Gut chitin synthase and sterols from larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae)

Gut chitin synthase was characterized and the sterols and ecdysteroids in the sugarcane rootstalk borer weevil, Diaprepes abbreviatus , were identified. An in vitro cell‐free chitin synthase assay was developed using larval gut tissues from D. abbreviatus . Subcellular fractionation experiments showed that the majority of chitin synthase activity was located in 10,000g pellets. The gut chitin synthase requires Mg 2+ to be fully active: 7–8‐fold increases in activity were obtained with 10 mM Mg 2+ present in reaction mixture. Calcium also stimulated activity (4–5‐fold with 10 mM Ca 2+ ), while Cu +2 completely inhibited at 1 mM. Other monovalent and divalent cations had little or no effect on activity. The pH and temperature optima were 7 and 25°C, respectively. Gut chitin synthesis was activated ca. 50% by trypsin treatments. GlcNAc stimulated chitin synthase activity, but Glc, GlcN and glycerin did not. Polyoxin D, UDP, and ADP inhibited the chitin synthase reaction with I 50 's of 75 μM, 2.3 mM, and 3.6 mM, respectively. Nikkomycin Z was a potent inhibitor of chitin synthase (91% inhibition at 10 μM). Tunicamycin and diflubenzuron had no effect on the enzyme. The apparent Km and V max for the gut chitin synthase were, respectively, 122.5 ± 7.4 μM and 426 ± 19.7 pmol/h/mg protein utilizing UDP‐GlcNAc as the substrate. Sterol analyses indicated that cholesterol was the major dietary and larval sterol. HPLC/RIA data indicated that 20‐hydroxyecdysone was the major molting hormone.