Thiamine as an integral component of brain synaptosomal membranes

Synaptic plasma membranes were prepared from rat cerebral cortex to determine if thiamine was localized in the membranes. The synaptosomes, prepared by discontinuous sucrose gradient centrifugation, were subjected to osmotic shock at pH 9.5 for 10--15 min and subfractionated on a discontinuous sucrose gradient. The two membrane fractions that were obtained were free of contamination by mitochondrial membrane and soluble fractions. In order to ensure specificity, thiamine was assayed fluorometrically before and after the addition of thiaminase I (thiamin:base 2-methyl-4-aminopyrimidine-5-methenyltransferase, EC 2.5.1.2). The thiamine content of the two membrane fractions was 9 and 10 pmol/mg protein.