首页> 外文OA文献 >ATPase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres: a real time phosphate assay.
【2h】

ATPase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres: a real time phosphate assay.

机译:ATP酶动力学对激活兔和青蛙的等渗肌纤维的实时性:实时磷酸盐检测。

代理获取
本网站仅为用户提供外文OA文献查询和代理获取服务,本网站没有原文。下单后我们将采用程序或人工为您竭诚获取高质量的原文,但由于OA文献来源多样且变更频繁,仍可能出现获取不到、文献不完整或与标题不符等情况,如果获取不到我们将提供退款服务。请知悉。

摘要

1. The rate of appearance of inorganic phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca2+. Pi appearance was monitored from the fluorescence signal of a Pi-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the phosphate-binding protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay. 2. On photolysis of NPE-caged ATP in the presence of Ca2+ at 20 degrees C, the fluorescence increase of MDCC-PBP was non-linear with time. ATPase activity was 41 s-1 in the first turnover based on a myosin subfragment 1 concentration of 150 microM. This was calculated from a linear regression of the fluorescence signal reporting 20-150 microM of Pi release. Tension was at 67% of its isometric level by the time 150 microM Pi was released. ATPase activities were 36 and 31 s-1 for Pi released in the ranges of 150-300 microM and 300-450 microM, respectively. The ATPase activity had a Q10 value of 2.9 based on measurements at 5, 12 and 20 degrees C. 3. An NADH-linked assay showed the ATPase activity had a lower limit of 12.7 s-1 at 20 degrees C. The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s-1. 4. On photolysis of NPE-caged ATP in the absence of Ca2+ the ATPase activity was 0.11 s-1 at 20 degrees C with no discernible rapid transient phase of Pi release during the first turnover of the ATPase. 5. To avoid the rigor state, the ATPase rate in the presence of Ca2+ was also measured on activation from the relaxed state by photolytic release of Ca2+ from a caged Ca2+ compound, nitrophenyl-EGTA. At 5 degrees C the ATPase rate was 5.8 and 4.0 s-1 in the first and second turnovers, respectively. These rates are comparable to those when NPE-caged ATP was used. 6. The influence of ADP and Pi on the ATPase activities was measured using the MDCC-PBP and NADH-linked assays, respectively. ADP (0.5 mM) decreased the initial ATPase rate by 23%. Pi (10 mM) had no significant effect. Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time. 7. The MDCC-PBP assay and NPE-caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog. At 5 degrees C in the presence of Ca2+ the ATPase activity was biphasic being 15.0 s-1 during the first turnover (based on 180 microM myosin subfragment 1). Tension was 74% of its isometric level by the time 180 microM Pi was released. During the third turnover the ATPase rate decreased to about 20% of that during the first turnover. 8. ATPase activity in isometric rabbit muscle fibres during the first few turnovers is about an order of magnitude greater than that when a steady state is reached. Possible reasons and the consequences for understanding the mechanism of muscular contraction are discussed.
机译:1.在激光闪光光解ATP的P3-1-(2-硝基苯基)乙酯之后,测量了开始较严格的无机磷(Pi)的出现速率,以及因此而最初在较严格的条件下测量的兔腰大肌在单个渗透性肌纤维中的ATPase活性。 (NPE笼养的ATP)存在和不存在Ca2 +的情况。从Pi敏感探针MDCC-PBP的荧光信号监测Pi的出现,MDCC-PBP是来自大肠杆菌的磷酸结合蛋白的香豆素标记的A197C突变体。将纤维浸入油中以优化荧光信号并消除扩散问题。还使用NADH联结的酶测定法在类似条件下根据NADH消失的速率测量了ATPase活性。 2.在20°C下在Ca2 +存在下NPE笼罩的ATP进行光解后,MDCC-PBP的荧光强度随时间呈非线性变化。基于肌球蛋白亚片段1的浓度为150 microM,第一次转换的ATPase活性为41 s-1。这是通过荧光信号的线性回归计算得出的,该信号报告了20-150 microM的Pi释放。到释放150 microM Pi时,张力达到其等轴测高度的67%。对于释放在150-300 microM和300-450 microM范围内的Pi,ATP酶活性分别为36和31 s-1。根据在5、12和20摄氏度下的测量,ATPase活性的Q10值为2.9。3.与NADH联用的测定结果表明,ATPase活性在20摄氏度下的下限为12.7 s-1。 ADP的释放表明NADH消失的速率部分地受到通过偶合反应的通量的限制。模拟表明,链接的测定数据与40 s-1的初始ATPase活性一致。 4.在不存在Ca2 +的条件下,NPE笼罩的ATP进行光解后,ATP酶的活性在20摄氏度时为0.11 s-1,并且在ATP酶的首次转换过程中没有明显的Pi释放快速瞬变阶段。 5.为了避免严格的状态,还通过从笼中的Ca2 +化合物硝基苯基-EGTA光解释放Ca2 +,从松弛状态激活时,测量了Ca2 +存在时的ATPase速率。在5摄氏度下,第一和第二次翻转的ATPase速率分别为5.8和4.0 s-1。这些比率与使用NPE笼养ATP时的比率相当。 6.分别使用MDCC-PBP和NADH-连锁测定法测定ADP和Pi对ATP酶活性的影响。 ADP(0.5 mM)使初始ATPase速率降低了23%。 Pi(10 mM)没有明显影响。 ATP水解过程中形成的ADP抑制作用导致ATPase活性随时间下降。 7.使用MDCC-PBP测定法和NPE-笼养的ATP来测量青蛙的半腱肌的单个透化肌纤维中的ATP酶速率。在5℃下,在存在Ca2 +的情况下,第一次转换期间ATPase活性是双相的,为15.0 s-1(基于180 microM肌球蛋白亚片段1)。到释放180 microM Pi时,张力是其等轴测水平的74%。在第三次周转期间,ATPase率下降至第一次周转期间的约20%。 8.等距兔肌肉纤维在最初的几次翻转中的ATPase活性比达到稳态时的ATPase活性大一个数量级。讨论了可能的原因和了解肌肉收缩机制的后果。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
代理获取

客服邮箱:kefu@zhangqiaokeyan.com

京公网安备:11010802029741号 ICP备案号:京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有
  • 客服微信

  • 服务号