Abstract
Esterification of fatty acids with the small polar molecule carnitine is a required step for the regulated flow of fatty acids into mitochondrial inner matrix. We have studied the interactions of acyl carnitines (ACs) with model membranes [egg yolk phosphatidylcholine (PC) vesicles] by (13)C-nuclear magnetic resonance (NMR) spectroscopy. Using AC with (13)C-enrichment of the carbonyl carbon of the acyl chain, we detected NMR signals from AC on the inside and outside leaflets of the bilayer of small unilamellar vesicles prepared by cosonication of PC and AC. However, when AC was added to the outside of pre-formed PC vesicles, only the signal for AC bound to the outer leaflet was observed, even after hours at equilibrium. The extremely slow transmembrane diffusion ("flip-flop") is consistent with the zwitterionic nature of the carnitine head group and the known requirement of transport proteins for movement of ACs through the mitochondrial membrane. The partitioning of ACs (8-18 carbons) between water and PC vesicles was studied by monitoring the [(13)C]carbonyl chemical shift of ACs as a function of pH and concentration of vesicles. Significant partitioning into the water phase was detected for ACs with chain lengths of 12 carbons or less. The effect of ACs on the integrity of the bilayer was examined in vesicles with up to 25 mol% myristoyl carnitine; no gross disruption of the bilayer was observed. We hypothesize that the effects of high levels of long-chain AC (as found in ischemia or in certain diseases) on cell membranes result from molecular effects on membrane functions rather than from gross disruption of the lipid bilayer.
Highlights
Esterification of fatty acids with the small polar molecule carnitine is a required step for the regulated flow of fatty acids into mitochondrial inner matrix
Our study focuses on the binding of acyl carnitine (AC) with different chain lengths (8–18 carbons) to model membranes composed of phospholipid bilayers
A series of six [octanoyl (8:0), decanoyl (10:0), lauroyl (12:0), myristoyl (14:0), palmitoyl (16:0), and oleoyl (18:1)] dl-O-acylcarnitine-(1Ј-13C) chloride analogs were prepared on scales of 100 to 300 mg from the corresponding carbonyl-labeled (99 atom% 13C) fatty acid (Cambridge Isotope Laboratories, Woburn, MA) and thionyl chloride followed by treatment of the intermediate fatty acid chloride with dl-carnitine chloride in trichloroacetic acid as solvent according to the reported method [23]
Summary
Esterification of fatty acids with the small polar molecule carnitine is a required step for the regulated flow of fatty acids into mitochondrial inner matrix. We hypothesize that the effects of high levels of long-chain AC (as found in ischemia or in certain diseases) on cell membranes result from molecular effects on membrane functions rather than from gross disruption of the lipid bilayer.—Ho, J. Two AC transferases (CPT I and CPT II) and a carnitine-AC translocase located within the inner mitochondrial membrane control the conversion between AC and acyl-CoA and regulate the flux of AC into the inner matrix [1, 2] This transport is regulated by both fatty acid and carbohydrate metabolism. Under ischemic and hypoxic conditions, levels of long-chain ACs may increase 8- to 10-fold in myocytes and may be elevated up to 100-fold in sarcolemma [7] Such levels of longchain ACs elicit adverse pharmacological effects on cellular functions and make the heart more susceptible to arrhythmias and other dysfunction [8]. Definitive data about the transmembrane movement of natural ACs in model membranes are lacking, there is direct [21] and indirect [22] evidence that palmitoyl carnitine has a slow rate of transbilayer movement in erythrocyte membranes
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