CHARACTERIZATION AND SUBCELLULAR-LOCALIZATION OF L-[H-3]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-[H-3] carnitine bound with relatively high affinity (K-D = 281 nM) and in a saturable manner to a finite number (apparent B-max value = 7.3 pmol/m g of protein) of binding sites. Binding was reversible and dependent o n 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 x 10(-3) min(-1) nM(-1) Binding was highest in spinal cord, followed by medull a oblongata-pons greater than or equal to corpus striatum greater than or equal to cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. L-[H-3] carnitine binding was stereoselective for t he L-isomers of carnitine, propionylcarnitine, and acetylcarnitine. Th e most potent inhibitor of L- [3H]carnitine binding was L-carnitine fo llowed by propionyl-L-carnitine. Acetyl-L-carnitine and isobutyryl-L-c arnitine showed an affinity similar to 500-fold lower than that obtain ed for L-carnitine. The precursor gamma-butyrobetaine had negligible a ctivity al 0.1 mM. L-Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glu tamate, aspartate, acetylcholine, dopamine, norepinephrine, epinephrin e, 5-hydroxytryptamine, histamine) at a final concentration of 0.1 mM, In addition, the binding of these neuroactive compounds to their rece ptors was not influenced by the presence of 0.1 mM L-carnitine. Finall y, a subcellular fractionation study showed that synaptic vesicles con tained the highest density of L-carnitine membrane binding sites where as L-carnitine palmitoyltransferase activity was undetectable, thus ex cluding the possibility of the presence of an active site for carnitin e palmitoyltransferase. This finding indicated that the localization o f the L-[H-3] carnitine binding site should be essentially presynaptic .