Pediatric Resident Golisano Children's Hospital at The University of Rochester Medical Center Rochester, New York, United States
Background: Large molecular weight supercomplexes of the electron transport chain in the inner mitochondrial membrane are proposed to increase the efficiency of electron transport, generation of the proton motive force, and ATP production. Assembly of supercomplexes is a dynamic process that responds to changes in energetic state of the cell and energy demands. Data from various labs over the last 50 years suggests that some enzymes of the tricarboxylic (TCA) cycle may also form supercomplexes called metabolons. Objective: We hypothesized that the composition and presence of TCA cycle metabolons in cardiac mitochondria respond to energetic states and anaplerotic/cataplerotic flux. Design/Methods: Assays were performed using frozen cardiac myocytes from a mouse model. The mitochondria of cardiac myocytes were isolated using differential centrifugation and protein concentration was calculated using spectrophotometry. Samples were then run via native gel electrophoresis followed by in-gel enzyme assays, 2D electrophoresis, and immunoblotting to determine the presence and relative prevalence of TCA cycle metabolomes. Results: Western blot analysis demonstrated the presence of multiple supercomplexes with molecular weights of greater than 500 kD using cardiac mitochondria. Bands with signals of aconitase (ACON), isocitrate (IDH) or malate dehydrogenase (MDH) activity were excised from the native gel and the proteins were separated by SDS electrophoresis/immunoblotting. A protein complex of about 1000 kD contained MDH, citrate synthase (CS), IDH, and ACON but not alpha-ketoglutarate dehydrogenase or succinate dehydrogenase. A band with MDH activity at 500 kD contained mostly MDH, IDH and CS, but less ACON.
Conclusion(s): Based on these preliminary studies, we propose a model where the balance of energetic state and cataplerotic flux dictates the levels of metabolons in the mitochondrial matrix and thus the efficiency of TCA cycle function.
