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Related Experiment Videos

Immunologically reactive multiple species of mitochondrial coupling factor B.

S S Kaplay, S Joshi, C Gopalswami

    The Journal of Biological Chemistry
    |August 25, 1984
    PubMed
    Summary

    Mitochondrial coupling factor B (FB) likely exists as a monomer in mitochondria. It appears to form higher molecular weight aggregates during extraction and purification processes.

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    Area of Science:

    • Biochemistry
    • Mitochondrial Function

    Background:

    • Mitochondrial coupling factor B (FB) has been previously reported in various molecular weight forms with differing activities.
    • These forms included a highly active 13,000 Da species and less active 29,200 Da and 46,000 Da forms.

    Purpose of the Study:

    • To investigate the molecular behavior and potential aggregation of mitochondrial coupling factor B (FB) during purification.
    • To determine the native state of FB within mitochondria.

    Main Methods:

    • Analysis of FB preparations using SDS-PAGE with silver staining and immunostaining.
    • Utilized rabbit anti-FB serum and monoclonal FB antibody for immunodetection.
    • Examined effects of lyophilization, freeze-thawing, and absence of reducing agents on FB forms.

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    Main Results:

    • Highly purified FB showed single bands on SDS-PAGE, but higher molecular weight bands (48,000 and 68,000 Da) appeared after specific treatments.
    • Freeze-thawing and absence of dithiothreitol/glycerol induced higher molecular weight forms.
    • Immunoblots confirmed the presence of multiple higher molecular weight forms in treated and partially purified samples.

    Conclusions:

    • Mitochondrial coupling factor B (FB) is likely a monomer in its native mitochondrial environment.
    • FB undergoes oligomerization or aggregation upon extraction and during purification procedures.
    • The observed variations in molecular weight are artifacts of sample handling and purification, not native forms.