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Buffer-induced rat liver mitochondrial aggregation during differential centrifugation.

M Petersons, J B Allred

    Physiological Chemistry and Physics
    |January 1, 1981
    PubMed
    Summary
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    Buffers in homogenization media can cause significant loss of enzyme activity. Tris chloride buffer addition precipitated mitochondrial enzymes, indicating aggregation and reduced assayable activity during differential centrifugation.

    Area of Science:

    • Biochemistry
    • Cell Biology
    • Biochemical Assays

    Background:

    • Homogenization media composition is critical for preserving enzyme activity.
    • Particulate enzyme activity can be lost during cellular fractionation processes.
    • Buffer selection impacts the integrity and recovery of cellular components.

    Purpose of the Study:

    • To investigate the effect of tris chloride buffer on enzyme activity during homogenization.
    • To determine the impact of buffer choice on mitochondrial integrity and recovery.
    • To assess the precipitation of mitochondrial enzymes during differential centrifugation.

    Main Methods:

    • Differential centrifugation of cellular homogenates.
    • Assay of specific mitochondrial and cytoplasmic marker enzymes.

    Related Experiment Videos

  • Photomicrographic analysis of mitochondrial morphology.
  • Resuspension of isolated mitochondria in different buffer solutions.

    • Main Results:

    • Tris chloride buffer addition precipitated 80-95% of mitochondrial enzyme activity (3-hydroxy-3-methylglutaryl CoA lyase, citrate synthase).
    • Cytoplasmic lactate dehydrogenase was not precipitated, confirming enzyme loss was not due to intact cells.
    • Photomicrographs revealed mitochondrial aggregation induced by tris chloride buffer.
    • Resuspension in tris chloride or potassium phosphate buffers caused mitochondrial aggregation and decreased enzyme activity.

    Conclusions:

    • Tris chloride buffer significantly reduces assayable mitochondrial enzyme activity due to aggregation.
    • Buffer selection is crucial for maintaining mitochondrial integrity during biochemical assays.
    • Careful consideration of buffer composition is necessary to avoid artifactual loss of enzyme activity in fractionation studies.