The malonyl-CoA-sensitive form of carnitine palmitoyltransferase is not localized exclusively in the outer membrane of rat liver mitochondria.
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.
Hoppel
C L
CL
Department of Veterans Affairs Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA. CLH5@po.cwru.edu
Kerner
J
J
Turkaly
P
P
Turkaly
J
J
Tandler
B
B
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D015415:Biomarkers; D002334:Carnitine O-Palmitoyltransferase / Q000187:drug effects / Q000302:isolation & purification*; D002451:Cell Compartmentation; D002458:Cell Fractionation / Q000379:methods; D003066:Coenzyme A Ligases / Q000302:isolation & purification; D006874:Hydrostatic Pressure; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000201:enzymology* / Q000648:ultrastructure; D007527:Isoenzymes / Q000187:drug effects / Q000302:isolation & purification; D008316:Malonyl Coenzyme A / Q000494:pharmacology*; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000648:ultrastructure; D010101:Oxygen Consumption / Q000187:drug effects; D051381:Rats; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013367:Submitochondrial Particles / Q000201:enzymology* / Q000648:ultrastructure; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D015415:Biomarkers; D007527:Isoenzymes; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D008316:Malonyl Coenzyme A; D000244:Adenosine Diphosphate; D002334:Carnitine O-Palmitoyltransferase; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase; D019297:2,4-Dinitrophenol