INCORPORATION OF [¹⁴C]GLUCOSAMINE INTO SYNAPTOSOMES IN VITRO

Abstract— Synaptosomes isolated from rat cerebral cortex by zonal centrifugation in‐corporated radioactive glucosamine into macromolecules in vitro as glucosamine, galactosamine, N‐acetylneuraminic acid, and glucuronic acid. The largest percentage of incorporated radioactivity was recovered in the particulate fraction in which radioactive carbohydrates were bound in covalent linkage requiring acid hydrolysis or enzymatic digestion for release. Less than 20 per cent of the particulate radioactivity represented incorporation into gangliosides. Some 20 per cent of the radioactivity was incorporated into proteins as glucosamine, identified in hydrolysates by paper chromatography and by the amino acid analyser. After incubation, radioactivity was demonstrable in the proteins as sialic acid by paper chromatography and specific enzymic digestion; and as glucuronic acid by chromatography, electrophoresis, and digestion with hyaluronidase. Incorporation of carbohydrate was stimulated by sodium and potassium at concentrations demonstrated to enhance incorporation of amino acids, and involved the macro‐molecules of all subsynaptosomal fractions. Significant incorporation of radioactivity was found in the synaptic plasma membrane. The synthesis of glycoproteins was suggested by simultaneous incorporation of [¹⁴C]glucosamine and [³H]leucine into glycopeptides subsequently hydrolysed and subjected to polyacrylamide gel electrophoresis and two‐dimensional paper chromatography and electrophoresis. Such studies demonstrated that amino acids and carbohydrates may be incorporated into glycoproteins of the synaptic membrane and suggest the possibility of local synthesis as well as modification of material brought to the nerve ending by axoplasmic flow.