Calponin-calmodulin interaction: properties and effects on smooth and skeletal muscle actin binding and actomyosin ATPases.
Calponin-calmodulin interaction: properties and effects on smooth and skeletal muscle actin binding and actomyosin ATPases.
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钙调蛋白-钙调蛋白相互作用：对平滑肌和骨骼肌肌动蛋白结合和肌动球蛋白 ATP 酶的特性和影响。
DOI：
10.1021/bi00211a046
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Johnson,JD
中科院分区：
文献类型：
--
作者：
Winder,SJ;Walsh,MP;Vasulka,C;Johnson,JD
1993年9月28日接收的修订版Mandalpt ®摘要：平滑肌钙调蛋白与具有生物活性的荧光钙调蛋白[2-（4/-马来酰亚胺苯胺基）萘-6-磺酸-钙调蛋白]（MIANS-CaM）结合，K ²为80 nM，并产生3.4倍的荧光增强。PKC磷酸化的钙调蛋白（1.3摩尔P¡/摩尔）与CaM结合的亲和力低约15倍。Calponin抑制Ca 2 +/CaM激活的环核苷酸磷酸二酯酶（PDE）的CaM（10 nM）激活，IC 50为138 nM。钙调蛋白-钙调素相互作用是钙依赖性的：半最大结合的钙调蛋白MIANS-CaM发生在pCa 6.6与希尔系数为2.4。停流荧光动力学分析表明，EGTA螯合Ca 2+使MIANS-CaM-calponin复合物以1 s-1的速率断裂。钙调蛋白以（6.0±1.8）× 106 M +/s +/s的速率结合MIANS-CaM，蜂毒肽和未标记的脑CaM均以0.3±0.1 s +/s的速率破坏MIANS-CaM-钙调蛋白复合物。这些研究表明，钙调蛋白结合钙调蛋白的亲和力比肌球蛋白轻链激酶低80倍，钙调蛋白与钙调蛋白的结合速度比与钙调蛋白或肌球蛋白轻链激酶的结合速度慢得多。通过分析Ca 2 +-CaM对（i）钙调蛋白与肌动蛋白的相互作用和（ii）钙调蛋白介导的肌动蛋白激活的肌球蛋白MgATP酶活性的抑制的影响来评估CaM-CaP相互作用的生理相关性。Ca 2 +-CaM分别在5.4和11 µ CaM浓度下对钙调蛋白（2 µ）与平滑肌肌动蛋白和骨骼肌肌动蛋白（9 µ）的结合产生半最大抑制作用。Ca 2 +-CaM未能逆转钙调蛋白抑制平滑肌或骨骼肌肌动蛋白激活的肌球蛋白MgATP酶，即使在钙调蛋白的摩尔比是超生理相对于肌动蛋白和钙调蛋白。与这些发现相一致，在ATP酶反应条件下，Ca ~（2+）-CaM不能将钙调蛋白从肌动蛋白中解离出来。我们得出的结论是，钙调蛋白的生理功能（抑制肌球蛋白跨桥循环）可能不受其与CaM的相互作用的调节。钙调蛋白是一种细丝相关蛋白，与平滑肌收缩的调节有关，因为分离的蛋白质抑制磷酸化平滑肌肌球蛋白的肌动蛋白激活的MgAT-Pase活性（Winder &沃尔什，1990）。钙调蛋白最初作为肌动蛋白结合蛋白分离，其以Ca 2+依赖性方式与CaM 1相互作用（Takahashi et al.，1986年）。还发现纯化的蛋白质与原肌球蛋白结合（Takahashi等人，1988年）。钙调蛋白对肌动球蛋白MgATPase的抑制作用是由于其与肌动蛋白的相互作用，因为它不依赖于原肌球蛋白和Ca 2+（Winder &沃尔什，1990）。有几种机制被认为可以调节钙调蛋白介导的肌动球蛋白抑制
Revised Manuscript Received September 28, 1993® abstract: Smooth muscle calponin bound to the biologically active fluorescent calmodulin [2-(4/-maleimidoanilino) naphthalene-6-sulfonic acid-calmodulin](MIANS-CaM) with a K¿ of 80 nM and produced a 3.4-fold fluorescence enhancement. PKC-phosphorylated calponin (1.3 mol of P¡/mol) bound to CaM with~15-fold lower affinity. Calponin inhibited CaM (10 nM) activation of the Ca2+-/CaM-activated cyclic nucleotide phosphodiesterase (PDE) with an IC50 of 138 nM. The calponin-CaM interaction was Ca2+-dependent: half-maximal binding of calponin to MIANS-CaM occurred at pCa 6.6 with a Hill coefficient of 2.4. Stopped-flow fluorescence kinetic analysis demonstrated thatEGTA chelation of Ca2+ from CaM disrupted the MIANS-CaM-calponin complex at a rate of 1 s-1. Calponin bound MIANS-CaM at a rate of (6.0±1.8) X 106 M™ 1 s™ 1, and melittin and unlabeled brain CaM both disrupted the MIANS-CaM-calponin complex at a rate of 0.3±0.1 s_1. These studies suggest that calponin binds CaM with 80-fold lower affinity than myosin light-chain kinase and that calponin associates with CaM much slower than it associates with caldesmon or myosin light-chain kinase. The physiological relevance of the CaM-calponin interaction was evaluated by analysis of the effects of Ca2+-CaM on (i) the interaction of calponin with actin and (ii) calponin-mediated inhibition of actin-activated myosin MgATPaseactivity. Ca2+-CaM half-maximally inhibited calponin (2 µ) binding to smooth and skeletal muscle actins (9 µ) at 5.4 and 11 µ CaM, respectively. Ca2+-CaM failed to reverse calponin inhibition of smooth or skeletal muscle actin-activated myosin MgATPases, even at molar ratios of CaM that were supraphysiological relative to actin and calponin. Consistent with these findings, Ca2+-CaM, under ATPase reaction conditions, failed to dissociate calponin from actin. We conclude that calponin’s physiological function (inhibition of myosin cross-bridge cycling) is probably not modulated by its interaction with CaM.Calponin is a thin filament associated protein that has been implicated in the regulation of smooth muscle contraction since the isolatedprotein inhibits the actin-activated MgAT-Pase activity of phosphorylated smooth muscle myosin (Winder & Walsh, 1990). Calponin was originally isolated as an actin-binding protein that interacted with CaM1 in a Ca2+-dependent manner (Takahashi et al., 1986). The purified protein was also found to bind to tropomyosin (Takahashi et al., 1988). The inhibitory effect of calponin on the actomyosin MgATPase is due to its interaction with actin, since it is independent of tropomyosin and Ca2+(Winder & Walsh, 1990). Several mechanisms have been considered that may regulate calponin-mediated inhibitionof the actomyosin