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Carrier-mediated Transport of Choline into Synaptic Nerve Endings

From the ¹ From the Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115

Synaptosomes from guinea pig brain were incubated with ³H-methylcholine and then subjected to a chromatographic assay which distinguishes nonspecific entry from specific transport. Nonspecific entry of labeled choline into synaptosomes was linear with added substrate, but specific uptake exhibited saturation kinetics. K_t, the concentration for the half-maximal specific transport of choline into synaptosomes was 8.3 x 10^-5 m. Under standard conditions the rate of specific transport of choline was linear with time and protein concentration, and there was a marked temperature dependence. The pH optimum was about 8.6. Choline transport did not require added monovalent nor divalent cations, some of which were potent inhibitors. Nucleotides did not have a specific effect. Choline uptake as measured in this system did not appear to be energy dependent. Reagents which attack sulfhydryl groups were effective inhibitors. Hemicholinium-3 was a competitive inhibitor of specific choline transport with a K_i of 4 x 10^-5 m. Nonspecific uptake was unaffected by hemicholinium-3.

After entry into synaptosomes, 60% of the radioactivity could be recovered as choline, 11% as phosphorylcholine, and 10% as betaine. Acetylcholine constituted less than 5% of the labeled cation. The conversion of ³H-choline to phosphorylcholine and betaine was largely abolished by hemicholinium-3.

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