He Z H, Ferenczi M A, Brune M, Trentham D R, Webb M R, Somlyo A P, Somlyo A V
National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
Biophys J. 1998 Dec;75(6):3031-40. doi: 10.1016/S0006-3495(98)77744-6.
The rate of release of inorganic phosphate (Pi) from cycling cross-bridges in rabbit portal-anterior mesenteric vein smooth muscle was determined by following the fluorescence of the Pi-reporter, MDCC-PBP (Brune, M., J. L. Hunter, S. A. Howell, S. R. Martin, T. L. Hazlett, J. E. T. Corrie, and M. R. Webb. 1998. Biochemistry. 37:10370-10380). Cross-bridge cycling was initiated by photolytic release of ATP from caged-ATP in Triton-permeabilized smooth muscles in rigor. When the regulatory myosin light chains (MLC20) had been thiophosphorylated, the rate of Pi release was biphasic with an initial rate of 80 microM s-1 and amplitude 108 microM, decreasing to 13.7 microM s-1. These rates correspond to fast and slow turnovers of 1.8 s-1 and 0.3 s-1, assuming 84% thiophosphorylation of 52 microM myosin heads. Activation by Ca2+-dependent phosphorylation subsequent to ATP release resulted in slower Pi release, paralleling the rate of contraction that was also slower than after thiophosphorylation, and was also biphasic: 51 microM s-1 and 13.2 microM s-1. These rates suggest that the activity of myosin light chain kinase and phosphatase ("pseudo-ATPase") contributes <20% of the ATP usage during cross-bridge cycling. The extracellular "ecto-nucleotidase" activity was reduced eightfold by permeabilization, conditions in which the ecto-ADPase was 17% of the ecto-ATPase. Nevertheless, the remaining ecto-ATPase activity reduced the precision of the estimate of cross-bridge ATPase. We conclude that the transition from fast to slow ATPase rates reflects the properties and forces directly acting on cross-bridges, rather than the result of a time-dependent decrease in activation (MLC20 phosphorylation) occurring in intact smooth muscle. The mechanisms of slowing may include the effect of positive strain on cross-bridges, inhibition of the cycling rate by high affinity Mg-ADP binding, and associated state hydrolysis.
通过跟踪无机磷酸（Pi）报告分子MDCC - PBP的荧光，测定了兔门静脉 - 肠系膜前静脉平滑肌中循环横桥释放无机磷酸（Pi）的速率（Brune, M., J. L. Hunter, S. A. Howell, S. R. Martin, T. L. Hazlett, J. E. T. Corrie, and M. R. Webb. 1998. Biochemistry. 37:10370 - 10380）。在处于僵直状态的Triton通透化平滑肌中，通过光解笼化ATP释放ATP来启动横桥循环。当调节性肌球蛋白轻链（MLC20）被硫代磷酸化时，Pi释放速率呈双相性，初始速率为80 μM·s⁻¹，幅度为108 μM，随后降至13.7 μM·s⁻¹。假设52 μM肌球蛋白头部有84%被硫代磷酸化，这些速率分别对应1.8 s⁻¹和0.3 s⁻¹的快速和慢速周转。ATP释放后，Ca²⁺依赖性磷酸化激活导致Pi释放更慢，这与收缩速率平行，收缩速率也比硫代磷酸化后慢，且同样呈双相性：51 μM·s⁻¹和13.2 μM·s⁻¹。这些速率表明，肌球蛋白轻链激酶和磷酸酶（“假ATP酶”）的活性在横桥循环过程中对ATP消耗的贡献小于20%。通透化使细胞外“胞外核苷酸酶”活性降低了八倍，在这种条件下，胞外ADP酶是胞外ATP酶的17%。然而，剩余的胞外ATP酶活性降低了横桥ATP酶估计的精度。我们得出结论，ATP酶速率从快到慢的转变反映了直接作用于横桥的性质和力，而不是完整平滑肌中发生的激活（MLC20磷酸化）随时间依赖性降低的结果。减慢的机制可能包括正应变对横桥的影响、高亲和力Mg - ADP结合对循环速率的抑制以及相关状态水解。
