ATPase and shortening rates in frog fast skeletal myofibrils by time-resolved measurements of protein-bound and free Pi
ATPase and shortening rates in frog fast skeletal myofibrils by time-resolved measurements of protein-bound and free Pi
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DOI：
10.1016/s0006-3495(98)78018-x
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发表时间：
1998-06-01
影响因子：
3.4
通讯作者：
Webb, MR
中科院分区：
文献类型：
--
作者：
Barman, T;Brune, M;Webb, MR
在4 ℃无负荷条件下，测量了蛙快肌Ca 2+激活的肌原纤维的缩短和ATP酶速率。使用磷酸盐结合蛋白法（游离磷酸盐）和猝灭流（总磷酸盐）测定ATP酶速率。通过在pH 3.5下淬灭50 ms至10 s的反应混合物并在光学显微镜下测量肌节长度来估计在接近零负荷（V-O）下的缩短速率。与兔腰肌肌原纤维（C。Lionne，F. Travers和T. Barman，1996，Biophys. J. 70：887-895），ATP酶进展曲线具有三个阶段：瞬时Pi爆发、快速线性阶段（k（F））和减速至缓慢阶段（k（S））。有证据表明，k（F）是缩短肌原纤维的ATP酶速率。与肌原纤维和肌纤维的力学测量结果一致。在相同条件下，ATP饱和时，V-o和k（F）分别为2.4 μ m半肌节s（-1）和4.6 s（-1），K-m值分别为33和200 μ M。这些参数高于兔腰大肌肌原纤维。肌原纤维k（F）高于先前在青蛙快肌中获得的纤维ATP酶速率，但显著低于通过磷酸盐结合蛋白法（Z. H.赫利河，巴西-地K.奇林沃思，M. J. E.布伦T.科里，D. R. Trentham，M. R. Webb和M. R. Ferenczi，1997，J.Physiol.50：125-148）。我们发现，与青蛙与兔肌原纤维ATP酶，磷酸盐释放是限速步骤。
Shortening and ATPase rates were measured in Ca2+-activated myofibrils from frog fast muscles in unloaded conditions at 4 degrees C. ATPase rates were determined using the phosphate-binding protein method (free phosphate) and quench flow (total phosphate). Shortening rates at near zero load (V-o) were estimated by quenching reaction mixtures 50 ms to 10 s old at pH 3.5 and measuring sarcomere lengths under the optical microscope. As with the rabbit psoas myofibrils (C. Lionne, F. Travers, and T. Barman, 1996, Biophys. J. 70:887-895), the ATPase progress curves had three phases: a transient Pi burst, a fast linear phase (k(F)), and a deceleration to a slow phase (k(S)). Evidence is given that k(F) is the ATPase rate of shortening myofibrils. V, is in good agreement with mechanical measurements in myofibrils and fibers. Under the same conditions and at saturation in ATP, V-o and k(F) are 2.4 mu m half-sarcomere(-1) s(-1) and 4.6 s(-1), and their K-m values are 33 and 200 mu M, respectively. These parameters are higher than found with rabbit psoas myofibrils. The myofibrillar k(F) is higher than the fiber ATPase rates obtained previously in frog fast muscles but considerably lower than obtained in skinned fibers by the phosphate-binding protein method (Z. H. He, R. K. Chillingworth, M. Brune, J. E. T. Corrie, D. R. Trentham, M. R. Webb, and M. R. Ferenczi, 1997, J. Physiol. 50:125-148). We show that, with frog as with rabbit myofibrillar ATPase, phosphate release is the rate-limiting step.