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Related Experiment Videos

Inside-out membrane vesicles isolated from spinach thylakoids.

B Andersson, H E Akerlund

    Biochimica Et Biophysica Acta
    |September 7, 1978
    PubMed
    Summary
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    Researchers isolated inside-out thylakoid vesicles from chloroplasts using a two-phase system. These vesicles exhibit unique properties, confirming their inverted orientation for studying photosynthetic processes.

    Area of Science:

    • Plant Biology
    • Biochemistry
    • Membrane Biophysics

    Background:

    • Chloroplast lamellae contain thylakoid membranes crucial for photosynthesis.
    • Disruption of chloroplasts yields membrane vesicles of varying orientations.
    • Separating inside-out vesicles is vital for targeted biochemical and biophysical studies.

    Purpose of the Study:

    • To isolate inside-out thylakoid vesicles from disrupted chloroplasts.
    • To characterize the unique properties of these isolated vesicles.
    • To validate the utility of a two-phase system for membrane vesicle separation.

    Main Methods:

    • Press disruption of chloroplast lamellae.
    • Separation of membrane vesicles using an aqueous dextran-polyethylene glycol two-phase system.

    Related Experiment Videos

  • Characterization of vesicle orientation via proton extrusion, pH changes, trypsin sensitivity, and freeze-fracture electron microscopy.

    • Main Results:

    • Successfully isolated inside-out thylakoid vesicles.
    • Demonstrated light-induced proton extrusion dependent on Photosystem II activity.
    • Observed internal pH changes correlating with external proton release.
    • Showcased distinct trypsin sensitivity and freeze-fracture morphology compared to right-side-out vesicles.

    Conclusions:

    • The dextran-polyethylene glycol two-phase system effectively separates inside-out thylakoid vesicles based on surface properties.
    • The isolated vesicles exhibit functional and structural characteristics consistent with an inverted orientation.
    • This method provides a valuable tool for investigating thylakoid membrane sidedness and function.