Perry 2011 Biochem J
|Perry CG, Kane DA, Lin CT, Kozy R, Cathey BL, Lark DS, Kane CL, Brophy PM, Gavin TP, Anderson EJ, Neufer PD (2011) Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle. Biochem J 437:215-22.|
Abstract: Assessment of mitochondrial ADP-stimulated respiratory kinetics in permeabilized fibre (pfi) 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 pfi bundles 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 pfi bundles respectively. In the absence of BLEB, pfi bundles contracted and the Km for ADP decreased ~2-10-fold in a temperature-dependent manner. pfi bundles were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30 °C but not 37 °C. In pfi bundles 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.
• Keywords: Bioenergetics, Blebbistatin, Creatine kinase, Myosin-ATPase, N-benzyltoluene sulfonamide, Skeletal muscle contraction
Labels: MiParea: Respiration
Organism: Human, Rat Tissue;cell: Skeletal muscle Preparation: Permeabilized tissue Enzyme: Complex I, Complex V;ATP synthase Regulation: ADP, Inhibitor, Temperature
Pathway: N HRR: Oxygraph-2k