High K(m) of oxidative phosphorylation for ADP in skinned muscle fibers: where does it stem from?

Area of Science:

• Mitochondrial function
• Cardiac bioenergetics
• Cellular respiration

Background:

• Mitochondria in skinned cardiac fiber bundles exhibit lower apparent ADP affinity than isolated mitochondria.
• This discrepancy was previously attributed to factors other than diffusion gradients due to short diffusion distances.
• The role of diffusion gradients in modulating mitochondrial ADP affinity in situ remained unclear.

Purpose of the Study:

• To investigate the presence and impact of ADP diffusion gradients within saponin-skinned cardiac fiber bundles.
• To test the hypothesis that increasing intracellular ADP production can reduce these diffusion gradients.
• To accurately determine the intrinsic ADP affinity of mitochondria within the cardiac fiber bundle environment.

Main Methods:

• Utilized saponin-skinned rat heart fiber bundles.
• Increased intracellular ADP production by incubating bundles with yeast hexokinase and glucose.
• Measured respiration rates and apparent Michaelis constant (Km) to ADP.
• Employed a mathematical model to estimate mitochondrial ADP affinity.

Main Results:

• Incubation with hexokinase and glucose significantly decreased the apparent Km to ADP from 216 ± 59 to 50 ± 9 μM.
• Mathematical modeling estimated the intrinsic Km of mitochondria within the bundles to be 25 μM.
• The results indicate substantial ADP diffusion gradients within the fiber bundles.

Conclusions:

• Significant ADP diffusion gradients exist within saponin-skinned cardiac fiber bundles.
• Increasing intrafiber ADP production effectively diminishes these diffusion gradients.
• The intrinsic affinity of cardiac mitochondria to ADP in situ is comparable to that of isolated mitochondria.