Integumental Chitin Synthase Activity in Cell-free Extracts of Larvae of the Australian Sheep Blowfly, Lucilia cuprina, and Two Other Species of Diptera

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Abstract
Chitin synthase activity was demonstrated in crude homogenates of larval integuments from L. cuprina and in similar preparations from Musca domestica and Calliphora erythrocephala. This is the 1st report of an insect integumental chitin synthase. This activity brings about the incorporation of radioactivity from UDP-N-acetyl-[14C]glucosamine into an ethanol- and alkali-insoluble form. A major part of this labeled product has been characterized as chitin by its insolubility in alkali, resistance to degradation by proteases and its susceptibility to digestion by chitinase and HCl. Most of the radioactivity solubilized during digestion by chitinase co-migrates with N-acetylglucosamine, glucosamine and chitobiose during paper chromatography. Some radioactivity also becomes incorporated into non-chitin products in this system. There is substantial evidence that incorporation is not brought about by whole epidermal cells or by microbial contamination in the homogenates. The extent of incorporation obtained with the homogenates is limited by the presence of degradative enzymes which rapidly break down the substrate (UDP-N-acetylglucosamine). The incorporation was partially inhibited (50-70%) by both polyoxin-D (apparent Ki 0.04 .mu.M) and diflubenzuron (apparent Ki 5-8 .mu.M). This is the 1st report of a cell-free chitin-synthesizing system derived from insect tissue which is sensitive to inhibition by diflubenzuron.