Synaptosomal plasma membrane glycoproteins: fractionation by affinity chromatography on concanavalin A

Abstract

Synaptosomal plasma membrane glycoproteins were solubilized in 0.08% sodium dodecyl sulfate (SDS) and separated by affinity chromatography on concanavalin A-Sepharose. Three fractions were obtained. Fraction CO (unadsorbed) contained 63% of the protein, but only 23% of the sugar and was rich in fucose, galactose and N-acetyl-neuraminic acid (NANA) relative to the other sugars. Many proteins were detected in this fraction by polyacrylamide gel electrophoresis, but only on band stained well for carbohydrate. Fraction CR (retarded) contained glycoproteins which reacted weakly with concanavalin A and stained poorly with periodic acid-Schiff reagent (PAS). There was no enrichment in total sugar/mg protein relative to the original fraction, but there was a marked enrichment in N-acetyl-glucosamine and NANA relative to the other sugars. The protein profile of this faction was complex, but only one major PAS-positive band was detected. However, most, if not all, the proteins seemed to be weakly PAS-positive. Fraction C1 (adsorbed) was markedly enriched (5-fold) in sugar/mg protein, particularly in mannose and N-acetyl-glucosamine. It had a relatively simple protein profile, and most of the protwin bands stained well with the PAS reaction. Glucose was detected in the initial fraction, and in all the subfractions, but it could not be shown definitely to be either a contaminant or an intrinsic constituent of synaptosomal plasma membrane (SPM) glycoproteins. Minor sugars, if present, could, at most, account for less than 0.25% of the carbohydrate.