Protonmotive force in muscle mitochondria.

Area of Science:

• Mitochondrial physiology
• Bioenergetics
• Cellular respiration

Background:

• Mitochondrial protonmotive force (delta p) is a key indicator of cellular energy production.
• Accurate measurement of delta p in muscle mitochondria is essential for understanding metabolic function.
• Existing methods for delta p measurement have limitations, particularly with coupled mammalian muscle mitochondria.

Purpose of the Study:

• To establish a reliable method for measuring the protonmotive force (delta p) in muscle mitochondria.
• To determine the membrane conductance (Cm,H+) of muscle mitochondria.
• To validate the new delta p assay using both rat and human muscle biopsy specimens.

Main Methods:

• Utilized 14C-labeled TPMP and 14C-labeled acetate to estimate delta p across the inner mitochondrial membrane.
• Determined matrix volume using 3H-labeled H2O and 3H-labeled mannitol.
• Employed rapid centrifugation through silicone oil for efficient mitochondria separation, avoiding anaerobic conditions and trapped media.
• Measured oxygen consumption to calculate membrane conductance (Cm,H+).

Main Results:

• Rat muscle mitochondria exhibited a state-4 delta p of 192 ± 26 mV and Cm,H+ of 0.49 ± 0.18 nmol H+/min/mg/mV.
• Human muscle biopsy specimens showed state-4 delta p values of 197 ± 30 mV and Cm,H+ values of 0.52 ± 0.12 nmol H+/min/mg/mV.
• Results are consistent with Mitchell's chemiosmotic theory.

Conclusions:

• A novel and effective assay for measuring protonmotive force (delta p) in coupled mammalian muscle mitochondria has been developed.
• This assay provides valuable insights into mitochondrial membrane function.
• The method is suitable for assessing mitochondrial bioenergetics in various experimental conditions and clinical settings.