Nascimento A A, Cheney R E, Tauhata S B, Larson R E, Mooseker M S
Department of Biology, Yale University, New Haven, Connecticut 06511, USA.
J Biol Chem. 1996 Jul 19;271(29):17561-9. doi: 10.1074/jbc.271.29.17561.
The actin binding and ATPase properties, as well as the functional domain structure of chick brain myosin-V, a two-headed, unconventional myosin, is reported here. Compared to conventional myosin from skeletal muscle, brain myosin-V exhibits low K-EDTA- and Ca-ATPase activities (1.8 and 0.8 ATP/s per head). The physiologically relevant Mg-ATPase is also low (approximately 0.3 ATP/s), unless activated by the presence of both F-actin and Ca2+ (Vmax of 27 ATP/s). Ca2+ stimulates the actin-activated Mg-ATPase over a narrow concentration range between 1 and 3 microM. In the presence of saturating Ca2+ and 75 mM KCl, surprisingly low concentrations of F-actin activate the Mg-ATPase in a hyperbolic manner (KATPase of 1.3 microM). Brain myosin-V also binds with relatively high affinity (compared to other known myosins) to F-actin in the presence of ATP, as assayed by cosedimentation. Digestion of brain myosin-V with calpain yielded a 65-kDa head domain fragment that cosediments with actin in an ATP-sensitive manner and a 80-kDa tail fragment that does not interact with F-actin. The 80-kDa fragment results from cleavage one residue beyond the proline-, glutamate-, serine-, threonine-rich region. Our data indicate that the Mg-ATPase cycle of brain myosin-V is tightly regulated by Ca2+, probably via direct binding to the calmodulin light chains in the neck domain, which like brush border myosin-I, results in partial (approximately 30%) dissociation of the calmodulin associated with brain myosin-V. The effect of Ca2+ binding, which appears to relieve suppression by the neck domain, can be mimicked by calpain cleavage near the head/neck junction.
本文报道了鸡脑肌球蛋白-V（一种双头非传统肌球蛋白）的肌动蛋白结合和ATP酶特性以及功能结构域结构。与骨骼肌中的传统肌球蛋白相比，脑肌球蛋白-V表现出较低的K-EDTA和Ca-ATP酶活性（每个头部每秒1.8和0.8个ATP）。生理相关的Mg-ATP酶也较低（约每秒0.3个ATP），除非同时存在F-肌动蛋白和Ca2+（Vmax为每秒27个ATP）。Ca2+在1至3 microM的狭窄浓度范围内刺激肌动蛋白激活的Mg-ATP酶。在饱和Ca2+和75 mM KCl存在的情况下，令人惊讶的是，低浓度的F-肌动蛋白以双曲线方式激活Mg-ATP酶（KATPase为1.3 microM）。通过共沉降分析，脑肌球蛋白-V在ATP存在下也以相对较高的亲和力（与其他已知肌球蛋白相比）结合F-肌动蛋白。用钙蛋白酶消化脑肌球蛋白-V产生一个65 kDa的头部结构域片段，该片段以ATP敏感的方式与肌动蛋白共沉降，以及一个80 kDa的尾部片段，该片段不与F-肌动蛋白相互作用。80 kDa的片段是由富含脯氨酸、谷氨酸、丝氨酸、苏氨酸区域之外的一个残基切割产生的。我们的数据表明，脑肌球蛋白-V的Mg-ATP酶循环受到Ca2+的严格调控，可能是通过直接结合到颈部结构域中的钙调蛋白轻链上，这与刷状缘肌球蛋白-I类似，导致与脑肌球蛋白-V相关的钙调蛋白部分（约30%）解离。Ca2+结合的作用似乎是解除颈部结构域的抑制，这可以通过头部/颈部交界处附近的钙蛋白酶切割来模拟。
