文摘
Chitosomes, small secretory vesicles with S-value of ca 100 that act as conveyors of proteolytically-activatable chitin synthase (ChS) to its site of action at the cell surface of fungi, display an unusually high affinity for concanavalin A (ConA), if present in a compacted milieu. The outstandingly strong bonding between the lectin and 100 S-ChS established under this condition in all probability results from a superposition of four types of interaction: ‘classical’ lectin recognition, hydrophobic complexation, extended lectin-receptor bridging, and lectin-mediated liposome/liposome aggregation. The following three-step-procedure for the purification of ChS, together with the result of lipo-selective affinity chromatography (AC) of the enzyme on heparin, allowed the identification of a single 60-kDa polypeptide as a UDPGlcNAc transferase with defining enzymic properties of ChS: (i) isolation of gradient-purified chitosomes according to a standard procedure; (ii) ConA-AC of chitosomes to remove non-binding as well as other contaminating proteins that interact with the lectin only at its saccharide binding site; (iii) selective desorption of ChS with digitonin, methyl mannoside or NaCl following dilution of the chitosome-loaded ConA-gel. The results support the notion of a glycoconjugate nature of ChS, provide a means of obtaining homogeneous preparations of ChS for structural and mechanistic analyses as well as for biotechnological applications, throw light on some hitherto unexplained findings encountered using the Scarborough method to label the plasma membrane with ConA, and weaken the experimental basis for the firmly entrenched tenet of the plasma membrane as the only locus operandi of ChS.
