Freeze-thawing causes masking of membrane-bound outer carnitine palmitoyltransferase activity: implications for studies on carnitine palmitoyltransferases deficiency.

Carnitine-dependent transport of fatty acids into mitochondria is believed to require participation of two carnitine palmitoyltransferase (CPT) activities, one outer, overt (CPTo) and the other inner, latent (CPTi). For exposing the CPTi and monitoring of the total CPT activity, freeze-thawing and sonication have been frequently employed as membrane-disruptive procedures, particularly when examining for CPT-deficiency diseases. Our evaluations have shown, however, that freeze-thawing and sonication yield misleading data for both the CPT activities owing to their previously unrecognized masking and unmasking effects on CPT activities. Formation of vesicular/sheath structures with mixed membrane orientation that prevents the access of medium substrate to enzymes on both aspects of the membrane at the same time appears responsible for these results. That such procedures can yield inexact data when monitoring the latency and sidedness of other membrane-bound biocatalysts as well needs to be recognized. We show that in muscle mitochondria also, a malonyl-CoA-inhibitable CPTo activity resides in the outer membrane, while a malonyl-CoA-insensitive, CPTi, activity is present in the inner membrane. Our results rationalize why Zierz and Engel ((1987) Neurology 37, 1785) were unable to obtain evidences for a latent CPT activity in mitochondria particularly of muscles. Although simple methods to allow an unambiguous quantitation of the two CPT activities in tissue extracts remain unavailable, evaluation of the possibility that two different CPT deficiencies occur appears justified.