Solubilisation et caractérisation d'une glycoprotéine : fucosyl-transférase cérébrale

Abstract

Il existe au niveau du cortex cérébral de mouton une glycoprotéine: fucosyl-transférase très active après solubilisation par le Triton X 100. La purification de l'enzyme solubilisée a été abordée par chromatographie hydrophobe sur éthyl-agarose. Cette technique permet d'obtenir une fraction enzymatique partiellement purifiée et complètement débarrassée des activités pyrophosphatasiques contaminantes. Dans ces conditions, il a été possible de déterminer les principales propriétés physico-chimiques de la fucosyl-transférase (pH, température, rôle du Mn 2+ et de la concentration en substrats). La fraction obtenue semble homogène en chromatographie sur Ultrogel AcA 22 et ultracentrifugation analytique (&-Mw = 280.000, S 20 = 10), mais l'abaissement de la force ionique et l'action de dénaturants montrent qu'il s'agit d'un agrégat formé au cours de l'élution sur éthyl-agarose. o 1. A glycoprotein: fucosyl-transferase activity was demonstrated in sheep cerebral cortex, using desialylated fetuin as exogenous acceptor and detergent Trition X 100 for solubilization. 2. Addition of Triton X 100 to the membrane suspension gave first an activation then a solubilization of the cerebral fucosyl-transferase. 3. Hydrophobic chromatography was investigated for purification of the enzyme. Binding was effective using alkyl-agarose chromatographic columns with two or more than two atoms of carbon, but elution was only possible with ethyl and butyl-agarose. 4. Combination of subcellular fractionation and hydrophobic chromatography on ethylagarose led to a 30 fold purification. 5. After ethyl-agarose chromatography, some properties of fucosyl-transferase were studied: the optimal temperature was 25°C. The optimum pH was about neutrality. Light activation was observed with Mn 2+ concentration below 1 mM. 6. Homogeneity was tested using Ultrogel chromatography, polyacrylamide gel electrophoresis and ultracentrifugation. 7. It was concluded that ethyl-agarose hydrophobic chromatography easily bind a few solubilized proteins (about 20 per cent of the ST supernatant). When elution was performed, these proteins, including fucosyl-transferase, were released from ethyl-agarose columns as a stable aggregate, only dissociated with lower ionic strength. Ethyl-agarose fraction (eluted with KCl 120 mM) showed homogeneity: u — with Ultrogel AcA 22 chromatography (Mw = 300.000). — with polyacrylamide gel electrophoresis without S.D.S. — with the analytical ultracentrifugo giving Mw = 280.000; S 20 = 10. But after dialysis overnight against a buffer without KCl, ultracentrifugation technics showed no homogeneity. Futhermore, SDS gel electrophoresis gave more than four bands.