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Isolation of intramitochondrial helical filaments appearing in outer compartment of mitochondria
Background:
Packets of helical filaments have been observed in the outer compartment of occasional mitochondria in many cell types in a variety of animals. The composition and function of these intramitochondrial helical filaments (IMHF) are unknown.
Methods:
IMHF were induced in a hepatic mitochondria by administration of ethanol in the drinking water of rats. Hepatic mitochondria were isolated and ruptured by osmotic shock, releasing the IMHF. To purify these structures, the IMHF-containing supernatant was further fractionated by ammonium sulfate precipitation, a 50-60% solution of this reagent being the most effective in this regard. Isolated IMHF were examined by electron microscopy and were analyzed by SDS-PAGE.
Results:
Isolated IMHF closely resembled their in situ counterparts: they had a right-handed helical structure with a 16 nm pitch. SDS-PAGE analysis revealed that they contained three polypeptides with molecular weight of 135, 98, and 53 kD, respectively.
Conclusions:
These observations will stand as a baseline for comparisons with IMHF that occur naturally or that are induced in other cell types by other kinds of experimental manipulation.
Insights
Ethanol induces intramitochondrial helical filaments (IMHF) in rat liver mitochondria. These purified filaments possess a right-handed helical structure and contain three distinct polypeptides, providing a baseline for future research.
Area of Science:
- Cell Biology
- Mitochondrial Research
- Biochemistry
Background:
- Intramitochondrial helical filaments (IMHF) are observed in mitochondria across various animal cell types.
- The composition and function of IMHF remain largely unknown.
Purpose of the Study:
- To induce and isolate IMHF from rat liver mitochondria.
- To characterize the structure and protein composition of induced IMHF.
Main Methods:
- Ethanol administration to rats to induce IMHF in hepatic mitochondria.
- Mitochondrial isolation, osmotic shock for IMHF release, and ammonium sulfate precipitation for purification.
- Electron microscopy and SDS-PAGE for structural and compositional analysis.
Main Results:
- Isolated IMHF exhibit a right-handed helical structure with a 16 nm pitch.
- SDS-PAGE analysis identified three polypeptides in IMHF with molecular weights of 135, 98, and 53 kD.
Conclusions:
- The study establishes a baseline for comparing naturally occurring or experimentally induced IMHF.
- Provides foundational data on the structure and protein components of IMHF.