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Functional studies on in situ-like mitochondria isolated in the presence of polyvinyl pyrrolidone
Abstract:
Mitochondria isolated and maintained in sucrose mannitol medium show a large intermembrane space and a condensed matrix unlike the appearance of in situ mitochondria. Mitochondria resembling in situ organelles are obtained when the isolation medium is supplemented with certain macromolecules such as polyvinyl pyrrolidone. We found that the in situ appearance was acquired also by the conventionally isolated mitochondria when they were exposed to 2% polyvinyl pyrrolidone supplemented medium. Paradoxically, however, these in situ looking mitochondria proved functionally inferior in that their brief incubation without substrates led to a marked loss of their ability to respire with subsequently added substrates such as pyruvate, acylcarnitines or glutamate. The oxidation of succinate was, however, not so affected. This phenomenon was shared by heart and skeletal muscle mitochondria of different animal species but not by rat liver mitochondria. The inhibition of respiration could not be related to the failure to oxidize NADH, to the tieing up of mitochondrial free CoASH, or to the increased matrix space of mitochondria that was observed in the presence of polyvinyl pyrrolidone. The polyvinyl pyrrolidone-exposed mitochondria regained their respiratory ability on being freed from polyvinyl pyrrolidone. The same phenomenon was seen also when the medium contained 2% albumin or 20% Ficoll.
Insights
Adding macromolecules like polyvinyl pyrrolidone to mitochondrial isolation media improves appearance but impairs function. This effect, seen in muscle mitochondria, is reversible and not due to specific metabolic failures.
Area of Science:
- Biochemistry
- Cell Biology
- Mitochondrial Research
Background:
- Mitochondrial isolation often results in altered organelle morphology compared to in situ mitochondria.
- Supplementing isolation media with macromolecules can restore a more native mitochondrial appearance.
Purpose of the Study:
- To investigate the functional consequences of achieving an in situ-like mitochondrial appearance using polyvinyl pyrrolidone (PVP).
- To determine the reversibility of functional impairment and identify affected mitochondrial subtypes.
Main Methods:
- Isolation of mitochondria from various tissues (heart, skeletal muscle, liver) using sucrose mannitol medium.
- Exposure of isolated mitochondria to polyvinyl pyrrolidone (PVP), albumin, or Ficoll.
- Assessment of mitochondrial respiratory function using different substrates (pyruvate, acylcarnitines, glutamate, succinate).
- Analysis of NADH oxidation, CoASH availability, and matrix space.
Main Results:
- Mitochondria exposed to PVP acquired an in situ-like morphology but exhibited impaired respiration with specific substrates (pyruvate, acylcarnitines, glutamate).
- Succinate oxidation remained largely unaffected.
- This functional deficit was observed in heart and skeletal muscle mitochondria but not liver mitochondria.
- The respiratory impairment was reversible upon removal of PVP and also observed with albumin and Ficoll.
Conclusions:
- Achieving an in situ-like mitochondrial morphology with macromolecules does not guarantee preserved function.
- Muscle mitochondria are particularly susceptible to respiration inhibition induced by PVP exposure.
- The observed functional defect is not linked to NADH oxidation, CoASH, or increased matrix volume and is reversible.