Crosbie R H, Chalovich J M, Reisler E
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024, USA.
J Muscle Res Cell Motil. 1995 Oct;16(5):509-18. doi: 10.1007/BF00126435.
The contribution of the extended and bent forms of caldesmon to its function was investigated by examining chemically modified forms of this protein. The bent 'hairpin' form of caldesmon was enhanced between pH 6.0 and 8.0 and at low ionic strengths, as reported by an increase in excimer fluorescence of pyrene-labelled caldesmon under these conditions. The presence of nucleotides also produced significant conformational changes in caldesmon, as detected by fluorescence measurements and protease digestions. Titrations of pyrene caldesmon with actin, heavy meromyosin, and calmodulin resulted in a decrease in excimer fluorescence. The function of the bent form of caldesmon was investigated by using intramolecular 1-ethyl-3-(3-dimethylamino propyl) carbodiimide-crosslinked caldesmon. The inhibition of acto-S-1 ATPase activity by crosslinked caldesmon was less efficient compared with that by pyrene modified and control caldesmons. Caldesmon's ability to switch from an activator to an inhibitor of actin-activated ATPase of myosin was also affected by the folding. Cosedimentation experiments revealed normal binding of crosslinked caldesmon to smooth muscle myosin. These results indicate the importance of caldesmon's transition from extended to folded forms and suggest possible functional roles for these different forms of caldesmon.
通过研究该蛋白质的化学修饰形式，探讨了钙调蛋白的延伸形式和弯曲形式对其功能的贡献。如在这些条件下芘标记的钙调蛋白的准分子荧光增加所报道的那样，在pH 6.0至8.0之间以及低离子强度下，钙调蛋白的弯曲“发夹”形式增强。通过荧光测量和蛋白酶消化检测，核苷酸的存在也在钙调蛋白中产生了显著的构象变化。用肌动蛋白、重酶解肌球蛋白和钙调蛋白对芘标记的钙调蛋白进行滴定，导致准分子荧光降低。通过使用分子内1-乙基-3-(3-二甲基氨基丙基)碳二亚胺交联的钙调蛋白，研究了钙调蛋白弯曲形式的功能。与芘修饰的和对照钙调蛋白相比，交联的钙调蛋白对肌动蛋白-S-1 ATP酶活性的抑制效率较低。钙调蛋白从肌球蛋白的肌动蛋白激活ATP酶的激活剂转变为抑制剂的能力也受到折叠的影响。共沉降实验揭示了交联的钙调蛋白与平滑肌肌球蛋白的正常结合。这些结果表明钙调蛋白从延伸形式转变为折叠形式的重要性，并暗示了这些不同形式的钙调蛋白可能的功能作用。
