Wills F L, McCubbin W D, Kay C M
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Biochemistry. 1994 May 10;33(18):5562-9. doi: 10.1021/bi00184a027.
Calponin inhibits actomyosin Mg2+ ATPase and is proposed to regulate smooth muscle contraction; however, the mechanism by which it exerts its effect and the regulation of its behavior is still under investigation. The proposed methods by which calponin regulation is effected include reversible phosphorylation of calponin which would allow contraction to occur and regulation by interaction with calcium-calmodulin. However, several investigators have been unable to find evidence of in vivo phosphorylation of calponin, and the affinity between calponin and calmodulin is not high enough to suggest that this interaction is biologically significant. In this paper, we present an alternative method of calponin regulation via calcium-caltropin and describe the calponin-caltropin complex for the first time. Caltropin, a calcium-binding protein isolated from smooth muscle, is a dimer under native conditions and interacts with calponin in a calcium-dependent fashion in the ratio of 2 mol of dimer: 1 mol of calponin. The formation of this complex can be monitored by following the fluorescence of an acrylodan label on cysteine 273 of calponin, which undergoes a 35-nm blue shift in wavelength peak from 505 to 470 nm when calponin becomes complexed with caltropin. This fluorescence change when titrated with calcium indicates that the concentration of calcium required for complex formation is approximately 10(-5) M, corresponding to the low-affinity calcium-binding sites of caltropin. This complex was further characterized by circular dichroism (CD).(ABSTRACT TRUNCATED AT 250 WORDS)
钙调蛋白抑制肌动球蛋白Mg2+ ATP酶，并被认为可调节平滑肌收缩；然而，其发挥作用的机制及其行为的调节仍在研究中。提出的钙调蛋白调节方法包括钙调蛋白的可逆磷酸化，这将允许收缩发生，以及通过与钙调蛋白相互作用进行调节。然而，几位研究人员未能找到钙调蛋白在体内磷酸化的证据，并且钙调蛋白与钙调蛋白之间的亲和力不够高，不足以表明这种相互作用具有生物学意义。在本文中，我们提出了一种通过钙藜芦碱调节钙调蛋白的替代方法，并首次描述了钙调蛋白-钙藜芦碱复合物。钙藜芦碱是一种从平滑肌中分离出的钙结合蛋白，在天然条件下是二聚体，并以2摩尔二聚体：1摩尔钙调蛋白的比例以钙依赖的方式与钙调蛋白相互作用。这种复合物的形成可以通过跟踪钙调蛋白半胱氨酸273上丙烯罗丹标记的荧光来监测，当钙调蛋白与钙藜芦碱结合时，其波长峰值从505纳米发生35纳米的蓝移至470纳米。用钙滴定这种荧光变化表明，形成复合物所需的钙浓度约为10（-5）M，这与钙藜芦碱的低亲和力钙结合位点相对应。这种复合物通过圆二色性（CD）进一步表征。（摘要截短于250字）
