Evidence that carnitine palmitoyltransferase I (CPT I) is expressed in microsomes and peroxisomes of rat liver

Abstract

Mitochondria, microsomes and peroxisomes all express overt (cytosol-facing) carnitine palmitoyltransferase activity that is inhibitable by malonyl-CoA. The overt carnitine palmitoyltransferase activity (CPTo) associated with the different fractions was measured. Mitochondria accounted for 65% of total cellular CPTo activity, with the microsomal and peroxisomal contributions accounting for the remaining 25% and 10%, respectively. In parallel experiments, rat livers were perfused in situ with medium containing dinitrophenyl (DNP)-etomoxir in order to inhibit quantitatively and label covalently (with DNPetomoxiryl-CoA) the molecular species responsible for CPTo activity in each of the membrane systems under near-physiological conditions. In all three membrane fractions, a single protein with an identical molecular mass of approximately 88 000 kDa (p88) was labelled after DNP-etomoxir perfusion of the liver. The abundance of labelled p88 was quantitatively related to the respective specific activities of CPTo in each fraction. On Western blots the same protein was immunoreactive with three anti-peptide antibodies raised against linear epitopes of the cytosolic N-and C-domains and of the inter-membrane space loop (L) domain of the mitochondrial enzyme (L-CPT I). However, the reaction of the microsomal protein with the anti-N peptide antibody (raised against epitope Val-14^Lys-29 of CPT I) was an order of magnitude stronger than expected from either microsomal CPTo activity or its DNP-etomoxiryl-CoA labelling. This suggests that the N-terminal domain of the microsomal protein differs from that in the mitochondrial or peroxisomal protein. This conclusion was confirmed using antibody backtitration experiments, in which the binding of anti-N and anti-C antibodies by mitochondria and microsomes was quantified.z 1999 Federation of European Biochemical Societies.

The malonyl-CoA–long-chain acyl-CoA axis in the maintenance of mammalian cell function

“…value for malonyl-CoA [27]. This property coincides with distinctive immunoreactive properties of the N-terminal domain of the microsomal protein [24].…”

“…Hence the terminology of CPT-I (for mitochondrial-outer-membrane CPTo) and CPT-II, for the inner-membrane CPT (extensively reviewed elsewhere [22,23]). However, malonyl-CoA-sensitive CPT activity also occurs on the cytosol-facing aspects of the endoplasmic reticulum and of peroxisomes [24]. An activity has also been described in the nucleus [25] [or associated endoplasmic reticulum (ER)] and in the plasma membrane of human erythrocytes [26].…”

“…This is especially relevant in view of the strong evidence that the ER and peroxisomal CPTo are very similar to, if not identical with, the mitochondrial CPTo (CPT-I). In the liver, CPTo activity in each of these membranes is positively and quantitatively related to the labelling of an 88 kDa protein by dinitrophenyletomoxir [24], a substrate analogue inhibitor that binds covalently to the protein.…”

“…In addition, microsomal and peroxisomal CPTo cross-react with several antibodies raised against linear epitopes of the mitochondrial outer membrane CPTo [24]. The mitochondrial and peroxisomal proteins have extremely similar kinetic properties, although the microsomal CPTo has a lower IC &!…”

“…The traffic of carnitine esters of chain-shortened very-long-chain fatty acids between peroxisomes and the ER and mitochondria [28] would appear to suggest that this is not the case, but does not explain the requirement for expression of the protein(s) at all three sites. It is possible that CPTo in membranes of different lipid composition or subtle differences in structure (such as those suggested by the observations of [24] on the N-terminal domain of CPTo) may affect the kinetic properties of the enzyme in different locations. Evidence supporting this has emerged from the work of Ramsay [27], who showed that the malonyl-CoA-sensitivity of microsomal CPTo is about 2-fold higher than that of the peroxisomal and mitochondrial enzyme, and that it does not show such a large change in kinetic characteristics as the others on fasting of the rats.…”