Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, SE-177 77, Sweden.
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, SE-177 77, Sweden.
Arch Biochem Biophys. 2022 Jun 15;722:109212. doi: 10.1016/j.abb.2022.109212. Epub 2022 Apr 6.
The biophysical function of myosin in vitro has been extensively investigated in different motility assays, but the study of myosin ATPase properties at the fiber level is insufficiently investigated. In this study, quantum dot (QD) mediated thermometry measurements were optimized to measure the efficiency of myosin extracted from muscle mini bundles. A reduction in fluorescent intensity of QD reflects an increase in temperature caused by the heat released during ATP hydrolysis and denotes the efficiency of the motor protein myosin. The procedure for extracting myosin was similar to the single fiber in vitro motility assay with some small modifications, and the concentration of myosin was represented by the extracted total protein since the ratio of extracted myosin to total protein was constant. Moreover, the efficiencies of myosin extracted from preparations containing different myosin heavy chain isoforms reveal lower efficiency of slow compared to fast myosin isoforms. Specifically, more heat was released in slow myosin enzymatic reaction, resulting in faster decay of QD fluorescence intensity. Hence, the optimized QD mediated thermometry provides a novel and sensitive approach to evaluate efficiency of myosin ATPase obtained from small muscle samples, representing a significant advantage in the clinical evaluation of neuromuscular disorders.
肌球蛋白在体外的生物物理功能已在不同的运动检测中得到广泛研究，但对纤维水平上肌球蛋白 ATP 酶性质的研究还不够充分。在这项研究中，优化了量子点（QD）介导的测温测量，以测量从小肌肉束中提取的肌球蛋白的效率。QD 荧光强度的降低反映了由 ATP 水解过程中释放的热量引起的温度升高，这表示了运动蛋白肌球蛋白的效率。提取肌球蛋白的程序与体外单纤维运动检测相似，但略有修改，由于提取的肌球蛋白与总蛋白的比例保持不变，因此肌球蛋白的浓度由提取的总蛋白表示。此外，从含有不同肌球蛋白重链同工型的制剂中提取的肌球蛋白的效率表明，与快肌同工型相比，慢肌同工型的效率较低。具体而言，在慢肌的酶反应中释放出更多的热量，导致 QD 荧光强度更快地衰减。因此，优化后的 QD 介导的测温法为从小肌肉样本中评估肌球蛋白 ATP 酶的效率提供了一种新颖而灵敏的方法，在神经肌肉疾病的临床评估中具有重要优势。
