Summary
Mitochondria, microsomes and peroxisomes all express overt (cytosol-facing) carnitine palmitoyltransferase activities that are inhibitable by malonyl-CoA and are collectively termed CPTo. In order to quantify the relative contribution of the different membrane systems towards overall hepatocyte activity, all three membrane fractions and a high-speed supernatant (soluble) fraction were prepared quantitatively from rat liver homogenates. The overt (malonyl-CoA-sensitive) carnitine palmitoyltransferase activity (CPTo) associated with the different fractions were measured. In parallel experiments, rat livers were perfused in situ with oxygenated medium containing dinitrophenyl (DNP)-etomoxir in order to label covalently (with DNP-etomoxiryl-CoA) and quantitatively the molecular species responsible for CPTo activity in each of the membrane systems under near-physiological conditions. Mitochondria accounted for only 65% of total cellular overt CPT activity, with the microsomal and peroxisomal contributions accounting for the remaining 25% and 10%, respectively. A single major protein with an identical molecular size (Mr 88,000) was labelled by DNP-etomoxir perfusion in all three membrane fractions. The abundance of this 88kDa protein in each fraction was quantitatively positively related to the respective specific activities of overt CPT. The same protein was immunoreactive with three anti-peptide antibodies raised against linear epitopes within the N- and C-terminal and loop (L) domains of the mitochondrial outer membrane CPT I of the liver mitochondrial outer membrane (L-CPT I). However, whereas reaction with anti-L and anti-C antipeptide antibodies were proportional to the respective overt CPT activities and DNP-etomoxir labelling in all three membrane fractions, reaction with anti-N peptide antibody was much stronger for microsomal CPT. We conclude that in all three membrane systems overt CPT activity is associated with the same or highly similar molecular species to mitochondrial outer membrane CPT I, but that the protein expressed in microsomes has a modified N-terminal domain, which gives the microsomal enzyme its higher malonyl-CoA sensitivity and may target the protein to its microsomal location.
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Fraser, F., Corstorphine, C.G., Zammit, V.A. (2002). Subcellular Distributuon of Mitochondrial Carnitine Palmitoyltransferase I in Rat Liver. In: Quant, P.A., Eaton, S. (eds) Current Views of Fatty Acid Oxidation and Ketogenesis. Advances in Experimental Medicine and Biology, vol 466. Springer, Boston, MA. https://doi.org/10.1007/0-306-46818-2_2
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DOI: https://doi.org/10.1007/0-306-46818-2_2
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