mitoplasts - large scale

Dean R. Appling appling at
Thu Mar 3 14:44:20 EST 1994

We are attempting to purify an enzyme from rat liver mitochondria that
resides in the matrix.  Unfortunately, there is a cytosolic isozyme of
identical size that binds to the outer membrane of isolated mitochondria
that we are unable to wash off.  We thus need to prepare large amounts of
mitoplasts for purification purposes.  We have successfully used the
digitonin procedure to subfractionate mitochondria on a small scale, but
have not had much success scaling it up.  Pedersen's Methods in Cell
Biology chapter (Vol. XX, 1978) mentions a French press method for
preparing mitoplasts that might be more amenable to scale up.  Does
anyone have experience with large scale mitoplast preps, or other ideas? 

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