Oligosaccharides of arterial basement membrane-like material studied on rabbit aortic myomedial cells in culture

Abstract

Oligosaccharides from myomedial basement membrane-like material were characterized in respect of size, attachment to protein and charge heterogeneity after metabolic labelling with[3h] glucosamine. Arterial basement membrane-like material was isolated from cultures of aortic myomedial cells by a sonication-differential centrifugation technique. Glycopeptides were then obtained by proteinase digestion and Sephadex G-50 chromatography. Alkaline borohydride treatment of the glycopeptides followed by Sephadex G-25 chromatography revealed that 1014% of the oligosaccharides were released by a β-eliminative reaction. The molecular weight of the alkali labile carbohydrate units was estimated to be approximately 750. Alkali stable oligosaccharides were released from the glycopeptides by hydrazinolysis. The liberated oligosaccharides were retarded by Sephadex G-50 chromatography corresponding to a molecular weight of 2700 using unit B thyroglobulin oligosaccharides as standard. The chromatographic pattern obtained by anion exchange chromatography of the glycopeptides after neuraminidase treatment showed that a major part of the glycopeptides contain one or more sialic acid residues, but alsoother negatively charged groups - possibly phosphomannosyl residues were present as suggested by [ 32PO 4 3-] labelling and concanavalin-A-Sepharose chromatography. Concanavalin-A-Sepharose chromatography combined with α- mannosidase treatment and gel filtration suggested that a minor part of the glycopeptides were of high-mannose- type.