FUNCTIONAL CHICKEN GIZZARD HEAVY-MEROMYOSIN EXPRESSION IN AND PURIFICATION FROM BACULOVIRUS-INFECTED INSECT CELLS
FUNCTIONAL CHICKEN GIZZARD HEAVY-MEROMYOSIN EXPRESSION IN AND PURIFICATION FROM BACULOVIRUS-INFECTED INSECT CELLS
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DOI：
10.1073/pnas.92.3.704
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发表时间：
1995-01-31
影响因子：
11.1
通讯作者：
FUJIWARA, K
中科院分区：
文献类型：
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作者：
ONISHI, H;MAEDA, K;FUJIWARA, K
在感染两种重组苜蓿银纹夜蛾杆状病毒混合物的草地贪夜蛾细胞中共表达了鸡砂囊重肌球蛋白（HMM）的重链、必需轻链和调节轻链。获得了由重链和两种类型的轻链组成的可溶性HMM。从病毒感染的细胞中纯化重组HMM并进行表征。分离的重组HMM的调节轻链被磷酸化的肌球蛋白轻链激酶在钙调蛋白的存在下，在Ca 2+依赖的方式。当反应介质中存在肌球蛋白轻链激酶、钙调蛋白和Ca 2+时，兔骨骼肌肌动蛋白激活重组HMM的ATP酶。肌球蛋白增强肌动蛋白激活的ATP酶活性。重组HMM修饰肌动蛋白丝，沿丝显示特征性的箭头图案沿着。本报告的功能性重组双头平滑肌肌球蛋白片段开辟了详细的分子研究的道路。
The heavy chain and the essential and the regulatory light chains of chicken gizzard heavy meromyosin (HMM) were coexpressed in Spodoptera frugiperda (fall army-worm) cells infected with a mixture of two recombinant Autographa californica baculoviruses. Soluble HMM consisting of the heavy chain and the two types of light chains was obtained. The recombinant HMM was purified from the virus-infected cells and characterized. The regulatory light chain of the isolated recombinant HMM was phosphorylated by myosin light chain kinase in the presence of calmodulin in a Ca2+-dependent manner. The ATPase of the recombinant HMM was activated by rabbit skeletal muscle actin when myosin light chain kinase, calmodulin, and Ca2+ were present in the reaction medium. Chicken gizzard tropomyosin enhanced the actin-activated ATPase activity. The recombinant HMM decorated actin filaments, displaying the characteristic arrowhead pattern along the filaments. This report on a functional recombinant double-headed smooth muscle myosin fragment opens the way to detailed studies on the molecule.