Jin Jian-Ping, Wu Di, Gao Jimin, Nigam Rita, Kwong Stephen
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Ave, Cleveland, OH 44106-4970, USA.
Protein Expr Purif. 2003 Oct;31(2):231-9. doi: 10.1016/s1046-5928(03)00185-2.
Three homologous calponin isoforms, named h1, h2, and acidic calponins, have been found in birds and mammals. Based primarily on studies of chicken gizzard smooth muscle (h1) calponin, calponin has been identified as a family of actin-associated proteins that inhibit actomyosin ATPase activity. Evolutionary divergence of the calponin isoforms suggests differentiated function. While the role of h1 calponin in smooth muscle contraction is under investigation, h2 calponin has been shown regulating the function of actin cytoskeleton. Using cloned cDNA, we expressed mammalian h1 and h2 calponins in Escherichia coli. We have developed effective methods to purify biologically active h1 and h2 calponin proteins from transformed bacterial culture. The purified calponin isoform proteins were used to generate monoclonal antibodies that reveal epitopic structure difference between h1 and h2 calponins. Together with their differential expression in tissues and during development, the structural diversity of h1 and h2 calponins suggests non-redundant physiological function. Nevertheless, h1 and h2 calponins bind F-actin with similar affinity, indicating a conserved mechanism for their role in regulating actin filaments in smooth muscle and non-muscle cells.
在鸟类和哺乳动物中发现了三种同源的钙调蛋白异构体，分别命名为h1、h2和酸性钙调蛋白。主要基于对鸡肌胃平滑肌（h1）钙调蛋白的研究，钙调蛋白已被鉴定为一类与肌动蛋白相关的蛋白质，可抑制肌动球蛋白ATP酶活性。钙调蛋白异构体的进化差异表明其功能有所分化。虽然h1钙调蛋白在平滑肌收缩中的作用正在研究中，但h2钙调蛋白已被证明可调节肌动蛋白细胞骨架的功能。我们利用克隆的cDNA在大肠杆菌中表达了哺乳动物的h1和h2钙调蛋白。我们开发了有效的方法，从转化的细菌培养物中纯化具有生物活性的h1和h2钙调蛋白。纯化的钙调蛋白异构体蛋白被用于产生单克隆抗体，这些抗体揭示了h1和h2钙调蛋白之间的表位结构差异。连同它们在组织和发育过程中的差异表达，h1和h2钙调蛋白的结构多样性表明它们具有非冗余的生理功能。然而，h1和h2钙调蛋白以相似的亲和力结合F-肌动蛋白，表明它们在调节平滑肌和非肌肉细胞中肌动蛋白丝的作用机制是保守的。
