[摘要]：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 K-m for ADP vary considerably (similar to 20-300 mu M) 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 K-m values for ADP of >similar to 250 and similar to 80 mu M in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the K, for ADP decreased similar to 2-10-fold in a temperature-dependent manner. PmFBs were sensitive to hyperoxia (increased K,) in the absence of BLEB (contracted) at 30 degrees C but not 37 degrees C. In PmFBs from humans, contraction elicited high sensitivity to ADP (K-m < 100 mu M), whereas blocking contraction (+ BLEB) and including a phosphocreatine/creatine ratio of 2:1 to mimic the resting energetic state yielded a K-m for ADP of 1560 mu M 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.
