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Fractionation and analysis of mitochondria with polycarbonate membrane filters
1Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
Abstract:
Polycarbonate membrane filters were used to fractionate mitochondrial populations depending on their aggregation or association with other subcellular structures. Isolated rat liver mitochondria penetrated through filters which have pore sizes larger than 1 micron. In contrast, mitochondria which were induced to aggregate in vitro by incubation at low pH were retained by the filters and thus could be separated from the single or small aggregates of mitochondria. Use of this membrane filtration method to analyze release of mitochondria from isolated hepatocytes showed that treatment with digitonin at concentrations only sufficient to lyse the plasma membrane did not release mitochondria. Homogenization or sonication following digitonin treatment released 25-50% of the mitochondria, but only a small fraction was intact. A high yield of intact mitochondria was released from digitonin-treated cells by a brief treatment with a low concentration of the proteolytic enzyme nagarse. Thus, this membrane filtration method provides a simple and rapid approach to analyze the extent of mitochondrial aggregation and association with other subcellular structures.
Insights
This study introduces a membrane filtration technique to separate aggregated mitochondria from single ones. This method aids in analyzing mitochondrial release from cells and their structural integrity.
Area of Science:
- Cell Biology
- Biochemistry
- Subcellular Fractionation
Background:
- Mitochondria play crucial roles in cellular energy production and apoptosis.
- Understanding mitochondrial release and aggregation is vital for studying cellular health and disease.
- Current methods for analyzing mitochondrial populations can be complex and time-consuming.
Purpose of the Study:
- To develop a simple and rapid method for fractionating mitochondrial populations based on aggregation.
- To analyze the release and integrity of mitochondria from isolated hepatocytes.
- To assess the association of mitochondria with other subcellular structures.
Main Methods:
- Utilized polycarbonate membrane filters with varying pore sizes (e.g., >1 micron) for mitochondrial fractionation.
- Induced mitochondrial aggregation in vitro using low pH incubation.
- Applied membrane filtration to analyze mitochondria released from digitonin-treated hepatocytes, with and without subsequent homogenization, sonication, or nagarse treatment.
Main Results:
- Isolated rat liver mitochondria passed through filters with pore sizes >1 micron.
- In vitro aggregated mitochondria were retained by the filters, enabling separation from single mitochondria.
- Digitonin treatment alone did not release significant amounts of mitochondria from hepatocytes.
- Homogenization or sonication released 25-50% of mitochondria, but with low intactness.
- A high yield of intact mitochondria was obtained using brief nagarse treatment on digitonin-permeabilized cells.
Conclusions:
- Membrane filtration is an effective technique for separating aggregated mitochondria.
- This method allows for the analysis of mitochondrial aggregation and release from cells.
- Proteolytic enzyme treatment (nargase) combined with membrane filtration offers a high-yield approach for isolating intact mitochondria from cells.