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Abstract
The data used to support the idea that malonyl-coenzyme A (CoA)-sensitive carnitine palmitoyltransferase (CPT-I) is localized on the outer mitochondrial membrane are based on harsh techniques that disrupt mitochondrial physiology. We have turned to the use of the French press, which produces a shearing force that denudes mitochondria of their outer membrane without the physiologically disruptive effects characteristic of phosphate swelling. Our results indicate that the mitoplasts contain just 15-19% of the outer membrane marker enzyme activity while retaining 85% of the total CPT activity and 50% of both CPT-I, as well as long-chain acyl-CoA synthase activity, the latter two supposed outer membrane enzymes. These mitoplasts were shown by electron microscopy to have the configuration of mitochondria that merely have been divested of their outer membranes. Carnitine-dependent fatty acid oxidation was retained in the mitoplasts, showing that they were physiologically intact. Moreover, protein immunoblotting analysis showed that CPT-I, as well as the inner CPT-II, was localized in the mitoplast fraction. The outer membrane fraction, which consisted of membrane "ghosts," contained most (50-60%) of marker enzyme activity, monoamine oxidase-B and porin proteins, but only about 27-29% CPT-I activity. Because CPT-I and long-chain acyl-CoA synthetase appear to be associated with both inner and outer membranes, we postulate that these enzymes reside in contact sites, which represent a melding of both limiting membranes.
Highlights
The two carnitine palmitoyltransferase (CPT)1 enzyme activities are essential in the mitochondrial oxidation of longchain fatty acids
The upper layer and the clear zone were removed and the lower brownish layer consisting of mitochondria collected
Unlike Decker and Greenawalt, who at a pressure of 1500 p.s.i. found that only 5% of the outer membrane marker enzyme, monoamine oxidase, was retained by the mitoplasts, we found with our French press 80% of the enzyme remained associated with the mitoplasts at this pressure (Fig. 1)
Summary
The two carnitine palmitoyltransferase (CPT) enzyme activities are essential in the mitochondrial oxidation of longchain fatty acids. Previous studies on the localization of CPT have relied upon techniques for separating inner and outer membranes that potentially could alter the membranes by producing breaks with subsequent annealing in a non-native configuration and which might not yield membranes of absolute purity. To overcome these potential pitfalls in localizing the malonyl-CoAsensitive CPT, we have utilized a method of mitochondrial fractionation that does not rely on methods that use either organellar swelling to rupture the outer membrane [1, 2], or exposure to detergents [3], or enzymatic digestion of membrane components [5]. Our data show that CPT-I and long-chain acyl-CoA synthase, supposed outer membrane enzymes, are associated with a component of the mitoplast fraction
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