Rovner A S, Freyzon Y, Trybus K M
Department of Molecular Physiology and Biophysics, University of Vermont, College of Medicine, Burlington 05405-0068, USA.
J Muscle Res Cell Motil. 1997 Feb;18(1):103-10. doi: 10.1023/a:1018689102122.
Smooth muscle myosin isoforms of the heavy chain and the essential light chain have been hypothesized to contribute to the different shortening velocities of phasic and tonic smooth muscles, and to their different affinities for MgADP. We used the baculovirus/insect cell system to express homogeneous heavy meromyosin molecules differing only in seven amino acid insert (QGPSFSY) in the motor domain near the active site, or in the type of essential light chain isoform. Myosin from tonic rabbit uterine smooth muscle lacks the heavy chain insert, while myosin from phasic chicken gizzard contains it. The properties of a mutant uterine heavy meromyosin with added insert, and a mutant gizzard heavy meromyosin with the insert deleted, were compared with their wild type progenitors. Phosphorylated heavy meromyosins with the insert have a twofold higher enzymatic activity and in vitro motility han heavy meromyosins without the insert. These functional properties were not altered by the essential light chain isoforms. The altered motility caused by the insert implies that it modulates the rate of ADP release, the molecular step believed to limit shortening velocity. The insert may thus account in part for both the lower sensitivity to MgADP and the higher shortening velocity of phasic compared to tonic smooth muscles.
重链和必需轻链的平滑肌肌球蛋白同工型被认为与相平滑肌和紧张性平滑肌的不同缩短速度以及它们对MgADP的不同亲和力有关。我们利用杆状病毒/昆虫细胞系统表达仅在活性位点附近的运动结构域中有七个氨基酸插入序列（QGPSFSY）不同，或必需轻链同工型类型不同的均一的重酶解肌球蛋白分子。来自紧张性兔子宫平滑肌的肌球蛋白缺乏重链插入序列，而来自相平滑肌鸡砂囊的肌球蛋白含有该序列。将添加了插入序列的突变型子宫重酶解肌球蛋白和删除了插入序列的突变型砂囊重酶解肌球蛋白的特性与其野生型亲本进行了比较。带有插入序列的磷酸化重酶解肌球蛋白比没有插入序列的重酶解肌球蛋白具有两倍高的酶活性和体外运动性。这些功能特性不受必需轻链同工型的影响。插入序列引起的运动性改变表明它调节了ADP释放的速率，这一分子步骤被认为限制了缩短速度。因此，插入序列可能部分解释了相平滑肌与紧张性平滑肌相比对MgADP较低的敏感性和较高的缩短速度。
