Butler T M, Pacifico D S, Siegman M J
Department of Physiology, Jefferson Medical College, Philadelphia, Pennsylvania 19107.
Am J Physiol. 1989 Jan;256(1 Pt 1):C59-66. doi: 10.1152/ajpcell.1989.256.1.C59.
The purpose of this study was to determine the nucleotide bound to myosin and its rate of release under relaxed and activated conditions in permeabilized rabbit portal veins. Incubation of the muscles in a relaxing solution containing [3H]-ATP resulted in the formation of 60-70 microM radiolabeled ADP in the muscle whether or not the myosin light chains had been thiophosphorylated. This value was similar to the estimate of the concentration of myosin subfragment 1. Upon transfer of the muscles to a chase solution containing no labeled ATP, there was a very slow loss of labeled ADP when the light chains were unphosphorylated, but a much faster release occurred when the light chains were thiophosphorylated. The results suggest that smooth muscle myosin exists primarily in a complex with ADP under both relaxed and phosphorylated conditions and that phosphorylation of all of the light chains results in a large increase in the rate of release of the products of ATP splitting from all of the myosin. Interestingly, the exponential release of ADP in relaxed muscle shows two components, one of which contains about two-thirds of the total ADP and is 5- to 10-fold faster than the other. If the difference in rates of ADP release observed in relaxed muscle persists when the myosin is phosphorylated, then it is possible that there is a 5- to 10-fold difference in rates of cycling for different phosphorylated cross bridges in smooth muscle.
本研究的目的是确定在松弛和激活条件下，通透的兔门静脉中与肌球蛋白结合的核苷酸及其释放速率。无论肌球蛋白轻链是否已被硫代磷酸化，将肌肉在含有[3H]-ATP的松弛溶液中孵育，都会在肌肉中形成60 - 70微摩尔的放射性标记ADP。该值与肌球蛋白亚片段1浓度的估计值相似。当将肌肉转移到不含标记ATP的追踪溶液中时，轻链未磷酸化时，标记ADP的损失非常缓慢，但轻链硫代磷酸化时，释放速度要快得多。结果表明，平滑肌肌球蛋白在松弛和磷酸化条件下主要以与ADP的复合物形式存在，并且所有轻链的磷酸化会导致ATP水解产物从所有肌球蛋白中释放的速率大幅增加。有趣的是，松弛肌肉中ADP的指数释放显示出两个成分，其中一个成分约占总ADP的三分之二，其速度比另一个成分快5至10倍。如果在肌球蛋白磷酸化时，松弛肌肉中观察到的ADP释放速率差异仍然存在，那么平滑肌中不同磷酸化的横桥循环速率可能存在5至10倍的差异。
