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Separation of subchloroplast membrane particles by counter-current distribution
Biochimica Et Biophysica Acta
|January 15, 1976
Summary
Counter-current distribution effectively fractionates chloroplast membrane particles by surface properties, yielding enhanced Photosystem II enrichment. Combining this technique with differential centrifugation offers superior separation of Photosystem I and II particles without detergents.
Area of Science:
- Plant Biology
- Biochemistry
- Photosynthesis Research
Background:
- Chloroplasts contain Photosystem I and Photosystem II, crucial for light-dependent reactions.
- Fractionating these photosystems is essential for understanding their distinct functions.
- Previous methods like differential centrifugation have limitations in achieving pure fractions.
Purpose of the Study:
- To fractionate membrane fragments from Class II chloroplasts using counter-current distribution.
- To analyze the photochemical activities, chlorophyll, and P-700 content of separated fractions.
- To compare the efficacy of counter-current distribution with differential centrifugation for photosystem enrichment.
Main Methods:
- Press treatment of Class II chloroplasts to obtain membrane fragments.
- Fractionation of membrane fragments using counter-current distribution in a Dextran-polyethylene glycol aqueous two-phase system.
- Analysis of fractions for photochemical activities, chlorophyll, and P-700 content.
Main Results:
- Counter-current distribution achieved better Photosystem II enrichment than differential centrifugation alone.
- Combining differential centrifugation with counter-current distribution yielded the best separation of Photosystem I and II enriched particles.
- Photosystem II particles partitioned to the dextran-rich phase, while Photosystem I particles favored the polyethylene glycol-rich phase.
- Detergent-free vesicles enriched in Photosystem II were obtained, exceeding enrichment in intact grana stacks.
Conclusions:
- Counter-current distribution is a highly effective method for fractionating chloroplast membrane particles based on surface properties.
- This technique, especially when combined with differential centrifugation, provides superior separation of Photosystem I and II.
- The results suggest that the outer membrane of Class II chloroplasts, exposed during fractionation, is rich in Photosystem I from stroma lamellae.