Chitin synthase in Coprinus cinereus
A rapid technique for the preparation of large quantities of solubilized chitin synthase from Coprinus cinereus is described. This enzyme preparation is very stable and has high specific and total activities. Chitin synthase has a high level of activity in a number of "Good" buffers between pH 7.0 and pH 9.0. The enzyme is rapidly inactivated by exogenous proteolysis and chitin synthase zymogen has not been detected. Chitin synthase is probably a lipoprotein. Chitin synthase has a propensity to form regular aggregates in vitro and may exist as a multimolecular aggregate at the cell membrane. Proteolytic activity is associated with solubilized chitin synthase preparations. Uridine diphosphatase activity is associated with the solubilized chitin synthase preparations, but can be purified away from the chitin synthase activity. UDPase activity is probably not intimately associated with chitin synthase at the cell membrane, as part of a multi-enzyme complex. Fibrillar structures in electron micrographs of chitin produced by chitin synthase in vitro closely resemble chitin microfibrils observed in electron micrographs from other sources. The presence of digitonin during gel permeation chromatography is essential for the elution of chitin synthase from columns. Solubilized chitin synthase can be purified 60 to 70-fold by a gel permeation and ion-exchange purification sequence. Ammonium sulphate precipitation and hydroxylapatite chromatography are unsuitable for the purification of solubilized chitin synthase. Chitin synthase activity can be detected on polyacrylamide gels but the enzyme activity is ephemeral. Affinity chromatography and inhibitor studies suggest that the initial interaction of chitin synthase with UDP-GlcNAc involves the uridine moeity of the substrate molecule. Digitonin is an inhibitor of chitin synthase activity. Synthetic derivatives of uridine impose considerable inhibition on chitin synthase activity. Tunicamycin is probably a competitive inhibitor of chitin synthase. The polyoxin-type compound nikkomycin inhibits the growth of the yeast form of Candida albicans. Nikkomycin is effectively non-toxic against mammalian cells when compared with other anti-fungal drugs.