Difference between revisions of "Perry 2011 Biochem J"
| Line 5: | Line 5: | ||
|year=2011 | |year=2011 | ||
|journal=Biochem J | |journal=Biochem J | ||
|abstract=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 (~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 ''K''m values for ADP of >~250 and ~80 μM in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the ''K''m for ADP decreased ~2-10-fold in a temperature-dependent manner. PmFBs were sensitive to hyperoxia (increased ''K''m) in the absence of BLEB (contracted) at 30 °C but not 37 °C. In PmFBs from humans, contraction elicited high sensitivity to ADP (''K''m<100 μM), 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 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. | |abstract=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 (~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 ''K''m values for ADP of >~250 and ~80 μM in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the ''K''m for ADP decreased ~2-10-fold in a temperature-dependent manner. PmFBs were sensitive to hyperoxia (increased ''K''m) in the absence of BLEB (contracted) at 30 °C but not 37 °C. In PmFBs from humans, contraction elicited high sensitivity to ADP (''K''m<100 μM), 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 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= | |keywords=Bioenergetics, Blebbistatin, Creatine kinase, Myosin-ATPase, N-benzyltoluene sulfonamide, Skeletal muscle contraction | ||
|mipnetlab=US_NC Greenville_Neufer PD, CA Toronto Perry CG, US_NC_Greenville_Anderson EJ, CA_Antigonish_Kane DA, US NC Greenville East Carolina Univ | |mipnetlab=US_NC Greenville_Neufer PD, CA Toronto Perry CG, US_NC_Greenville_Anderson EJ, CA_Antigonish_Kane DA, US NC Greenville East Carolina Univ | ||
}} | }} | ||
| Line 14: | Line 14: | ||
|tissues=Skeletal muscle | |tissues=Skeletal muscle | ||
|preparations=Permeabilized tissue | |preparations=Permeabilized tissue | ||
|kinetics=Temperature | |substratestates=CI | ||
|enzymes=Complex I, Complex V; ATP Synthase | |||
|kinetics=Inhibitor; Uncoupler, Temperature | |||
|topics=ATP; ADP; AMP; PCr | |||
}} | }} | ||
Revision as of 10:45, 12 February 2013
| 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: 215-222. |
Perry CG, Kane DA, Lin CT, Kozy R, Cathey BL, Lark DS, Kane CL, Brophy PM, Gavin TP, Anderson EJ, Neufer PD (2011) Biochem J
Abstract: 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. • Keywords: Bioenergetics, Blebbistatin, Creatine kinase, Myosin-ATPase, N-benzyltoluene sulfonamide, Skeletal muscle contraction
• O2k-Network Lab: US_NC Greenville_Neufer PD, CA Toronto Perry CG, US_NC_Greenville_Anderson EJ, CA_Antigonish_Kane DA, US NC Greenville East Carolina Univ
Labels:
Organism: Human, Rat
Tissue;cell: Skeletal muscle
Preparation: Permeabilized tissue
Enzyme: Complex I, Complex V; ATP Synthase"Complex V; ATP Synthase" is not in the list (Adenine nucleotide translocase, Complex I, Complex II;succinate dehydrogenase, Complex III, Complex IV;cytochrome c oxidase, Complex V;ATP synthase, Inner mt-membrane transporter, Marker enzyme, Supercomplex, TCA cycle and matrix dehydrogenases, ...) of allowed values for the "Enzyme" property.
Regulation: ATP; ADP; AMP; PCr"ATP; ADP; AMP; PCr" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property.
HRR: Oxygraph-2k
