Characterization and Subcellular Localization of l ‐[ 3 H]Carnitine Binding Sites in Rat Brain

In the present study, we investigated the existence of a binding site for l ‐carnitine in the rat brain. In crude synaptic membranes, l ‐[ 3 H]carnitine bound with relatively high affinity ( K D = 281 n M ) and in a saturable manner to a finite number (apparent B max value = 7.3 pmol/mg of protein) of binding sites. Binding was reversible and dependent on protein concentration, pH, ionic strength, and temperature. Kinetic studies revealed a K off of 0.018 min −1 and a K on of 0.187 × 10 −3 min −1 n M −1 . Binding was highest in spinal cord, followed by medulla oblongata‐pons ≥ corpus striatum ≥ cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. l ‐[ 3 H]Carnitine binding was stereoselective for the l ‐isomers of carnitine, propionylcarnitine, and acetylcarnitine. The most potent inhibitor of l ‐[ 3 H]carnitine binding was l ‐carnitine followed by propionyl‐ l ‐carnitine. Acetyl‐ l ‐carnitine and isobutyryl‐ l ‐carnitine showed an affinity ∼500‐fold lower than that obtained for l ‐carnitine. The precursor γ‐butyrobetaine had negligible activity at 0.1 m M . l ‐Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glutamate, aspartate, acetylcholine, dopamine, norepinephrine, epinephrine, 5‐hydroxytryptamine, histamine) at a final concentration of 0.1 m M . In addition, the binding of these neuroactive compounds to their receptors was not influenced by the presence of 0.1 m M l ‐carnitine. Finally, a subcellular fractionation study showed that synaptic vesicles contained the highest density of l ‐carnitine membrane binding sites whereas l ‐carnitine palmitoyltransferase activity was undetectable, thus excluding the possibility of the presence of an active site for carnitine palmitoyltransferase. This finding indicated that the localization of the l ‐[ 3 H]carnitine binding site should be essentially presynaptic.