Lu Xiaoying, Tobacman Larry S, Kawai Masataka
Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA, USA.
Biophys J. 2006 Dec 1;91(11):4230-40. doi: 10.1529/biophysj.106.084608. Epub 2006 Sep 15.
The effect of temperature on isometric tension and cross-bridge kinetics was studied with a tropomyosin (Tm) internal deletion mutant AS-Delta23Tm (Ala-Ser-Tm Delta(47-123)) in bovine cardiac muscle fibers by using the thin filament extraction and reconstitution technique. The results are compared with those from actin reconstituted alone, cardiac muscle-derived control acetyl-Tm, and recombinant control AS-Tm. In all four reconstituted muscle groups, isometric tension and stiffness increased linearly with temperature in the range 5-40 degrees C for fibers activated in the presence of saturating ATP and Ca(2+). The slopes of the temperature-tension plots of the two controls were very similar, whereas the slope derived from fibers with actin alone had approximately 40% the control value, and the slope from mutant Tm had approximately 36% the control value. Sinusoidal analysis was performed to study the temperature dependence of cross-bridge kinetics. All three exponential processes A, B, and C were identified in the high temperature range (30-40 degrees C); only processes B and C were identified in the mid-temperature range (15-25 degrees C), and only process C was identified in the low temperature range (5-10 degrees C). At a given temperature, similar apparent rate constants (2pia, 2pib, 2pic) were observed in all four muscle groups, whereas their magnitudes were markedly less in the order of AS-Delta23Tm < Actin < AS-Tm approximately Acetyl-Tm groups. Our observations are consistent with the hypothesis that Tm enhances hydrophobic and stereospecific interactions (positive allosteric effect) between actin and myosin, but Delta23Tm decreases these interactions (negative allosteric effect). Our observations further indicate that tension/cross-bridge is increased by Tm, but is diminished by Delta23Tm. We conclude that Tm affects the conformation of actin so as to increase the area of hydrophobic interaction between actin and myosin molecules.
采用细肌丝提取和重组技术，研究了温度对牛心肌纤维中一种原肌球蛋白（Tm）内部缺失突变体AS-Delta23Tm（丙氨酸-丝氨酸-Tm Delta(47-123)）的等长张力和横桥动力学的影响。将结果与单独重组肌动蛋白、心肌来源的对照乙酰-Tm和重组对照AS-Tm的结果进行了比较。在所有四个重组肌肉组中，对于在饱和ATP和Ca(2+)存在下激活的纤维，等长张力和刚度在5-40摄氏度范围内随温度呈线性增加。两个对照的温度-张力图的斜率非常相似，而仅含肌动蛋白的纤维的斜率约为对照值的40%，突变体Tm的斜率约为对照值的36%。进行正弦分析以研究横桥动力学的温度依赖性。在高温范围（30-40摄氏度）中识别出所有三个指数过程A、B和C；在中温范围（15-25摄氏度）中仅识别出过程B和C，而在低温范围（5-10摄氏度）中仅识别出过程C。在给定温度下，在所有四个肌肉组中观察到相似的表观速率常数（2pia、2pib、2pic），但其大小顺序明显为AS-Delta23Tm < 肌动蛋白 < AS-Tm ≈ 乙酰-Tm组。我们的观察结果与以下假设一致，即Tm增强肌动蛋白和肌球蛋白之间的疏水和立体特异性相互作用（正变构效应），但Delta23Tm降低这些相互作用（负变构效应）。我们的观察结果进一步表明，Tm增加张力/横桥，但Delta23Tm降低张力/横桥。我们得出结论，Tm影响肌动蛋白的构象，从而增加肌动蛋白和肌球蛋白分子之间的疏水相互作用面积。
