Inhibition of two family 18 chitinases by various allosamidin derivatives

The inhibitory activities of several allosamidin derivatives on two family 18 chitinases, an insect enzyme from the epithelial cell line from Chironomus tentans, and a bacterial enzyme, chitinase A from Serratia marcescens, were evaluated. The following structural requirements are necessary for inhibition of the Chironomus enzyme: 1. One N-acetylallosamine residue can be omitted without impairment of enzyme inhibition. 2. At least one N-acetylallosamine sugar must be present. 3. Glucosamine can replace the allosamine moiety without a negative effect on the inhibitory activity. 4. The spatial arrangement of the allosamizoline moiety is important for inhibition. 5. If one sugar is omitted and the arrangement of the cyclitol residue is changed, the inhibitory effect is diminished further. For purified chitinase A from Serratia marcescens the arrangement of the aglycone moiety is equally important, but recognition of the sugar is different: 1. Omission of one allosamine residue decreases the inhibitory activity considerably. 2. Inhibition is improved if the remaining N-acetylallosamine is replaced by the epimer N-acetylglucosamine. Only endochitinase activity is affected, since chitin formation (up to 10(-4) M) and N-acetylglucosaminidase activity (up to 10(-3) M) are not impaired, at least in Chironomus cells. (C) 1998 SCI

FlAsH labeling of a nuclear receptor domain (D domain of ultraspiracle) fused to tetracysteine tag

Biarsenical fluorescein compounds feature unique fluorescence characteristics and special binding mechanism to tetracysteine tags with certain structures and these dyes offer a feasible method for site specific labeling of heterologously expressed proteins. We aimed FlAsH fluorescent labeling of tetracysteine fused hinge region of the ultraspiracle from Drosophila melanogaster (DmUSP-D domain) to facilitate functional studies of this receptor domain. A CCPGCC tetracysteine motif was integrated between His6, Gateway attB1, and Flag tags and attached to the N-terminus of the DmUSP-D. The fusion protein was expressed in Esherichia coli and the FlAsH labeling was performed in bacterial extracts, under conditions which are compatible with receptor function. The dye was bound to the tetracysteine tag with high affinity and complex stability and the labeling proved to be specific for the target fusion protein. Results indicate that FlAsH labeling of the internal CCPGCC motif can be a valuable tool for the functional characterisation of any nuclear receptor domains