East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, USA.
Biochem J. 2011 Jul 15;437(2):215-22. doi: 10.1042/BJ20110366.
Assessment of mitochondrial ADP-stimulated respiratory kinetics in PmFBs (permeabilized fibre bundles) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (20-300 μM) and tend to overestimate respiration at rest. Noting that PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. BLEB (blebbistatin), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >250 and ~80 μM in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the Km for ADP decreased ~2-10-fold in a temperature-dependent manner. PmFBs were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30 °C but not 37 °C. In PmFBs from humans, contraction elicited high sensitivity to ADP (Km<100 μM), whereas blocking contraction (+BLEB) and including a phosphocreatine/creatine ratio of 2:1 to mimic the resting energetic state yielded a Km for ADP of ~1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate that the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state.
在临床诊断和基础研究环境中，越来越多地使用评估 PmFB（透化纤维束）中 ADP 刺激的呼吸动力学。然而，ADP 的 Km 估计值差异很大（20-300 μM），并且往往会高估休息时的呼吸。注意到 PmFB 在呼吸实验过程中会自发收缩，我们系统地确定了收缩、温度和氧合作用对 ADP 刺激的呼吸动力学的影响。BLEB（blebbistatin），一种肌球蛋白 II ATP 酶抑制剂，在所有条件下都能阻断收缩，并分别在红色和白色大鼠 PmFB 中产生 ADP 的高 Km 值>250 和~80 μM。在没有 BLEB 的情况下，PmFB 收缩，ADP 的 Km 值在没有 BLEB（收缩）的情况下以温度依赖性的方式降低约 2-10 倍。在 30°C 下，没有 BLEB（收缩）的情况下，PmFB 对高氧（增加 Km）敏感，但在 37°C 下则不然。在来自人类的 PmFB 中，收缩引起对 ADP 的高敏感性（Km<100 μM），而阻断收缩（+BLEB）并包含 2:1 的磷酸肌酸/肌酸比以模拟休息时的能量状态，则 ADP 的 Km 值约为 1560 μM，与体内休息时呼吸率<1%最大的估计值一致。这些结果表明，肌肉对 ADP 的敏感性在与收缩状态和细胞能量电荷有关的广泛范围内变化，这为骨骼肌内能量传递的酶偶联在工作状态下变得更有效的证据提供了依据。
