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.
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Research Article|
June 28 2011
Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle
Christopher G. R. Perry
;
*
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, U.S.A.†
Department of Exercise and Sport Science, East Carolina University, Greenville, NC 27858, U.S.A.2
To whom correspondence should be addressed (email perrych@ecu.edu).
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Daniel A. Kane
;
*
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, U.S.A.†
Department of Exercise and Sport Science, East Carolina University, Greenville, NC 27858, U.S.A.
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Chien-Te Lin
;
Chien-Te Lin
*
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, U.S.A.†
Department of Exercise and Sport Science, East Carolina University, Greenville, NC 27858, U.S.A.
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Rachel Kozy
;
Rachel Kozy
*
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, U.S.A.†
Department of Exercise and Sport Science, East Carolina University, Greenville, NC 27858, U.S.A.
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Brook L. Cathey
;
Brook L. Cathey
*
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, U.S.A.†
Department of Exercise and Sport Science, East Carolina University, Greenville, NC 27858, U.S.A.
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Daniel S. Lark
;
Daniel S. Lark
*
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, U.S.A.†
Department of Exercise and Sport Science, East Carolina University, Greenville, NC 27858, U.S.A.
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Constance L. Kane
;
Constance L. Kane
*
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, U.S.A.†
Department of Exercise and Sport Science, East Carolina University, Greenville, NC 27858, U.S.A.
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Patricia M. Brophy
;
Patricia M. Brophy
*
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, U.S.A.†
Department of Exercise and Sport Science, East Carolina University, Greenville, NC 27858, U.S.A.
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Timothy P. Gavin
;
Timothy P. Gavin
*
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, U.S.A.†
Department of Exercise and Sport Science, East Carolina University, Greenville, NC 27858, U.S.A.‡
Department of Physiology, East Carolina University, Greenville, NC 27858, U.S.A.
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Ethan J. Anderson
;
Ethan J. Anderson
4
*
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, U.S.A.†
Department of Exercise and Sport Science, East Carolina University, Greenville, NC 27858, U.S.A.
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P. Darrell Neufer
P. Darrell Neufer
*
East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, U.S.A.†
Department of Exercise and Sport Science, East Carolina University, Greenville, NC 27858, U.S.A.‡
Department of Physiology, East Carolina University, Greenville, NC 27858, U.S.A.
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Biochem J (2011) 437 (2): 215-222.
Article history
Received:
March 09 2011
Revision Received:
May 09 2011
Accepted:
May 10 2011
Accepted Manuscript online:
May 10 2011
Citation
Christopher G. R. Perry, Daniel A. Kane, Chien-Te Lin, Rachel Kozy, Brook L. Cathey, Daniel S. Lark, Constance L. Kane, Patricia M. Brophy, Timothy P. Gavin, Ethan J. Anderson, P. Darrell Neufer; Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle. Biochem J 15 July 2011; 437 (2): 215–222. doi: https://doi.org/10.1042/BJ20110366
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