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The isolation of coupled mitochondria from Physarum polycephalum and their response to Ca2+
Abstract:
A method for the isolation of coupled mitochondria from the acellular slime mould Physarum polycephalum is described. The mitochondria oxidize respiratory substrates at rates comparable to those of mitochondria from other microorganisms and show similar responses to respiratory inhibitors. ADP/O values approach similar values to those obtained with mitochondria from higher organisms: 3 with NAD-linked substrates, 2 with succinate, and 1 with ascorbate-TMPD. Mitochondria actively take up low concentrations of Ca2+ with stimulation of their respiration. With succinate or pyruvate-malate as substrates respiratory responses are depressed by Ca2+ concentrations in excess of 200 micron in the presence or absence of phosphate. Exogenous NADH is unique in supporting the uptake of large amounts of Ca2+ in the presence of phosphate and in showing an unusual 'uncoupled' response in the absence of phosphate. A sigmoidal relationship occurs between initial velocity of Ca2+ uptake and Ca2+ concentration with a maximum velocity of approx. 15 nmol/s per mg protein and half maximum velocity occurring at approx. 50 micron Ca2+.
Insights
Researchers isolated coupled mitochondria from Physarum polycephalum, demonstrating their capacity for respiration and calcium uptake. These mitochondria exhibit functional similarities to those from other organisms, offering insights into cellular energy production.
Area of Science:
- Biochemistry
- Cell Biology
- Mycology
Background:
- Mitochondria are crucial for cellular energy production.
- Understanding mitochondrial function in diverse organisms like slime molds is important.
- Physarum polycephalum offers a unique model for studying cellular processes.
Purpose of the Study:
- To describe a method for isolating coupled mitochondria from Physarum polycephalum.
- To characterize the respiratory function and calcium handling of these isolated mitochondria.
Main Methods:
- Isolation of coupled mitochondria from acellular slime mold Physarum polycephalum.
- Measurement of substrate oxidation rates and response to respiratory inhibitors.
- Analysis of ADP/O ratios with various substrates.
- Investigation of calcium (Ca2+) uptake kinetics and its modulation by substrates and phosphate.
Main Results:
- Isolated mitochondria exhibit respiratory rates comparable to other microorganisms.
- ADP/O values are consistent with those from higher organisms (3, 2, and 1 with specific substrates).
- Mitochondria actively take up Ca2+, with respiration stimulated at low concentrations.
- High Ca2+ concentrations depress respiration, while exogenous NADH uniquely supports Ca2+ uptake.
- A sigmoidal relationship was observed between Ca2+ concentration and uptake velocity.
Conclusions:
- A reliable method for isolating functional mitochondria from Physarum polycephalum has been established.
- These mitochondria possess robust respiratory capabilities and complex calcium handling mechanisms.
- The findings provide valuable data on mitochondrial bioenergetics and ion transport in a unique eukaryotic model.