A high-yield preparative method for isolation of rat liver mitochondria

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Abstract

A differential centrifugation method for the preparation of rat liver mitochondria is described, which results in final mitochondrial yields of at least 35 to 40 mg of mitochondrial protein per gram wet weight of liver. These mitochondria are shown to be functionally and ultrastructurally intact. They exhibit acceptor control ratios of 6 to 8 and ADPO ratios near 2.0 with succinate as a substrate. In addition, they appear homogeneous by electron microscopy criteria. This method can be used to prepare rat liver mitochondria in high yield on either a large- or a small-scale basis.

References (16)

  • W.A. Catterall et al.

    J. Biol. Chem

    (1971)
  • S.H.P. Chan et al.

    Biochim. Biophys. Acta

    (1976)
  • W.C. Schneider et al.

    J. Biol. Chem

    (1950)
  • R.M. Kaschnitz et al.

    Arch. Biochem. Biophys

    (1976)
  • D. Johnson et al.
  • W.C. Schneider

    J. Biol. Chem

    (1948)
  • R.W. Estabrook
  • A. Gornall et al.

    J. Biol. Chem

    (1949)
There are more references available in the full text version of this article.

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