenal gland that we had previously cloned [Zhu, T., & Ikebe, M. (1994) FEBS Lett. 339, 31-36], which is classified as myosin I beta. The expressed myosin I beta was well extracted when calmodulin was coexpressed in Sf9 cells. The recombinant myosin I beta cosedimented with actin in an ATP dependent manner. The purified myosin I beta was composed of one heavy chain and three calmodulins. The electron microscopic image of myosin I beta confirmed its single-headed structure with a short tail, which is similar to that of brush border myosin I (BBMI). Myosin I beta showed high K+,EDTA-ATPase activity (similar to 0.14 mu mol/min/ mg) and Ca2+-ATPase activity (similar to 0.32, mu mol/min/mg), and the KCl/pH dependence of these activities was different from that of conventional myosin. Mg2+-ATPase activity of myosin I beta alone was increased above pCa 6, while the actin dependent activity was not affected by Ca2+. Actin sliding velocity of myosin I beta in the absence of Ca2+ was 0.3-0.5 mu m/s at 25 degrees C, which is much greater than that of BBMI (<0.05 mu m/s). The actin sliding activity was abolished above pCa 6, and the sliding activity was restored when exogenous calmodulin was added in the absence of Ca2+. Within similar Ca2+ concentrations, one of the three calmodulins was dissociated from myosin I beta. The results suggest that Ca2+ dependent association of calmodulin may function as a regulatory mechanism of myosin I beta motor activity and that the motor activity of mammalian myosin I is largely different among distinct myosin I isoforms.
机译：脊椎动物非常规肌球蛋白的运动功能尚不十分清楚。在这项研究中，我们启动了杆状病毒表达系统，以表征先前克隆过的来自牛肾上腺的新型肌球蛋白I [Zhu，T.，＆Ikebe，M.（1994）FEBS Lett.。 339，31-36]，其被分类为肌球蛋白I beta。当钙调蛋白在Sf9细胞中共表达时，表达的肌球蛋白I beta可以很好地提取。重组肌球蛋白Iβ与肌动蛋白以ATP依赖性方式结合。纯化的肌球蛋白Iβ由一个重链和三个钙调蛋白组成。肌球蛋白Iβ的电子显微镜图像证实了其具有短尾巴的单头结构，类似于刷状边界肌球蛋白I（BBMI）。肌球蛋白I beta表现出较高的K +，EDTA-ATPase活性（约0.14μmol/ min / mg）和Ca2 + -ATPase活性（约0.32μmol/ min / mg），这些活性的KCl / pH依赖性为与传统的肌球蛋白不同。单独的肌球蛋白Iβ的Mg2 + -ATPase活性在pCa 6以上增加，而肌动蛋白依赖性活性不受Ca2 +影响。在不存在Ca2 +的情况下，肌球蛋白I beta的肌动蛋白滑动速度在25摄氏度下为0.3-0.5μm/ s，远大于BBMI（<0.05μm/ s）。在pCa 6以上消除了肌动蛋白的滑动活性，在不存在Ca 2+的情况下加入外源钙调蛋白时，恢复了滑动活性。在相似的Ca2 +浓度下，三种钙调蛋白之一与肌球蛋白Iβ分离。结果表明钙调素的Ca 2+依赖性缔合可能充当肌球蛋白Iβ运动活性的调节机制，并且哺乳动物肌球蛋白I的运动活性在不同的肌球蛋白I同工型之间存在很大差异。

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