Isolation of a novel N-acetylglucosamine-specific lectin from fresh sclerotia of the edible mushroom Pleurotus tuber-regium
Section snippets
Isolation procedure
Fresh sclerotia of P. tuber-regium collected in Mainland China (1.5 kg) were extracted with saline. After addition of (NH4)2SO4 to 30% saturation and removal of the precipitate, (NH4)2SO4 was added to 80% saturation. The precipitate was collected by centrifugation, dissolved, and then dialyzed before ion exchange chromatography on a DEAE–cellulose
Purification and N-terminal sequence of lectin
Hemagglutinating activity which could be inhibited by N-acetyl-d-glucosamine, hereinafter referred to as lectin activity, was eluted in peak D1 which was unadsorbed on DEAE–cellulose. Subsequent chromatography of D1 on immobilized N-acetyl-d-glucosamine fractionated it into a large unadsorbed peak N1 devoid of lectin activity and a small unadsorbed peak N2 with lectin activity (Fig. 1). FPLC-gel filtration of N2 on Superdex 75 yielded a large peak SU1 and a small peak SU2 (Fig. 2). Lectin
Discussion
Pleurotus tuber-regium lectin can be isolated with a relatively simple procedure involving (NH4)2SO4 precipitation, ion exchange chromatography on DEAE–cellulose, affinity chromatography on N-acetylglucosamine–agarose, and FPLC-gel filtration on Superdex 75.
Not many N-acetyl-d-glucosamine-specific lectins have been reported in the literature. What germ agglutinin is one of them. However, it also demonstrates specificity for sialic acid. It is composed of subunits with a molecular mass of 36 kDa
Acknowledgements
The award of a direct grant by the Medicine Panel, Research Committee, The Chinese University of Hong Kong and the expert secretarial assistance of Miss Fion Yung are gratefully acknowledged.
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