Permeabilized muscle fibers: Difference between revisions
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== Chemicals and Media == | == Chemicals and Media == | ||
''What is the optimum ADP concentration to stimulate maximum muscle fibre respiration?'' | '''''What is the optimum ADP concentration to stimulate maximum muscle fibre respiration?''''' | ||
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Especially in muscle fibers an ADP concentration up to 5 mM needs to be applied to be in the saturating range, an ADP titration might be required to evaluate, whether the applied concentration is saturating. | Especially in muscle fibers an ADP concentration up to 5 mM needs to be applied to be in the saturating range, an ADP titration might be required to evaluate, whether the applied concentration is saturating. | ||
Revision as of 14:15, 9 August 2011
References
The following references describe in detail the basis for working with muscle fibers in the O2k.
- Pesta D, Gnaiger E (2011) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. In: Mitochondrial bioenergetics: methods and protocols (Series Editor: Sir John Walker), edited by Carlos Palmeira and Antonio Moreno. In press.
- MiPNet14.14 PermeabilizedFibrePreparation|Pesta D, Gnaiger E. Preparation of permeabilized muscle fibres for diagnosis of mitochondrial respiratory function. Mitochondr. Physiol. Network 14.14.]]
- Gnaiger E. Isolated mitochondria or permeabilized tissues and cells. Mitochondr. Physiol. Network 11.05.
- Fasching M, Renner K, Gnaiger E. Mitochondrial respiration medium - MiR06. Mitochondr. Physiol. Network 14.13.
Chemicals and Media
What is the optimum ADP concentration to stimulate maximum muscle fibre respiration?
Especially in muscle fibers an ADP concentration up to 5 mM needs to be applied to be in the saturating range, an ADP titration might be required to evaluate, whether the applied concentration is saturating.
Pesta 2011 Protocols:At a high apparent Km for ADP of 0.5 mM, flux at 2.5 and 5 mM ADP is ADP-limited by 13% and 7% (assuming L/P=0.2). 2.5 mM ADP is saturating in many cases, yet a further increase of ADP concentration provides a test for saturating [ADP]. This is particularly important for evaluation of OXPHOS versus ETS capacity (P versus E; Fig. 5).
High oxygen concentration
Why is it necessary to measure muscle fiber respiration in an oxygen concentration range from 380 to 200 nM?
Respiratory flux of muscle fibre bundles is limited by oxygen diffusion even above 50% air saturation (O2 concentration of 100 ΞM). This problem is carefully controlled and avoided by application of a high oxygen regime.
See: Pesta 2011 Protocols, Gnaiger 2003 AdvExpMedBiol.