Histochemical and biochemical demonstration of sialic acid and sulphate in vesicles and membranes isolated from nerve endings of rat brain.

The reaction of colloidal iron hydroxide (CIH) with acidic groups was applied for an ultrastructural study of the distribution of sulphuric acid monoesters and sialic acid in synaptic vesicles and external nerve ending membranes isolated from rat brain. At pH 1-7 CIH was precipitated as electron-dense granules with a uniform size of 6—7 nm specifically labelling the carboxyl group of sialic acid and the sulphate group of monoesters of sulphuric acid. The differentiation of these 2 groups was achieved by treatment with neuraminidase and methylation followed by saponification. After preincubation with neuraminidase, which released 90— 100 % of the sialic acid from the membranes of the synaptic vesicles and the nerve endings, the electron-dense deposits marked the reaction sites of sulphate with CIH. The sulphate groups which were present at a concentration of 2-3 and 2-2 /jmol/mg protein for the synaptic vesicle and nerve ending membrane preparations, respectively, were rendered soluble as methyl monosulphate by trans-esterification with acid/methanol and quantitatively removed from the structures. By this treatment membrane-bound sialic acid was blocked as sialic acid methyl ester and partly split off by acid hydrolysis. About 55 % of the sialic acid found in the nerve ending membranes remained attached to the structure as compared with about 35 % of the sialic acid of the vesicles. The acid-resistant proportion of the sialic acid could be localized with CIH after saponification of the esterified preparations. The method described allows the electron-microscopical demonstration of acid-resistant, neuraminidase-sensitive sialic acid in synaptic structures and the discrimination from sulphated mucopolysaccharides.