Small-scale protocols to characterize mitochondrial Complex V activity and assembly in peripheral blood mononuclear...

Area of Science:

• Biochemistry
• Cell Biology
• Genetics

Background:

• Complex V (ATP synthase) is crucial for cellular energy production via oxidative phosphorylation.
• Defects in Complex V subunits lead to severe metabolic and neurodegenerative disorders.
• Assessing Complex V function and assembly is vital for diagnosing related diseases.

Purpose of the Study:

• To establish complementary assays for evaluating Complex V activity and assembly in peripheral blood mononuclear cells (PBMCs).
• To enable small-scale, minimally invasive investigations for patients with suspected Complex V deficiency.
• To explore the utility of PBMCs as a platform for mitochondrial function biomarker research.

Main Methods:

• Developed spectrophotometric and in-gel activity assays for Complex V.
• Utilized cytochemical assessment of mitochondrial transmembrane electrochemical gradient (∆Ѱm) to differentiate ATP synthase vs. ATPase activity.
• Employed Western blot analysis of clear native gels to evaluate Complex V assembly.
• Validated assays using 2 × 10^6 PBMCs from approximately 2 ml of blood.

Main Results:

• Successfully established three complementary assays to assess Complex V activity and assembly.
• Demonstrated the feasibility of using PBMCs for these assessments.
• Showcased the ability to distinguish forward (ATP synthase) and reverse (ATPase) enzyme function.
• Confirmed the utility of the assays for evaluating Complex V assembly.

Conclusions:

• Peripheral blood mononuclear cells (PBMCs) provide a viable platform for studying Complex V.
• The developed assays are suitable for small-scale, minimally invasive investigations of Complex V deficiency.
• These methods can support biomarker research for mitochondrial function and related diseases.