Evidence that carnitine palmitoyltransferase I (CPT I) is expressed in microsomes and peroxisomes of rat liver - Distinct immunoreactivity of the N-terminal domain of the microsomal protein

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 tot al cellular CPTo activity, with the microsomal and peroxisomal contribution s accounting for the remaining 25% and 10%, respectively, In parallel exper iments, rat livers were perfused in situ with medium containing dinitrophen yl (DNP)-etomoxir in order to inhibit quantitatively and label covalently ( with DNP-etomoxiryl-CoA) the molecular species responsible for CPTo activit y in each of the membrane systems under near-physiological conditions. In a ll three membrane fractions, a single protein with an identical molecular m ass of approximately 88000 kDa (p88) was labelled after DNP-etomoxir perfus ion 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 antibodie s raised against linear epitopes of the cytosolic N- and C-domains and of t he 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 m agnitude 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 peroxisoma l protein. This conclusion was confirmed using antibody back-titration expe riments, in which the binding of anti-N and anti-C antibodies by mitochondr ia and microsomes was quantified. (C) 1999 Federation of European Biochemic al Societies.